physiological and pathological regulation of thyroid cell … · 2017-10-19 · thyroid maintains...

PHYSIOLOGICAL REVIEWS Vol. 72, No. 3, July 1992

Printed iT2 U.S.A.

Physiological and Pathological Regulation of Thyroid Cell Proliferation and Differentiation by Thyrotropin

and Other Factors J. E. DUMONT, F. LAMY, P. ROGER, AND C. MAENHAUT

Institut de Recherche Interdisciplinaire, Faculte’ de Mdecine, H6pital Erasme, Universite’ Libre de Bruxelles, Campus Erasme, Brussels, Belgium

I. Cell Proliferation in Normal Thyroid Tissue In Vivo ............................................... A. Physiological situation ........................................................................... B. Growth under stimulatory conditions ........................................................... C. Coordination between various thyroid cell types ................................................ D. Stem cells ........................................................................................

II. Methodology and Model Systems for Study of Thyroid Growth ................................... A. Model systems ................................................................................... B. Methodology: indexes of cell proliferation ...................................................... C. Working definition: growth control ratio ........................................................

III. Differentiation, Determination, Differentiation Expression ....................................... A. Thyroid system .................................................................................. B. Model for thyroid ................................................................................

IV. Extracellular Signals Involved in Control of Proliferation of Thyroid Cells ....................... A. Thyrotropin ...................................................................................... B. Iodine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Other hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Pathological signals .............................................................................. E. Local signals .....................................................................................

V. Pathways Regulating Thyroid Cell Proliferation ................................................... A. General considerations .......................................................................... B. Mitogenic and differentiating action of thyrotropin ............................................ C. Effect of factors other than thyrotropin on proliferation and differentiation ................. D. Role of adenosine 3’,5’-cyclic monophosphate, phosphatidylinositol bisphosphate, and tyrosine

protein kinase cascades in thyrocyte proliferation ......................................... E. Kinetics of biochemical events in various thyroid mitogenic pathways ........................ F. Role of autocrine loop ............................................................................

VI. Apparently Opposite Programs: Proliferation and Differentiation Expression Are Induced by Thyrotropin and Adenosine 3’,5’-Cyclic Monophosphate Cascade ..............................

VII. Growth Control and Goitrogenesis .................................................................. A. General concepts ................................................................................. B. Graves’ disease ................................................................................... C. Congenital defects ............................................................................... D. Thyroid adenoma ................................................................................ E. Sporadic goiter ...................................................................................

VIII. Hypothetical and Existing Examples of Constitutive Activation of Adenosine 3’,5’-Cyclic Monophosphate Cascade ........................................................................

A. Adenylate cyclase-coupled receptors ............................................................ B. Cyclase-activating guanosine triphosphate-binding protein .................................... C. Adenosine 3’,5’-cyclic monophosphate-dependent protein kinase ............................... D. Negative controls of adenosine 3’,5’-cyclic monophosphate cascade .............................

IX. Conclusions ..........................................................................................

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I. CELL PROLIFERATION IN NORMAL THYROID TISSUE cles and of their supporting mesenchymal tissue and IN VIVO cells, the endothelial cells of the capillaries (20% ) and

the fibroblasts (10%) (78). Scarce calcitonin-secreting A. Physiological Situation parafollicular cells are located at the periphery of the

follicles. After its differentiation in the fetus, the tissue The thyroid tissue is mainly composed of thyroid grows roughly in parallel with body weight and remains

follicular cells, the thyrocytes (70954, arranged in folli- at the same size throughout adult life. As the fetal thy-

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668 DUMONT, LAMY, ROGER, AND MAENHAUT Volume 72

roid weighs -0.2 g at 20-25 wk in the fetus and -15 g in the adult and assuming a grossly similar tissue composition, this growth requires at least six to seven cell divisions. Human thyroid cells divide about five times in adulthood, which demonstrates that there is a slow constant turnover of these cells, with cell division and cell death compensating each other. When corrected for the life of the animal, the evaluated cell turnover is about five in adulthood for humans and for animals as different as dogs, rats, and mice (49). The calculation of the cell turnover in the whole gland does not depend on any assumption about the homogeneity of the cell population. However, if only a fraction of the population was involved in the turnover (e.g., f = 0.4) (45,298), the number of possible divisions in these cells (life span) would be correspondingly higher (n = 5/f = 12.5).

Although in adults, under constant conditions, the thyroid maintains its size with a slow cell turnover, it retains the capacity to grow by cell hypertrophy and proliferation in response to a stimulus. The size and function of the thyroid are controlled by a physiological negative feedback mechanism: the thyroid cell secretes thyroid hormones that inhibit the secretion by pituitary thyrotrophs of thyrotropin (TSH), the thyroid-stimulating hormone. Whenever thyroid hormone secretion decreases, as in iodine metabolism defects, iodine deficiency, or after goitrogen or antithyroid drug administration, TSH secretion increases, causing an activation of thyroid function and growth (77, 82, 88, 177).

B. Growth Under Stimulator-y Conditions

In vivo growth, as induced by goitrogen administration in rats, is followed by a progressive increase in thyroid weight, which plateaus after 3 mo (at Xl2 the origi- nal volume). In terms of relative components of the tissue, it first involves a fall in follicular lumen space and nonvascular stroma and a rise in epithelial cells and blood vessel space, with no further changes after 7-10 days (366). The proliferation of endothelial cells pre- cedes that of the thyrocytes (ZOl), and capillary vascularization varies more than follicular cell mass with the level of thyroid stimulation (140, 299, 357). Global increase in epithelial cell space is due to cell hypertrophy and cell multiplication; it is accompanied by folliculo- genesis (69, 70). The thyroid capsule also grows (356).

The fact that growth stops after 3 mo despite per- sistent high levels of TSH suggests an inherent limitation in the cells or, more simply, a desensitization of the cells to the effect of TSH (246, 258, 364, 365). Mitogenic effect of wounding in such a tissue shows that the desensitization is TSH specific (362, 367). As the mitotic rate at the plateau level remains elevated, the cell turnover itself remains higher than in control tissue. How- ever, if goitrogen is withdrawn from the diet for 3 wk, bringing back TSH levels to normal, the mitotic rate returns to normal, and follicular cell number slightly decreases. Restimulation by goitrogens brings back all parameters to their values before withdrawal. This does not fit in with a simple TSH desensitization mechanism, which should be fully reversible (364). The desensitiza-

tion is specific to TSH, since the in vitro effect of insulin at high concentrations but not of TSH persists (302). Other effects of TSH are not desensitized, which shows that the desensitization affects only the growth-promoting action of TSH (308,365). Indeed, the TSH receptor itself desensitizes little, and the expression of the gene is not downregulated by chronic stimulation (Maenhaut and Dumont, unpublished observations). Growth desensitization is observed in cultured cells from treated animals, which excludes the hypothesis of a chalone (308). Similarly, under constant stimulation after partial thyroidectomy, rat thyroids do not recover their normal size (77). This shows an inherent, albeit relative, limitation of the growth potenti al of the thyrocyte POP ulation as in other cells ( 114). It might reflect a limited life span (number of possible divisions) of the thyrocytes: in primary cell cultures no more than five to six divisions can be obtained (P. P. Roger, M. Baptist, and J. E. Dumont, unpublished observations). Also in thyroidectomized rats, 2 X lo3 incubated thyrocytes cannot restore normal function, whereas 6 X IO4 can (76). However, there are several arguments against this “very limited life span” hypothesis. In the constantly stimulated rat thyroid, although the weight and presumably the total number of cells do not increase further after 3 mo, the labeling index, although lower than at peak growth, is still elevated, which rather suggests a dynamic equilibrium, i.e., continuous stimulated cell proliferation compensated by increased cell death rate (366). Similarly, in dogs chronically stimulated by goitrogens the labeling index is still 8% after 2 mo, corresponding to a doubling time of 4-5 days, whereas the weight of the gland is barely increased (Maenhaut and Dumont, unpublished observations). In transgenic mice expressing a physiologically constit ut ive adenosine receptor activating adenylate cyclase, a labeling index of 6-8s (control ~0.5%) is found after 3-7 mo (corresponding doubling time ~4 days), and thyroid weights up to 400 mg (normal weight 1 mg) are observed after 9 mo (C. Ledent and J. E. Dumont, unpublished observations). It is interesting that this labeling index is normal and is barely increased in very young transgenic mice. The inherent limitation of thyroid cell number is therefore tighter than the limitation of the cell’s life span. This suggests increased cell death in chronically stimulated gland. Cell death or cell loss has been demonstrated in normal and chronically stimulated rat thyroids (44). The role and mechanism of such a process is unexplained, although it is interesting to note that in leukemia cells, adenosine 3’,5’-cyclic monophosphate (CAMP), which in the thyroid mediates TSH effects, induces programmed cell death (apoptosis) (176). In rat thyroid cells in culture, TSH decreases the persistence of [3H]DNA, which suggests it induces cell death (305). Thus, although the life span of the thyrocyte is certainly limited, it is probably higher than the limit reached under in vivo physiological situations or in vitro.

C. Coordination Between Various Thyroid Cell Types

Parenchymal, stromal, and endothelial cells (in thyroid capillaries, in the proximal part of veins and lym-

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FIG. 1. Schematic representation of possible cell interrelations in thyroid tissue. Input, known extracellular signals acting on thyrocyte. Output, release of extracellular signals by thyrocyte. These can act on thyrocyte itself (autocrine loops) or on fibroblasts, endothelial, or calci tonin-secreting (C) cells (paracrine loops). Products of those cells may also influence thyrocytes. cGRP, calcitonin growth factor; FGF, fibroblast growth factor; GH, growth hormone; I-, iodide; IGF, insulin-like growth factor (somatomedin); ILs, interleukin-6; IT, iodotyrosine; PGE, prostaglandin E; T,, triiodothyronine; T,, thyroxin; TG, thyroglobulin; TGF-P, transforming growth factor-b; TGT;, thyroxin and iodotyrosine of thyroglobulin; TSH, thyrotropin; X, putative unknown signal; ?, hypothetical.

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phatics, and in the distal segment of thyroid arteries) all proliferate during growth (53, 201, 299, 357, 365). Even the capsule greatly increases in thickness in stimulated rat thyroid (356). After iodide treatment the vascularization of the hyperstimulated thyroids regresses earlier than thyrocyte function in rats (274). These facts imply a cross signaling between these cells. Presumably, it is the thyrocyte that must coordinate the response of the other cell populations of the thyroid (70,115; Fig. 1). It is the obvious site of control of the information about TSH concentration and action, iodine supply and metabolism, and thyroid hormone formation. The role of the thyrocyte is supported by the induction by grafted do- nor thyrocytes of angiogenesis from cells of the recipient nude mice (215).

The intercellular paracrine signals generated by the follicular cells are not known, although several potential candidate molecules are secreted by thyroid cells in culture: insulin-like growth factor I (IGF-I) (14,196), plasminogen activator (199), fibroblast growth factor (FGF) (92,117), transforming growth factor-p (TGF-P) (120,217), interleukin-6 (345), and probably atria1 natriuretic factor (6,292). Other candidates should be considered: adenosine, the oxygen-sensitive signal (3,289), nitric oxide (216), and angiogenic factors (101). The possible paracrine relations between calcitonin-secreting (C) cells and thyrocytes have not been studied, although effects of calcemia on thyrocytes may well be mediated through C-cells (113, 157), and the parallelism between mitotic index of C-cells and follicular cells during the circadian rhythm suggests a relationship (156).

D. Stem Cells

Although, as in the liver (291), most of the thyrocytes in the thyroid appear to be able to respond by a few divisions to a proliferation stimulus in vivo and in vitro,

the existence of a population of stem cells able to replen- ish the pool of fully differentiated and proliferation- limited thyrocytes has been postulated. This hypothesis rests on indirect but suggestive evidence: I) the growth of thyroid transplants in recipient animals requires the injection of a minimal number of cells (219), which might measure the relative frequency of stem cells; and 2) in cloning assays in radiobiological experiments, foci formation occurs but with very low efficiency. From both types of experiments the frequency of stem cells would be estimated at most to be l/1,000. Such stem cells could be primary candidates for transformation and tumorigenesis, but the arguments in favor of their existence are indirect, and until now, we and presumably others have failed to demonstrate it directly. Cer- tainly in dog thyroid cells in primary culture, in the absence of stimulation, double labeling with [3H]thymidine and bromodeoxyuridine at several days interval is exceedingly rare (P. P. Roger, M. Baptist, and J. E. Du- mont, unpublished observations).

II. METHODOLOGY AND MODEL SYSTEMS FOR STUDY

OF THYROID GROWTH

A. Model Systems

In the study of organ function and growth the in- vestigator may resort to many different systems, from humans in vivo, who are our ultimate subject, to the purified enzyme. Often when we choose one particular experimental model we tend to be skeptical about the others. This should not necessarily be so if we respect two caveats. Going down the ladder from the complex physiological in vivo system to the clean in vitro purified systems, at each step the data become more precise, the relations simpler, and therefore the conclusions


clearer. However, at each step we lose some of the characteristics of the in vivo situation (in terms of controls, environment, and structure). Data collected in vivo may lead to erroneous conclusions because of the complexity of the systems involved; data obtained on simplified preparations may have no relevance to the in vivo situation.

Clinical experience with patients treated with antithyroid drugs or thyroid hormones, now backed by sophisticated and precise instrumentation, demonstrates every day the basics of thyroid growth control in humans in vivo. In vivo work on animals in which [3H]thymidine has been injected has provided us our concepts on the in vivo regulation of growth as well as the parameters of the cell cycle of thyroid cells. Exoge- nous transplants of human tissue in nude mice are in an in vivo environment, subject to endocrine but not neural control, with a reconstituted normal follicular architecture. They have allowed researchers to distinguish hyperfunction and growth due to the environmental milieu (Graves’ disease) and to an intrinsic cell property (autonomous adenoma) (152).

In vitro perfusion and the slices system lose the nervous control, and probably the paracrine controls, as extracellular signal molecules are diluted in the medium. However, such systems retain the normal tissue architecture and some physiological properties (secretion, thyroid hormone synthesis, TSH control). Studies of such systems have allowed the demonstration of the rich diversity of the patterns of neurotransmitter action from one species to another but also of the intracellular signals and cascades involved in the regulation of function. Although some first steps in the promotion of growth can be studied (such as ornithine decarboxylase induction), the short lifetime (4-7 h) of the system never allows researchers to verify if the end result, true growth, DNA synthesis, or mitosis, would have oc- curred. However, it must be said that in many studies on growth control in cell cultures this is not verified either. In the literature, the relationship of so-called early mitogenic events to proliferation is often only the fact that they are induced by growth factors.

Primary cell cultures have allowed for the demonstration of the direct mitogenic effect of TSH through CAMP in dog, rat, and human thyroid cells. The time limitations of these systems (-2 wk) have not allowed more than a few rounds of mitoses to be demonstrated in these cells. If cultured in the right conditions, these cells can regain their polarity and the asymmetry of their normal tissue architecture. They become then the best model for studies of transport.

In in vitro primary culture of dog thyroid cells, TSH triggers, in the absence of serum, or enhances, in the presence of serum, the proliferation of thyrocytes. Under maximal stimulation [i.e., with TSH, serum, and epidermal growth factor (EGF)], almost all the thyrocytes synthesize DNA, which shows that, as in vivo, most thyrocytes are able to respond (270). However, even under such stimulation, they do not divide more than four to six times. This limitation could be due to an

artifact (e.g., a necessary factor is missing) of the system. However, it is tempting to relate this limited life span to the known in vivo limits.

Thyroid cell lines are obviously very remote from physiology. The Fisher rat thyroid cell line, FRTL-5, that is mostly used (8) loses, for instance, the growth control by EGF, the Ca2+-phosphatidylinositol (PI) response to TSH, and iodide binding to proteins (89, 91). Cell lines may also have acquired new non-physiologically relevant controls, for example, P-receptors (93). Nevertheless, such cells are very useful not only for their simplicity and reproducibility but because they allow permanent transfections and genetic experiments. Moreover, they are perfectly suitable for the study of the metabolisms and the controls that they have re- tained, such as the control of growth by the CAMP pathway.

In cell cultures, except perhaps for whole follicles in suspension or polarized monolayers, the control of growth by the extracellular matrix is probably dis- turbed or nonexistent. It should also be realized that culture per se has a profound effect on cell proliferation: while in vivo the labeling index of dog thyrocytes is 4 X

10s4 and in quiescent cultures it is 5 X 10m3 (49).

B. Methodology: Indexes of Cell Proliferation

The various ways to test a growth-promoting effect on thyroid cells have been reviewed (87). The cytochemi- cal assay of glucose-6-phosphate dehydrogenase activity (80) was based on the unproven assumption that the flux of glucose through the pentose phosphate pathway reflects growth and on the erroneous postulate that this flux depends only on the activity of the first enzyme of this pathway.

The measurement of [3H]thymidine incorporation into DNA as such reflects DNA synthesis but also all the variations of the thymidine phosphate precursor pool specific activity that itself depends on the uptake and phosphorylation of exogenous thymidine, on the relative rate of endogenous thymidine phosphate synthesis, and on the degradation of thymidine phosphate (87,174, 225). There are numerous examples of discrepancies between measurements of thymidine incorporation in tri- chloroacetic acid precipitate of cells and true DNA synthesis (28,87,300). However, even glaring discrepancies between measurements of [3H]thymidine uptake and cell proliferation are sometimes not noticed in their own data by overenthusiastic researchers (19).

Cell number measures the net rate of cell proliferation resulting from the balance of proliferation and cell death. The DNA content gives the same information if the cells are diploid. Any change in the balance may result from a change in the proliferation rate or in the death rate. Although the latter is often minor in cell cultures, this should be checked. As mentioned, the steady state of hyperstimulated goiters results from an increased proliferation rate and death rate.

The number of mitoses (measured in the presence

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or absence of colchicine), the fraction of nuclei labeled with [3H]thymidine or bromodeoxyuridine, or the fraction of the cells between the diploid and tetraploid stage (as observed with cell sorter) measure the number of cells reaching a defined stage of the cell cycle (mitosis, DNA synthesis) after the no-return decision to go through one round of divisions. Applying these techniques on in vivo-obtained specimens immediately after removal gives information on the fraction of the cells that are at a particular stage of the mitotic cycle in vivo. However, such data as well as histological examination are snapshots; they only give information at one point in time. Similar examination 1 h or 1 mo after could reveal that the same cell or follicle that looked active at one time may be quiescent later (139).

In general all the techniques used to measure cell proliferation have drawbacks; therefore none should be used alone in the assessment of proliferation rate (87, 121). These methodological considerations are now accepted by a growing fraction of workers in the thyroid field, if not in others (it is striking to see how many so-called reputable journals accept articles on proliferation based only on [3H]thymidine uptakes in DNA).

Apparently negative results or discrepancies between results obtained by valid methodology with different models should be examined critically. I) In dog and rat thyroid cells and in other cell cultures the mito- genie effects of TSH and EGF require the presence in the medium of IGF-I and/or insulin. This shows that an absence of effect in a system may always be due to the absence of a required factor. 2) The concentration-effects relationship of TSH is often biphasic. 3) Due to polarity of cells in some systems, receptors may not be accessible to the culture medium. 4) For the control of function as well as of growth, species differences may exist. In dog thyroid, the CAMP system is negatively controlled by norepinephrine through Ni at the level of the cyclase and the Ca2’ -PI cascade is activated by ace- tylcholine through muscarinic receptors. The converse situation is found in mouse thyroids. Thyrotropin activates Ca2’-PI cascade in human but not in dog thyroid cells or in the FRTL-5 cell line. 5) The history of the investigated cells may modify their responses to a given agent. Depending on the pretreatment of pig thyroid cells in primary culture, receptors or GTP-binding transducing proteins appear or disappear (206,284). One should therefore, as always, remain very cautious about negative results. It is easy not to observe a phenomenon.

C. Working De3nition: Growth Control Ratio

When mitochondria are incubated with ADP their respiration is increased. The ratio of respiration in the presence versus in the absence of ADP is called the respiratory control. In other systems this ratio of activity in the presence of maximal concentrations of stimulant versus in its absence is called the stimulation ratio. The respiratory control and the stimulation ratio define the tightness of a control. Damaged mitochondria have a

higher basal respiration and a lower respiratory control than intact mitochondria (loose coupling). The damage is not characterized by a lower activity but by loss of control or uncoupling. In systems like proliferating cells or cells expressing genes, the coupling or tightness of control better characterizes the performance of the system than the basal activity of the measured process. In thyroid cell culture systems, the ratio of growth with or without TSH (growth control ratio) may vary between 1 and 100. In some cases (dog thyroid cells in the absence of serum, FRTL-5 cells) the control is almost all or none.

III. DIFFERENTIATION, DETERMINATION,

DIFFERENTIATION EXPRESSION

A. Thyroid System

In many systems proliferation and differentiation are antagonistic processes. As used in cell culture work these concepts may introduce confusion. In embryology when a cell, by an epigenetic mechanism, begins a new more differentiated lineage the process is called determination. On the other hand, a determined cell may express more or less the characteristics of its type; we call this modulation, differentiation expression. In thyroid cell culture, incubation with U-O-tetradecanoylphorbol-13-acetate (TPA) or EGF shuts off the specialized thyroid genes (thyroglobulin, thyroperoxidase). How- ever, these cells remain and remember that they are thyroid cells. Treated with TSH or with any agent enhancing CAMP accumulation, they will again fully express their specialized genes. When treated with EGF and TPA the cells remain determined but lose their differentiation expression and recover the latter when their CAMP increases. These concepts find their coun- terpart at the level of the proteins involved in specific thyroid cell control and of the chromatin. To remain determined and inserted in their physiological regulatory network the follicular cells must recognize the signals addressed to them and respond properly. Indeed, the TSH receptor mRNA levels are remarkably stable in thyroid cells in vivo and in vitro. Dedifferentiation with EGF and phorbol esters in vitro never abolishes TSH receptor mRNA expression, whereas it suppresses thyroperoxidase and thyroglobulin gene expression (Maen- haut and Dumont, unpublished observations). Simi- larly, in thyroid tumors, TSH receptor mRNA remains present when the other two have disappeared (234; G. Brabant, C. Maenhaut, and J. E. Dumont, unpublished observations). One would expect the same stability for the key proteins and genes involved more distally in the specific response of the thyroid cell to its physiological signals [e.g., thyroglobulin transacting factor 1 (TTF-1)] (46). At the level of chromatin, active differentiation- expressing dog thyroid cells exhibit two DNase-sensitive sites in the promoter of thyroglobulin. When treated with EGF they lose the downstream site; the upstream site remains open in the cells that are still


determined; it is not present in the chromatin of nonthyroid cells (122).

The stage of determination without differentiation expression is different from the undifferentiated state preceding differentiation in the embryo. It is probably during the transition between these states that the upstream DNase-sensitive site of the thyroglobulin promoter, specific for thyroid tissue, appears (122).

In vivo the integrity or even the presence of the hypothalamic hypophysis system is not necessary for the full differentiation and early growth of the thyroid in the embryo. Complete normal morphological development of the thyroid (e.g., follicular structure, with colloid) is observed in the absence of TSH in vitro (368) in anencephalic human fetuses missing the hypothalamus and hypophysis, in decapitated rabbit fetuses (153), and in chick embryos (134). In the latter case, thyroid growth and accumulation of thyroglobulin were present but diminished in the absence of the pituitary. This suggests that pituitary hormones stimulate but do not induce development of the thyroid and do not affect its differentiation in vivo. However, it should be pointed out that in none of these experiments were blood pituitary hormones (TSH, growth hormone) in the fetus actually measured, and therefore the remote possibility of such an action by extrapituitary factors has not been excluded.

B. Model for Thyroid

Our present knowledge of the various stages of thyroid cell differentiation in vivo and in vitro can be sum- marized in the scheme outlined in Figure 2. This scheme should be considered only as a working model, but it has the merit to provide a conceptual framework for further investigations. It certainly grossly oversimplifies the very complex and multistage pathway of differentiation. It distinguishes the stages of the totipotent embryonic cell (I), the determined cell that has acquired its thyroid differentiation (V), the cell expressing this differentiation after the start of thyroid function in the fetus (VI), the hyperfunctioning cell under intense physiological or pathological (e.g., hyperthyroidism) conditions (VIc), or the resting cell relieved of TSH positive control (e.g., in pituitary myxoedema) (Via).

The totipotent embryonic cell (I) has by definition no characteristics of the thyroid cell. The determined cell is a postulated stage (V) in which the genes of the proteins that normally insert the thyroid in its physiological network are expressed, e.g., the TSH receptor and the transacting factors conferring specific thyroid gene expression (such as TTF-1) (46, 296). This cell knows or “remembers” its differentiation (344). How- ever, none of the specific functional genes (e.g., thyroglobulin) or functions are expressed. Dog thyroid cells in primary culture can be led to a similar stage by treatment with dedifferentiating agents, such as EGF or phorbol esters (42, 273). In cultured cells at this stage the upstream DNase-sensitive site of thyroglobulin is open; it remains inaccessible in other cell tvpes (122).

Thus this postulated embryonic stage is proposed to present well-defined characteristics of an experimen- tally obtained cell culture situation. It is realized of course that the transition between the totipotent and the determined cell most probably involves multiple steps (II, III, IV?). Whether they are irreversible or require continuous regulation remains an open question (26). Transformation by vKi-ras, which fully represses TTF-1 and thyroglobulin expression may correspond to a reversion from the determined cell stage to a less differentiated (I to V) stage (13, 21). The transition from the determined (V) to the “physiological cell” (VI) reflects the expression of the acquired differentiation, i.e., a “modulation of differentiation” (344).

The cell in physiological conditions (VIb), for instance, in the normal adult, expresses, albeit at an intermediate level, all the thyroid-specific genes (thyroglobulin, thyroperoxidase) and functions (iodide transport, thyroid hormone synthesis, and secretion). Depending on the level of stimulation, these cells can be brought to quiescence (Via) or hyperfunction (VIc). In vivo, quiescence is obtained by TSH suppression (by thyroid hormone treatment, hypophysectomy); hyperfunction results from activation of the TSH receptor and its dependent regulatory cascade. The differences between these three latter situations are merely quantitative in terms of level of gene expression and function. In the rat in vivo a thyroid-specific gene such as thyroglobulin is already maximally expressed under physiological conditions but can be repressed by inhibition of TSH secretion by a treatment with thyroid hormone (287, 288, 335). In the dog the level of expression of the thyroglobulin gene is very low after triiodothyronine (T3) treatment, intermediate under physiological conditions, and appears enhanced in animals with high TSH plasma levels resulting from a treatment with antithyroid drugs (Maenhaut and Dumont, unpublished observations). Dog thyroid cells in primary culture incubated without TSH correspond to the “resting” stage, and those incubated with TSH correspond to the hyperfunctioning stage. Slices from the thyroids of experimental animals, reflecting their in vivo situation, correspond to the physiological conditions but can (333) in the presence of TSH reproduce the stimulation in vivo or in culture by this hormone for a short time period (hours).

The phosphorylations of many proteins in cells or slices take place within minutes. They represent the rapid posttranslational effects of TSH involved in the acute regulation of function. Phosphorylations of his- tone Hl(l69a), H3, or high-mobility group (HMG) 14/17 proteins (55, 56) also observed under these conditions may reflect a first step of the more delayed transcriptional effects of TSH that lead to growth and chronic hyperfunction. Phosphorylation of HMG 14 inhibits its interaction with nucleosomes and therefore modifies the structure of chromatin (304).

In cell culture the passage from the determined cell to the cell expressing differentiation requires several days (86, 261, 262, 266, 273, 334) and in the case of the thvroglobulin gene is enhanced bv insulin (108). In slices

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TGmRNA 0 0 + +++ +++ Tg gene tmn8cription

0 0 + +++ +++ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................................................. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

TG 0 0 + +++ +++ T4 SWlHESlS 0 0 0 + +++ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..................................................................................... . . . . . . . . . ..-............................................................................

. . ..SEGRrnQN 0 0 0 + +++ . . . TA I - 0 0 0 + +++ Hi8tone H phospho ation d - 0 0 ++

FIG. 2. Schematic representation of hypothetical model of stages of thyroid cell differentiation. Stages are I, embryonic totipotent cell; II, III, IV, putative intermediate stages between I and V; V, determined thyrocyte not expressing differentiation; VI, functional thyrocyte expressing differentiation; Via, quiescent; VIb, physiological level of activity; VIc, hyperstimulated. Arrows indicate transitions between stages (determination, differentiation expression, stimulation, hyperstimulation) with agents inducing these transitions in vitro or for stimulation in vivo or in vitro and delays involved. For each stage, level of expression or functional activity is indicated. EGF, epidermal growth factor; TA I-, iodide transport; Tg, TG, thyroglobulin; TPA, 1%0-tetradecanoylphorbol-13-acetate; TSH with line through it, not controlled by TSH; TSH-R, thyrotropin receptor; TTF-1, thyroglobulin transacting factor 1; ?, not known or hypothetical.

in vitro or in rats in vivo the activation of thyroglobulin expression only requires 1 h (108,109). Thus the delays involved in the transition from quiescent to stimulated states are much faster than the transition from determined cell to the cell expressing differentiation. Until now, in dog thyroid cells at least, all the effects of TSH on this differentiation pathway were reproduced by the activation of the CAMP cascade. They are assumed to represent similar CAMP-mediated steps of differentiation in the embryo (247). Both EGF and phorbol esters when applied to cells pretreated with TSH or forskolin dedifferentiate them to the “determined cell” stage (V).

IV. EXTRACELLULAR SIGNALS INVOLVED IN CONTROL OF PROLIFERATION OF THYROID CELLS

A. Thyrotropin

In general the major element controlling thyroid growth in vivo is the level of TSH (82). In addition to the classic endocrinological evidence, much new experimental physiology supports this concept. Proliferation, as evaluated by mitotic activity in young rats, follows TSH levels: I) it increases by a factor of five in goitrogen- treated rats (TSH X 54) (366), 2) it follows the circadian

rhythm of TSH (363), and 3) its circadian rhythm disap- pears with the TSH rhythm in goitrogen-treated rats (363). The growth of thyroid grafts in recipient animals greatly increases in thyroidectomized animals (greatly decreasing the influence of donors’ age). It increases and then decreases with the age of the recipient in parallel with serum active TSH levels. It increases in male versus female recipients but not in gonadectomized animals in parallel with TSH levels (341).

Thyrotropin-increasing treatments markedly enhance the growth of human thyroid tissue transplanted in nude mice (244). In several species, including humans, TSH promotes the proliferation of thyrocytes in culture (27.2). The lack of stimulatory or even the inhibitory effects observed on porcine and beef thyroid cells remain exceptions (106,110,130,342) perhaps due to artifacts of the culture system or the absence of comitogenic factors.

There is some in vivo evidence that the stimulation of thyroid growth by TSH is partly dependent on other hormones. Growth effects of TSH are reduced in hypophysectomized (145) or adrenalectomized rats (150). In vitro, optimal growth effects of TSH require IGF or insulin as comitogenic factors in several systems (269,270, 272, 303, 325-328, 353). Moreover the stimulus of iodine deficiency causes goiter in normal populations but not in pygmees, who are congenitally deficient in IGF-I (B.


Contempre and J. E. Dumont, unpublished observations).

B. Iodine

Iodine deficiency in hypophysectomized animals also induces some thyroid growth. Iodine, as such, thus exerts a negative endogenous control on the thyrocyte growth. Moreover iodine deficiency increases the sensitivity of the thyroid to the goitrogenic effects of TSH (30, 204). Thus iodine deficiency may render a normal TSH concentration goitrogenic. That this occurs in the physiological range is shown by the inhibitory effects of moderate iodine supplementation in humans (41, 151). The relief of direct effects of iodine thus partly accounts for the goitrogenic action of antithyroid drugs in vivo (309). Conversely the stimulated thyroid is more sensitive to iodide (47). The inhibitory effect of iodide has been partially reproduced in FRTL-5 cells (279). An iodolactone has been proposed as a putative intermediate in iodide action, as it inhibits the proliferation of rat and porcine thyroid cells in vivo and in vitro, respectively (81,249). However, because these cells require exogenous arachidonate to synthesize the iodinated derivative, the role of the iodolactone in this process remains debatable. The role of the iodinated lipid normally present in thyroid (2iodohexadecanal) has not yet been investigated (243).

C Other Hormones

It would be interesting to investigate whether thyroid hormones by themselves exert a direct control on the growth of the thyroid gland. Thyroxine certainly increases the growth of transplanted autonomous thyroid tumors in rats in vivo (297), and thyroid cells contain many T, receptors (64).

Growth hormone, perhaps through IGF-I as an intermediate (14), induces thyroid growth but does not markedly enhance function as demonstrated in acromegaly (107,168,212), although some degree of autonomy in the goitrous acromegalic has been reported (359). In Snell dwarf mice, growth hormone induces cell proliferation but, contrary to TSH, no cell hypertrophy (68,190).

Human chorionic gonadotropin and thus luteiniz- ing hormone at high concentrations activate the CAMP cascade and consequently proliferation in FRTL-5 cells (62, 373, 374) and human thyroid cells (241). Because these effects are inhibited by TSH receptor-blocking antibodies, they are mediated by this receptor (372). The concentrations reached in patients with trophoblastic tumors or even in pregnancy (241,374) are sufficient to activate the human thyroid (132, 159, 371).

D. Pathological Signals

Other plasma signals appear only in disease, such as the autoimmune immunoglobulins directed against

thyroid cell membrane receptors. Thyroid-stimulating antibodies (TSAbs) and thyroid-blocking antibodies (TBAbs) bind to the adenylate cyclase-coupled TSH receptor. The TSAbs activate (4) and TBAbs block the stimulation by this receptor of function and growth. The TSAbs are responsible for Graves’ disease hyperthyroidism; TBAbs are responsible for some idiopathic myx- oedemas (192,379).

The concept of specific thyroid growth immunoglobulins (TGI) arose in the 1980 (79,351) from the acknow- ledgement that some TSH effects were not mediated by CAMP, which might be explained by the existence of different TSH receptors or effecters (83). Since then TGI activities have been reported by some groups, but the methodologies used raise questions (87). Although the concept may remain valid, its demonstration would require an unquestionable double-blind study using accepted methodologies (379). If accepted, the concept should provide an explanation for the thyroid specificity of TGI. Indeed all known growth factors and their receptors are remarkably ubiquitous, specificity being in- sured by local delivery through autocrine or paracrine mechanisms.

E. Local Signals

As shown, compensatory hypertrophy after thyroidectomy and goitrous hyperplasia in iodine deficiency or after goitrogens administration are caused by the oper- ation of the classic thyroid hormone-pituitary-TSH feedback and thus are prevented by thyroid hormone treatment. However, other local controls of thyroid cell proliferation exist (77).

In the embryo the thyroid develops before TSH secretion, even in anencephalic, i.e., hypopituitary, fetuses (134, 153, 306). Similarly a lesion of the thyroid (wound or cell death and necrosis) provokes an important local wave of cell divisions (307) even in thyroids desensitized to TSH action (362). Growth occurs in some cases (iodine treatment after iodine deficiency) when TSH levels are actually decreasing or in other cases in the absence of TSH (ZOO, 275). Compensatory hyperplasia in hemithyroidectomized mice also takes place, although at a reduced level, in dwarf mice that have a hereditary lack of growth hormone, prolactin, and TSH and in hypophysectomized animals (68, 188, 189). These growth processes are obviously independent from pituitary control.

Thyrocytes, as other cells, also respond in vitro to a number of paracrine factors, i.e., factors secreted by neighboring cells. Some of these factors are also synthesized and secreted by the thyrocytes themselves (autocrine secretion). Several growth factors have been shown to be mitogenic or comitogenic (permissive) for thyrocytes (29,92,96,98,110,160,196,235,261,263,265, 269,270,272,273,303,325-329,347,349,353): EGF,FGF, IGF-I, the secondary factor secreted in response to growth hormone, IGF-II, and insulin, even at physiological concentrations. Insulin-like growth factor-I is produced by sheep thyroid (14), IGF-II by FRTL-5 cells

July 19% REGULATION OF THYROID CELL PROLIFERATION AND DIFFERENTIATION 675

(196), and FGF by porcine thyrocytes (92,117). A possible role of insulin and/or IGF-I in modulating thyroid growth response in vivo has been known for a long time (149). The action of growth hormone on thyroid cell proliferation in Snell dwarf mice congenitally deficient in pituitary hormones is probably mediated by IGF-I (189). The removal of submaxillary glands in mouse, which presumably greatly decreases serum EGF, has been reported to lead to thyroid regression (311), whereas perfusion of fetal sheep with EGF results in a considerable enlargement of the thyroid gland (324). Thyroxine increases plasma EGF levels and increases them in the thyroid, which rather suggests a functional negative feedback in mice in vivo (237).

At least one local hormone has been shown to inhibit growth, TGF-0 (120, 217, 330, 360), as in other epithelial cells (195). It also induces extracellular matrix deposition, cell spreading, and the organization of the thyrocyte actin microfilaments network in bundles (104).

The panel of factors active on thyrocyte growth may vary from one species to another. Of course, such local hormones (IGF-I, FGF) also act on the endothelial cells and fibroblasts of the thyroid and may thus represent the mediators of the cross signaling that must exist to allow balanced growth of the gland. As the number of known growth factors increases, one may expect the progressive unraveling of a complex network of cell cross signaling.

In pathological situations, cytokines such as interleukin-1, interferon-y, and tumor necrosis factor might be secreted in the gland by cells of the immune system (345). These cytokines strongly influence FRTL-5 and human thyroid cell proliferation and metabolism (94, 162, 166, 167, 211, 256, 369, 375, 377).

As in other cell types the extracellular matrix also probably exerts a local control (presumably negative). This is suggested by the growth response after wounding and by the inverse relation of proliferative response versus cell density in primary thyrocyte cultures. Such local controls would have great importance in pathology, generating diverse patterns from one area or one cell to another, i.e., tissue heterogeneity (111, 142144). It is known that in thyrocytes (118), as in other cell types (381), a modulation of cytoskeleton organization modifies the activity of the CAMP cascade.

V. PATHWAYS REGULATING THYROID CELL

PROLIFERATION

A. General Considerations

It is probable that the key machinery in the immediate control of the decision of a cell to divide will turn out to be very general. Indeed the first studies of comple- mentation of proliferation-deficient yeasts with mammalian genes have already allowed the identification in mammalian cells of genes able to perform the same

function. One of these genes, cdc 2+ kinase has been shown to be a part of the well-known maturation promoting factor involved in the triggering of meiosis in oocytes (233). On the other hand, the regulatory circuits allowing the control of mammalian cells by extracellular signals vary in importance and significance from one cell type to another and within one cell type from one species to another. It is therefore highly improbable that the role of these circuits in the control of the decision to divide, i.e., in the control of the key machinery, will be conserved in all mammalian cells. Indeed, examples of opposite effects of the same cascade on proliferation and differentiation in different cell types abound: the phorbol ester protein kinase C pathway, which in the thyroid induces proliferation and dedifferentiation, exerts the opposite effects in keratinocytes and in colon cancer cells (40). The CAMP cascade that negatively regulates proliferation in fibroblasts and lymphocytes stimulates it in keratinocytes and thyrocytes. Thus the role of a given cascade in the control of cell proliferation and differentiation has to be considered for each cell type of each species. Generalizations are unwarranted in this field.

Reviews on the control of vertebrate cell proliferation generally consider two signal cascades as the main pathways between an extracellular mitogenic signal and mitogenesis itself: the growth factor receptor-protein tyrosine kinase pathway and the PI-Ca2+-diacylglycerol-protein kinase C cascade (38, 293). In such reviews, the first demonstrated signal cascade, the CAMP system, is either not considered or merely ascribed a role of inhibitory pathway (16). This general picture de- rives from the fact tha t grow th and proliferation have mostly been studied on “easy experimental objects,” fibroblasts and cell lines of mesenchymal origin. As often in science, the choice of experimental subject has biased the outlook on the whole subject. In fact, in several epithelial cells and in yeast cells the CAMP cascade is a positive regulatory pathway of cell growth and proliferation (84, 86). In the thyroid we consider at least three well-defined distinct pathways (84, 170, 197): the hormone receptor-adenylate cyclase-CAMP protein kinase system, the hormone receptor-tyrosine protein kinase pathway, and the hormone receptor-phospholipase C- diacylglycerol protein kinase C-calcium calmodulin protein kinase cascade (Fig. 3). In dog thyroid cells, the receptor-tyrosine kinase pathway may be subdivided in at least two main branches: some growth factors, such as EGF, induce proliferation and repress differentiation expression, whereas others, such as FGF or IGF-I, are mitogenic or are necessary for the proliferation effect of other factors without being mitogenic by themselves, but they do not inhibit differentiation expression (250).

ent As we might have guessed when we look a complexity of the regulation of a simple

,t the pres- organism

like the phage, this simple scheme is now daily expanding and becoming more sophisticated. First, new regulatory pathways are being defined, e.g., the atria1 natriuretic factor-guanylate cyclase-guanosine 3’,5’-cyclic monophosphate, the hormone-phospholipase A,-arachi-

676 DUMONT, LAMY, ROGER, AND MAENHAUT

FGF, IGF, T’SH, 751 EGF ATP, ACEIYLCHOUNE, TSH

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Volume 72

FIG. 3. Mitogenic pathways in thyroid. Data from thyroid cell system are integrated into present general scheme of cell proliferation cascades. In first line, known activators of various cascades in dog and human thyroid cells are shown. Various levels indicate a time sequence and postulated causal relations from initial interaction of extracellular signal with its receptor to end points: proliferation and differentiation expression. Cyclin is now called PCNA, proliferating cell nuclear antigen; DAG, diacylglycerol; IP,, inositol 1,4,&triphosphate; PI, phosphatidylinositol; TSI, thyroid-stimulating immunoglobulins; =, overlapping patterns; arrow with plus, stimulation; arrow with bar, inhibition; line with dot, time sequence; Z, different patterns; T, stimulation; 1, inhibition.

donate, and the hormone-phospholipase C-phosphatidylcholine cascades (24). These pathways have not yet been considered in the regulation of thy ,roid cell proliferation. Second, at each level, any of the regulatory pathways may be directly controlled by any step of this or the other pathways. Thus, in the dog thyroid, calcium through calmodulin activates a cyclic nucleotide phos- phodiesterase and diacylglycerol inhibits phospholipase C and the prostaglandin E, stimulation of adenylate cyclase and potentiates TSH activation of this enzyme. Third, the level of the proteins involved in each pathway may be controlled at gene transcription or distal to it by the same or other pathways. Fourth, the sti mulation of a regulatory pathway in one cell may stimul .ate the production and release of factors that control these same cells (autocrine mechanisms) or neighboring cells (paracrine mechanisms). A possible example of such interactions is the secretion by pig thyroid cells of an FGF that can act on thyrocytes, on fibroblasts, and on endothelial cells (105). Therefore different mechanisms con- curring to the same end will probably coexist in each system, and the demonstration of one such mechanism does not exclude the possibility of another. Moreover, at each level the circuits may be different from one species to another (257). Therefore if data should be coherent within o another.

ne system, they may vary from one system to

B. Mitogenic and Di.$ierentiating Action of Thyrotropin

Although there is no doubt that TSH in vivo stimulates the proliferation of thyroid cells, there was in the 1970s little evidence that this was a direct effect (82).

Indeed, the adrenocorticotropic hormone (ACTH) trophic effect on the adrenal appears to be indirect (136). The study of early steps of growth in slices (213) showed that TSH enhances ornithine decarboxylase activity in dog thyroid cells as it does in vivo and in vitro in rat thyroid (205, 286), which is generally considered as a preliminary to growth. The effect was mimicked by CAMP analogues and inhibited by agents inhibiting CAMP accumulation. As these results were against the current dogma, they were generally ignored.

To get at the problem of proliferation, it was necessary to use primary cultures. A technique derived from References 99,164, and 255 was used, with a serum-free medium supplemented as proposed by Ambesi-Impiom- bato et al. (8). With the use of several methods, it was demonstrated that TSH stimulates proliferation of the dog thyroid cells (262, 265, 269). More recently this result was confirmed in normal human thyroid cells (272). Despite earlier negative studies using inadequate culture conditions or pathological tissues (96,331,332,346), the mitogenic effect of TSH on human thyroid cells is now well established in vitro (137, 352, 353). Other results obtained in various culture systems were sometimes contradictory. Although in dog thyroid cells in primary culture, in rat thyroid follicles in suspension (232,303), in ovine cell lines (OVNI) (98), and in rat cell lines FRTL-5 (8,73) and WRT (29) TSH has been demonstrated to enhance or induce cell proliferation, to our knowledge no such effect has been obtained in porcine (97,106,130,342), calf (llO), or ovine (90) thyroid cells in primary culture. Whether this is due to inaccessibility of the TSH receptor(s), lack of an essential element in the culture medium, alteration of cell program in culture, or true unresponsiveness to direct TSH action is

July 1992 REGULATION OF THYROID CELL PROLIFERATION AND DIFFERENTIATION 677

not known. The latter hypothesis is plausible, as in the very similar system of the adrenal cells the stimulating effects of ACTH on the proliferation in vivo (240) have not been convincingly reproduced in vitro (112). In this case, there are even arguments that the stimulating effect may be indirect: ACTH would induce the synthesis and secretion of growth factors by the adrenal cells, which would then, acting as extracellular signals, trig- ger the cell proliferation.

In porcine thyroid cells, TSH through CAMP induces EGF receptors, making these cells more responsive to EGF (12348,350). In rat thyroid, propylthiouracil-induced goitrogenesis is accompanied by an increased concentration of IGF-II receptors (251). In FRTL-5 cells TSH greatly increases the effects of IGF-I, serum, and TPA on [3H]thymidine incorporation into DNA (314). In the control of thyroid cell proliferation, complementary mechanisms as well as differences of strategy from one species to another are possible (23).

In dog (269,270) and human (236,272, 353) thyroid cells, IGF-I or insulin is generally required for the mito- genie action of TSH or EGF but does not by itself stimulate proliferation. It has been known for a long time that TSH by itself does not stimulate thyroid growth in hypophysectomized rats (145). In rats, full growth response to TSH requires adequate levels of another hormone(s) that can be replaced by insulin (149, 150). In FRTL-5 cells IGF-I potentiates the activation of adenylate cyclase by TSH (31). In FRTL-5 and rat cells, IGF-I is weakly stimulatory per se (303,326), although this has not been found by another group (283). In pig thyroid cells, IGF-I produces a stronger mitogenic signal (281).

In the promotion of growth by TSH, increased protein content results from increased protein synthesis (82) and decreased protein catabolism (75).

It should be noted that in dog thyroid cells TSH directly stimulates proliferation while maintaining the expression of differentiation. Differentiation expression, as evaluated by iodide transport or thyroperoxidase and thyroglobulin mRNA content or nuclear transcription, is induced by TSH, forskolin, cholera toxin, and CAMP analogues (39,108,109,224,250,263,273). A negative report in FRTL-5 cells is unconfirmed and unexplained (27). Similar results, albeit partial, have been obtained in human, FRTL-5, calf, and porcine cells (90, 110,146,161,173,224,272,380). In some species,such as sheep and calf, hydrocortisone potentiates this differentiating effect (10, 110). These effects are obtained in all the cells of a culture, as shown by in situ hybridization experiments (250). They are reversible; they can be obtained either after the arrest of proliferation or during the cell division cycle (250). Moreover the expression of differentiation, as measured by iodide transport, is stimulated by lower concentrations of TSH than those required for proliferation (263, 272). Although the mito- genie action of TSH and CAMP has not been observed in the thyrocytes of all species, its effects on gene expression appear to be general (e.g., calf and pig cells) (37, 110). The effects of TSH and CAMP on differentiation expression, i.e., on gene expression, belong to the two

quite different categories defined by Roesler et al. (260). I) In the case of thyroperoxidase, gene transcription and mRNA accumulation do not require prior protein synthesis, i.e., they are not inhibited by cycloheximide. They are rapid (transcription is enhanced in 1 h) (108, 109). The effect does not require the presence of insulin in the medium. Such early transcriptional effects of CAMP are often mediated through CAMP regulatory element (CRE) and the phosphorylation of CRE binding protein (CREB). However, there is no classic CRE in the thyroperoxidase promoter (1). 2) In the case of thyroglobulin, gene expression requires prior protein synthesis; it is inhibited by cycloheximide. It is delayed by several hours. It is stimulated by insulin or IGF-I. Such effects are not mediated through CRE or CREB (43, 108).

C. Eflect of Factors Other Than Thyrotropin on Proliferation and Diflerentiation

Iodide inhibits many metabolic steps in thyroid cells. Most of these effects, such as the inhibition of adenylate cyclase, are relieved by treatments that block the penetration of iodide in thyroid cells (e.g., perchlorate) or its oxidation (e.g., methimazole). They are therefore ascribed to a postulated iodinated inhibitor (336). It inhibits both the activation of the CAMP cascade and the phosphatidylinositol bisphosphate (PIP,) cascade (178, 336) but also, independently, other steps, such as protein iodination and iodothyronine synthesis. This negative role also extends to proliferation in some thyroid cells (such as the FRTL-5 cells) and to gene expression. An indication for the latter effects is the stimulation by methimazole of thyroglobulin and thyroperoxidase mRNA accumulation in FRTL-5 (183).

Epidermal growth factor also induces proliferation of dog thyroid cells (261, 263, 270, 273) as well as other cells (36). It also stimulates the growth of thyroid cells from other species in culture (e.g., porcine, ovine, bovine, and human but not of the FRTL cell line that lacks EGF receptors) (96, 110, 319, 347, 349). This effect is often weaker than the effect of TSH. However, the action of EGF is accompanied by a general and reversible loss of differentiation expression (50, 90, 173, 252, 261, 263,272,273,347) assessed as described. Similar results have been obtained in sheep in vivo (57,324) and in newborn rat thyroids transplanted in nude mice (238). The effects of EGF on differentiation can be dissociated from their proliferative action. Indeed, they are obtained in cells that do not proliferate in the absence of insulin (172,250), in human cells in which the proliferative effect is weaker (173), and in pig cells at concentrations lower than the mitogenic concentrations (343). Fi- nally other growth factors, FGFs, and serum induce dog thyroid cell proliferation without exerting the dedifferentiating effects of EGF (264). A similar effect of IGF-I in TSH-primed FRTL-5 cells has been reported (313). However, FGF and serum in calf and porcine thyroid cells act as EGF in dog cells, inducing proliferation but repressing differentiation (92, 110).


Finally, the tumor-promoting phorbol esters, the pharmacological probes of the protein kinase C system, and analogues of diacylglycerol also enhance the proliferation and inhibit the differentiation of dog as well as other thyroid cells (15, 125, 191, 266, 272, 319). These effects are transient because of desensitization of the system by protein kinase C inactivation (266). The activation of the PIP, cascade by physiological agents, such as carbamylcholine and bradykinin, in dog thyroid cells does not reproduce all the effects of phorbol esters. In particular, prolonged stimulation of the cascade inhibits rather than stimulates proliferation (E. Raspe and J. E. Dumont, unpublished observations) as well as induction of ornithine decarboxylase (213). Thus we cannot necessarily equate effects of phorbol esters and prolonged stimulation of the PIP, cascade. Similarly, prolonged enhancement of the intracellular Ca2’ level might explain the mitogenic effects of IGF-I on FRTL-5 cells (313) but does not stimulate growth in dog thyroid cells (Raspe and Dumont, unpublished observations). The dedifferentiating effects of phorbol esters do not require their mitogenic action either. Thus the effects of TSH, EGF, and phorbol esters on differentiation expression are largely independent of their mitogenic action.

In several thyroid cell models, very high insulin concentrations are necessary for growth even in the presence of EGF (8,269,270,272,303,326). We now know that this mainly reflects a requirement for IGF-I (58, 272). It is interesting that in the FRTL-5 cell line (196), as in cells from thyroid nodules (354), this requirement may disappear as the cells secrete their own somatome- dins and thus become autonomous with regard to these hormones. However, low physiological concentrations of insulin can replace IGF-I to allow growth stimulation by TSH in dog (270) but not in human thyrocytes (272). Serum and FGF also induce growth in dog and calf thyroid and in FRTL-5 cells (25,110,263). In addition, IGF-I per se also stimulates the proliferation of FRTL-5 cells or WRT rat cells (277, 326). Although serum fully inhibits differentiation expression in calf thyrocytes and FRTL-5 cells (110, 378) and partially inhibits it in dog thyroid cells, IGF-I and insulin have no such effect. In fact IGF-I and insulin acting through IGF-I receptors have some positive effects on specialized gene expression in FRTL-5 cells (283), and insulin even at low concentrations is a moderate inducer of thyroglobulin gene expression in dog cells (108,250). In FRTL-5 cells IGF-I also potentiates the stimulation of adenylate cyclase by TSH (31). This therefore represents another type of receptor-tyrosine protein kinase pathway that leads to mitogenesis and to some extent to differentiation expression.

D. Role of Adenosine 3’,5’-Cyclic Monophosphate, Phosphatidylinositol Bisphosphate, and Tyrosine Protein Kinase Cascades in Thyrocyte Proliferation

The TSH effects on the proliferation and differentiation of thyroid cells are mediated by CAMP. Thyrotro-

pin induces within minutes a striking morphological change in dog thyroid cells in culture, a rounding up following the disruption of the actin network (231, 255, 263,268). All the cells are affected. In addition, TSH also enhances the accumulation of CAMP in these cells within <5 min; it remains elevated for 48 h in the continuous presence of the hormone. In the dog thyroid cells, analogues of CAMP as well as general cyclase activators (forskolin, cholera toxin) reproduce all the effects of TSH: acute morphological changes, proliferation, expression of differentiation (263, 265, 267, 269, 271, 273). Microinjection of the catalytic subunit of CAMP-dependent protein kinase also reproduces the disruption of the actin network (267). All the protein phosphorylations induced by TSH are also caused by forskolin (54, 181, 337). The pattern of protein synthesis induced by TSH is also fully reproduced by cholera toxin (171). Moreover, combinations of CAMP analogues that are synergistic on the two CAMP-dependent kinases isoenzymes are also synergistic on these effects (338). There- fore CAMP is a general intracellular positive signal for function, proliferation, and differentiation in the dog thyroid cells.

For proliferation, similar results have been obtained with human (165, 272) and rat thyroid cells in culture (361) and, despite a first contradictory report (331,332), in FRTL-5 cells (73,89,148,280). In the latter cells, thyroid blocking antibodies inhibit in parallel TSH binding and TSH-induced CAMP accumulation and [3H]thymidine uptake (34). It is interesting that in cloned dedifferentiated tumorigenic FRTL-5-derived cells, CAMP, as in fibroblasts, inhibits proliferation (93). Thus changing the phenotype of these cells may reverse the role of CAMP. In other cloned mutated FRTL-5 cells, growth becomes independent of TSH and CAMP (329).

The stimulation of proliferation by CAMP is mediated by the activation of CAMP-dependent protein kinases. Indeed analogues of CAMP that are selective for the two sites of each kinase and for each kinase must be used in the combination that activates these kinases to elicit the mitogenic effects in dog thyroid cells (338). The data even suggest a predominant role for type I CAMP kinase in this process. This is also supported by the fact that desensitization to the CAMP growth effect coin- cides with type I kinase downregulation (32) and other in vivo results in other systems supporting a positive role of this kinase in growth (52). The inhibitory effect of iodide on thyroid cell proliferation in vivo can also be explained by its well-documented inhibition of thyroid adenylate cyclase (336).

One argument that CAMP may be the mediator of rat thyroid cell proliferation in vivo is the fact that methylxanthines, inhibitors of CAMP phosphodiester- ases, even at doses that do not further enhance serum TSH levels or decrease serum thyroid hormones, greatly potentiate the goitrogenic action of propylthiouracil (355). The action of methylxanthines is abolished by a high-iodine diet or hypophysectomy (355). The effect of methylxanthine could be due to inhibition of adenosine receptors. As thyroid mostly contains A,-receptors negatively coupled to adenylate cyclase, the goitrogenic ef-

July mei? REGULATION OF THYROID CELL PROLIFERATION AND DIFFERENTIATION 679

feet of methylxanthines in vivo would in this framework also support the role of CAMP. Analogues of CAMP or CAMP injected in rats or mice in vivo has also been reported to cause thyroid growth even though an indirect effect is not excluded (82, 187, 248).

The most compelling argument for the roles of the CAMP cascade in vivo is the effects of TSAbs in humans and of adenosine AZ-receptor expression in the thyroids of transgenic mice. The TSAbs are antibodies against the TSH receptor that activate it. Their chronic stimulatory effects lead to hyperthyroidism. At the concentrations reached in vivo these immunoglobulins only activate the CAMP cascade in human thyroid cells (180). The cloned adenosine AZ-receptor behaves in transfected cells as a physiologically constitutive activator of adenylate cyclase. In dog thyroid cells its expression is sufficient to elicit DNA synthesis (198). When expressed in the thyroids of transgenic mice it induces hyperthyroidism (i.e., hyperfunction) and goiter (i.e., growth). Even- tually the mice die from hyperthyroidism and, if treated with antithyroid drugs, from the consequences of their huge goiter (e.g., tracheal compression) (Ledent and Dumont, unpublished observations). In the same way, expression of the active subunit of cholera toxin in somatotrophs of transgenic mice induces pituitary hyperplasia and gigantism (35). It should be noted that in a few species no stimulation of thyroid cell proliferation by TSH and by the CAMP cascade has been demonstrated, but when TSH enhances proliferation, this effect is mimicked by CAMP analogues or enhancers. A case in point is the porcine thyroid cell in which TSH and forskolin neither enhance proliferation nor c-myc and c-fos expression. In these cells iodide stimulates both (128).

That TSH can act on the growth of human thyroid cells by pathways other than the CAMP cascade is suggested by the facts I) that in human thyroid cells in primary culture the mitogenic effect of the hormone under some conditions is not fully reproduced by CAMP enhancers (272) and 2) that the hormone is mitogenic in a human hybrid cell line GEJ (158) and in primary cultures of differentiated cancers in which it does not enhance CAMP accumulation. Similarly, clones of FRTL-5 cells with low CAMP but a normal mitogenic response to TSH have been reported (63). If these results cannot be explained otherwise, another pathway of TSH action might be involved. We know that TSH in human thyrocytes but not in dog thyroid cells nor in the FRTL-5 cell line activates both the CAMP and the PI cascades (179).

The effects of EGF on dog thyroid cell (proliferation, inhibition of differentiation expression) are mimicked by phorbol ester tumor promoters (266). However, these compounds also inhibit EGF action: the effect of phorbol esters, which is less than the effect of EGF, is not increased by EGF. In several cell types, EGF not only activates a tyrosine-specific protein kinase but also induces a rapid rise in cytoplasmic free Ca2’ concentration. This rise in Ca2’ concentration following EGF stimulation has been linked to an activation of the PI-Ca2+ cascade, although it has been suggested recently that it might result from an entry of extracellular Ca2+

through the plasma membrane. It would therefore be conceivable that EGF action on the thyroid cell might result from an increase in Ca2+ entry or from an activation of the PI-Ca2’ cascade with the generation of diacylglycerol, the action of which is mimicked by phorbol esters. However, it should be noted that this activation of the PIP, cascade by EGF apparently only occurs in cells in which EGF receptors are overexpressed (186). Epidermal growth factor induces a rise in intracellular Ca2’ and an alkalinization in porcine thyroid cells (317, 321). Insulin-like growth factor I, which also enhances the proliferation of these cells, activates the PIP, cascade and raises Ca2’ levels in these cells (316,320); however, such an effect, if existent, is minor in dog cells. Moreover carbamylcholine, the most potent activator of the Ca2+-PI cascade in these cells, is not mitogenic (Raspe and Dumont, unpublished observations). On the other hand, neither EGF nor phorbol esters enhance CAMP accumulation in dog thyroid cells. It is therefore likely that EGF acts through the phosphorylation of key proteins on tyrosyl residues. In FRTL-5 cells adenosine inhibits TSH CAMP-induced thymidine incorporation into DNA while enhancing the effect of the IGF-I protein tyrosine phosphorylation cascade (218). The three cascades are therefore fully distinct at the level of their primary intracellular signal and/or of the first signal- activated protein kinase (54). It is interesting that iodide, through as yet unknown oxidized derivative(s), acutely inhibits the CAMP and the Ca2’-PI cascades and in a more delayed and chronic effect decreases the sensitivity of the thyroid to the TSH growth response. In FRTL-5 cells it inhibits the TSH-, IGF-I-, and TPA-induced cell proliferation (18, 279); these effects are relieved, according to our general paradigm (336), by perchlorate and methimazole.

E. Kinetics of Biochemical Events in Various Thyroid Mitogenic Pathways

The kinetics of the induction of thymidine incorporation into nuclear DNA of dog thyroid cells are very similar for TSH, forskolin, EGF, and phorbol esters (TPA) (270). Whatever the stimulant, there is a similar minimal delay of 16-20 h before the beginning of the labeling, i.e., of DNA synthesis. This is the minimal time required to prepare the necessary machinery. Only for the combination of TSH, EGF, and serum is the delay shortened to 14 h. For the CAMP pathway, the stimulatory agent has to be present during this whole prereplicative period: any interruption of the stimulation (e.g., by forskolin washing) greatly delays the start of DNA synthesis (271).

The time sequence of biochemical phenomena between the stimulation and the beginning of DNA synthesis in these dog thyroid cells in primary culture suggests a causal sequence (Fig. 3). However, many such postulated causal relationships remain to be proved (239).

The phenomenology of EGF, TPA, and TSH proliferative action on dog quiescent cells has been studied in detail with the aim to identifv steps in this action. Three


biochemical aspects of the proliferative response occurring at different times of the prereplicative phase have been considered. The pattern of protein phosphorylation induced within minutes by TSH is reproduced by forskolin and CAMP analogues (54). The phosphorylation of at least 11 proteins is increased or induced. Treatment of the gels with NaOH does not reveal any remaining phosphorylation on these proteins suggestive of tyrosine phosphorylation. The phosphorylation of seven proteins is decreased. In EGF-stimulated cells, the phosphorylation of five proteins is stimulated, two of them are phosphorylated on tyrosines (42,000 mol wt). These two proteins are similar (isoelectric points, approximate molecular weight, composition in phosphorylated amino acids) (54) to the two 42,000-mol wt proteins described in other systems, which have been implicated in the mitogenic response to diverse agents and recently identified as the mitogen-activated protein (MAP)-kinase (276). This kinase phosphorylates the S6 kinase II that is involved in the control of protein synthesis at the ribosome level. Phorbol esters induce the phosphorylation of 19 proteins, including the tyrosine- phosphorylated proteins mentioned. There is no overlap in the patterns of protein phosphorylation induced by TSH and CAMP enhancers, on the one hand, and by EGF and phorbol esters, on the other hand (54). Similarly the stimulation by EGF of Na’-H’ exchange in porcine thyroid cells has not been found in TSH-stimulated dog thyroid cells (318; E. Rasp& unpublished observations).

The expression of c-myc and c-fos has been studied by Northern analysis of RNA extracts (259). As in other types of cells (155), growth factors, EGF, and TPA enhance first c-fos and then c-rnyc mRNA expression (130, 259). On the other hand, TSH or forskolin enhances strongly but for a short time c-myc mRNA concentration and, with the same kinetics as for EGF/TPA, c-fos mRNA concentration. In fact, CAMP first enhances and then decreases c-m/ye mRNA accumulation. This second phenomenon is akin to what has been observed in fibroblasts in which CAMP negatively regulates growth (129). The enhancement has been observed for TSH and agents increasing intracellular CAMP in FRTL-5 cells, but the second phase has not been investigated or observed (71,325). In pig thyroid cells, TSH and CAMP are not mitogenic and do not enhance c-myc and c-fos gene expression (130). The effect of TSH and CAMP on c-fos gene expression in FRTL-5 cells is transcriptional (60). It is easily explained by the presence of two CAMP-dependent response elements in the promoter of c-fos gene. The dyad symmetry element accounts for the effects of EGF, serum, and phorbol esters. In dog thyroid cells the effects of TSH on c-myc and c-fos expression do not require the presence of insulin but are potentiated by insulin. The absence of growth in the absence of insulin thus shows that the TSH enhancement of c-myc and c-fos expression may be necessary but is not sufficient for the mitogenic response. The fact that expression of antisense c-fos inhibits FRTL-5 cells proliferation suggests that indeed expression of c-fos is necessary (102). The expression of c-jun is enhanced by EGF and phorbol

esters but inhibited by TSH and forskolin. This is similar to results obtained with NIH 3T3 cells in which phorbol esters stimulate proliferation while CAMP inhibits it (S. Reuse and J. E. Dumont, unpublished observations). Enhancement of c-jun expres sion is therefore not required for the induction of proliferation by TSH and CAMP. Thyrotropin, through CAMP, also enhances the mRNA expression of another protooncogene, C-ras, in rat cells (72).

In general, although initially distinct, the mito- genie pa thways activated by the growth factor receptor tyrosine kinases and by the PI cascade overlap and con- verge on several early events: stimulation of the PI cascade in some systems, but not in dog thyrocytes, in response to receptor tyrosine kinase activation; phosphorylation of 42,000-mol wt proteins on tyrosine following protein kinase C activation; common stimulation of Na+-H+ antiport; and similar control of c-fos and c-myc protooncogene expression. In contrast, as delineated in the dog thyrocyte system, the activation of the CAMP cascade is sufficient to induce DNA synthesis but is es- sentially distinct from the CAMP-independent pathways. Differences include divergent patterns of protein phosphorylations and thus utilization of different protein kinases and second messengers and different kinetics of protooncogene expression.

The pattern of proteins synthesized in response to the various proliferation stimuli has been studied (172, 182). Again two patterns emerge. Both TSH and forskolin induce the synthesis of at least eight proteins and decrease the synthesis of five proteins. Epidermal growth factor, phorbol ester, and serum induce the synthesis of at least two proteins and decrease the synthesis of two proteins. The only overlap between the two patterns concerns the decrease in the synthesis of a protein (18,000 mol wt) that is also reduced by EGF after proliferation has stopped. Only one protein has been shown to be synthesized in response to the three pathways, proliferating cell nuclear antigen (PCNA), the auxiliary protein of DNA polymerase 6 (17). However, the kinetics of this synthesis are very different, with an early synthesis in the CAMP cascade (consistent with a role of signal) and a late (S phase) synthesis in the other cascades (172). Finally the synthesized proteins satisfy- ing several criteria for signal proteins involved in the induction of DNA synthesis (biphasic kinetics, no induction in cells at confluence, synergy of induction by effec- tors of the other pathways), i.e., PCNA and protein 1, are specifically induced in G, by TSH and CAMP and by EGF and phorbol esters, respectively (171, 172). Thus obviously two different phenomenologies are involved in the proliferation response to TSH through CAMP, on the one hand, and EGF and phorbol ester, presumably through protein tyrosine phosphorylation, on the other hand. Although this conclusion needs to be further sub- stantiated, it certainly suggests that the proliferation of dog thyroid cells is controlled by at least two largely independent pathways. The effects of TSH, EGF, and phorbol esters on protein synthesis can be obtained in the absence of insulin in the medium, except for the


induction of PCNA synthesis; some are enhanced by in- and CAMP through protein kinase C activators or EGF sulin. Thus these effects also are not sufficient to induce is also very unlikely in dog thyroid cells. The kinetics of mitogenesis (172). action of TSH or forskolin are similar for the end point

of DNA synthesis for the three types of agents. More-

F. Role of Autocrine Loop over, the kinetics of protooncogene c-myc and c-fos expression are not delayed for TSH and CAMP. Finally the

The studies of protein phosphorylation, protoonco- patterns of protein phosphorylation and protein synthe-

gene expression, and protein synthesis in dog thyrocytes sis induced by EGF and phorbol esters show partially

allow for discrimination between two models of CAMP common responses, whereas there was no overlap with th

action on proliferation in this system, a direct effect on e pattern of TSH or CAMP action. There is no evidence

the thyrocyte or an indirect effect through the secretion either that, as in Swiss 3T3 cells, the effect of TSH

and autocrine action of another growth factor. If the might be secondary to a TSH-induced release of prosta-

effect of TSH through CAMP involved such an autocrine glandins. Thyrotropin does not induce such a release,

loop, one would expect a faster kinetics of action of the and indomethacin does not inhibit the mitogenic effect

growth factor and at least some common parts in the of TSH (264). Thus there is no evidence in favor of the

patterns of protein phosphorylation and protein synthe- involvement of an autocrine loop with a growth factor or

sis induced by CAMP and the growth factor. The results prostaglandin in the action of TSH and CAMP on dog thyroid cell proliferation. In FRTL-5 cells a CAMP-in&-

do not SuPPort such an hYPothesi% at least for the pendent TSH stimulation of arachidonate release has growth factors we have tested (Fig. 4). been reported. Arachidonate derivatives were therefore

It has been suggested, for example, that part or all suggested to play a role in the induction of proliferation the TSH growth effects on FRTL-5 cells are secondary by TSH (312). However, in this system the action of TSH to the autocrine secretion of IGF-II and other factors is fully reproduced by activation of the CAMP cascade. (314). This is not general as IGF-II, which is secreted by This does not exclude that such a mechanism may oper- FRTL-5 cells and is mitogenic for them (196), is not a ate in thyroids of other species, as suggested by the in- growth factor for dog thyroid cells by itself. Moreover, duction by TSH of EGF receptors in porcine thyroid dog thyrocyte cultures are in general carried out in the cells (12,348,350). presence of high concentrations of insulin that would saturate the IGF-I receptor (270). In the FRTL-5 cells VI. APPARENTLY OPPOSITE PROGRAMS: PROLIFERATION themselves, anti-IGF antibody (Sm 1.2) only inhibits the effects of low concentrations of TSH (196) and does

AND DIFFERENTIATION EXPRESSION ARE INDUCED

not inhibit the synergism between TSH and high con- BY THYROTROPIN AND ADENOSINE 3’,5’-CYCLIC

centrations of insulin. Moreover, there is no evidence MONOPHOSPHATE CASCADE

that TSH does stimulate the production of IGF-II or The incompatibility at the cell level of a prolifera- other autocrine factors in these cells. An effect of TSH tion and differentiation program is commonly accepted

FIG. 4. Models of mitogenic activation by CAMP cascade. ModeZ 1: direct activation of mitogenic pathway in cell. MbdeZ 2: induction or secretion of growth factor or growth factor receptor that by autocrine process will activate mitogenic pathway of this growth factor. Predicted consequences of modeZ 2 are outlined. They are not realized in case of stimulation by TSH of dog thyrocyte. CA, CAMP; cAPK, CAMP-dependent protein kinase; GF, growth factor; GFR, growth factor receptor; H, hormone; PDGF, platelet-derived growth factor; Prot, protein; Protoonc, protooncogene; R, rat; broken arrow with plus, stimulation; broken arrow with bar, inhi- bi tion.

-1 CA acta directly on prolifemtion pl’;lgmm

I

I”- 0

OTHER EFFECTS

# ~ / P/ROT. PHO~PHORYMON

/ ‘+

\

1 I \ I

\ \

PROTOONC. I -\ EXPRESS(ON I \ \

\ I \ I

\ I

2 I

J 0

IG ‘+ \ t

H 0 4

OTHER EFFECTS DNA SYNTHESIS

MOOEL - DELAY OF cA PATHWAY - SOME COMMON PARTS IN

PHOSPHORYlATlON PAlTERNS PROTElN SYNTHESIS PAITERN PROTOONCOCENE EXPRESSION

/ /

GF IGF,

PDGF EGF

cA induce8 a GF or GFR that r act8 on the pmlifemtion +

pmgmm t

CA

-+

t PROT. PHPSPHORYMlON

+

t NEW PROFN FESlS GF

H+

t

0 / PROT. PHO~PHORYMTlON

H ) +’ \

2:’ ) I \ I

\ \

OTHER PROTOONC. I \ EFFECTS FPRESS(ON I \

FlTM 00

KERATlNOM (R) THYROCYIE (R)

(PORCINE)

OTHER EFFECTS DNA SYNTHESIS

682 DUMONT,LAMY,ROGER,AND MAENHAUT Volume 72

in biology. In general, cells with a high proliferative capacity are poorly differentiated, and during development such cells lose this capacity as they progressively differentiate. Some cells even lose all potential to divide when reaching their full differentiation; this is called terminal differentiation. Conversely, in tumor cells there is an inverse relationship between proliferation and differentiation expression. It is therefore not sur- prising that in thyroid cells the general mitogenic agents and pathways, phorbol esters and the protein kinase C pathway, and EGF (and in calf and porcine cells FGF) and the protein tyrosine kinase pathway induce both proliferation and the loss of differentiation expression (92, 110, 263, 273, 266). The effects of the CAMP cascade are in striking contrast to this general concept. Indeed TSH and CAMP induce proliferation of the dog thyrocytes while maintaining differentiation expression: both proliferation and differentiation programs can be triggered by TSH in the same cells at the same time (250). This is by no means unique: neuro- blasts in the cell cycle may simultaneously differentiate (74). It is tempting to relate this apparent paradox to the role and expression of protooncogenes in these cells. The expression of c-fos is enhanced in a great variety of cell stimulations, leading to either proliferation or differentiation expression (220). On the other hand, if there is one generalization that could be made on protooncogenes, it is the dedifferentiating role of c-myc. A rapid and dramatic decrease in c-myc mRNA by antisense rnyc sequences induces differentiation of a variety of cell types (119,126,254). In rat thyroid cell lines, expression of m/ye oncogene abolishes differentiation expression (22). The dedifferentiation following establish- ment of thyroid cells in primary culture is accompanied by an increased c-myc expression (135). It is therefore striking that in the case of the thyrocyte in which the activation of the CAMP cascade leads to both proliferation and differentiation, the kinetics of the c-myc gene appear tightly controlled. After a first phase of 1 h of higher level of c-myc mRNA, c-myc expression is decreased below control levels. In this second phase, CAMP decreases c-myc mRNA levels, as it does in proliferation-inhibited fibroblasts. It even depresses EGF-induced expression (259). The first phase could be necessary for proliferation, whereas the second phase could reflect the stimulation of differentiation by TSH. This downregulation is suppressed by cycloheximide, which suggests the involvement of a neosynthesized or labile inhibitory protein at the transcriptional level or at the level of mRNA stabilization. Surprisingly the secondary downregulation of c-myc (51) has not been observed in FRTL-5 cells.

Preliminary results indicate that TSH downregu- lates at a posttranscriptional level the c-myc mRNA expression: as soon as TSH is in the medium, a destabilization of c-m/ye mRNA is observed in cells in which transcription is blocked by actinomycin D. It can therefore be hypothesized that the first rise of c-myc mRNA expression reflects a very high induction of transcription combined to a destabilization mechanism. Later, the pos-

itive transcription effect is repressed and the destabilization mechanism persists, leading to a downregulation of the c-myc mRNA level. The transcription could be repressed either at the initiation (124,242,322) or at the elongation level (209). It would be interesting to test whether cloned tumorigenic FRTL-5 cells, in which CAMP inhibits proliferation (93), have lost the first positive control of c-myc expression. In the feedback mechanism, the inhibitory neosynthetic protein could even be the c-myc protein itself, specifically modified at the posttranslational level by the CAMP pathway. Such an autoregulatory mechanism of blockade of transcriptional initiation requires additional transacting factors and could act as a homeostatic regulator of c-myc expression in vivo (242). It is interesting that transformed FRTL-5 cells have lost the positive effect of TSH and CAMP on proliferation and express only a negative control (93). This suggests that in normal cells a dual control exists with a dominant positive regulation.

In many cells stimulation of proliferation is accompanied by an increased expression of c-jun protooncogene. In cells in which proliferation is inhibited by the CAMP cascade, c-jun expression is also inhibited. The expression of c-jun has therefore been considered as a general marker of cell proliferation. In fact, in dog thyroid cells, TSH and CAMP enhancers both induce proliferation and inhibit c-jun expression (Reuse and Du- mont, unpublished observations). There is therefore no parallel between c-jun expression and proliferation in the dog thyroid cells, but it is possible that c-jun could be another factor involved in the negative control of differentiation expression. The effects of CAMP on c-jun expression are difficult to fit with the current concept that expression of c-jun is inhibited by CREB and that this inhibition is relieved by the CAMP-dependent phosphorylation of CREB.

VII. GROWTH CONTROL AND GOITROGENESIS

A. General Concepts

Goiter and therefore abnormalities in thyroid growth control are common to most thyroid diseases. Whatever its cause, goiter is the long-term end result of a disease. The thyroid has become extremely heterogeneous, and the primary lesion, even if still present, is swamped by a whole set of secondary and late consequences. This is well exemplified by the heterogeneous nature of many tumors, presumably of monoclonal origin, or of the thyroid after long-standing Graves’ disease, iodine deficiency, or congenital defects, all of which originate from a single cause. The normal thyroid already presents, at a given time, heterogeneity from follicle to follicle and cell to cell in terms of function (as demonstrated by iodine radioautography), of functional response to stimulatory agents (as demonstrated by induced macropinocytosis or by the presence or absence of colloid droplets), and of proliferation (as shown by the

July 1ewz REGULATION OF THYROID CELL PROLIFERATION AND DIFFERENTIATION 683

fact that, at a given time, only a few cells synthesize DNA) (8285). However, this heterogeneity, known for a long time but reinvented periodically, should be inter- preted with caution. The picture observed at one point in time is not necessarily permanent; in fact histological sections and autoradiographies may be compared with snapshots of a rapidly evolving scene. The demonstration that apparently “cold” follicles in aged mice contain the same amount of iodine as hot follicles is evidence that these cold follicles must have at one time taken up iodine (208). Similarly in the case of proliferation, in stimulated dog thyroid cells in primary culture it can be shown by double-label autoradiography that cells that divide at day 4 may not divide at day 8 and vice versa (Roger and Dumont, unpublished observation). As is well known in cancerology (230), given the dynamic heterogeneity of the thyroid, even a simple primary defect may lead to permanent heterogeneity due to many factors. 1) One is the different kinetics of general controlling factors (TSH, iodine) and of cell sensitivity (e.g., the cells that are more sensitive to TSH at a given time will react more to a TSH surge). Such differences in sensitivity to growth factors are even observed in cultures of homogeneous cell lines such as the Swiss 3T3 cells. These differences are not heritable in the long term but represent an unstable property lost after a few generations in high-serum medium (33). 2) In addition there are local factors, such as cell-to-cell contact (370), blood flow availability, inflammation, hemorrhage, necrosis, and scarring. Cell necrosis in thyroids in involution is associated with cell multiplication and folliculo- genesis (ZOO). In thyroids, toxic or autoimmune reactions induced by iodine or other factors would also contribute to heterogeneity (70). 3) There is also the cell’s immediate environment: cytokines and growth factors induce cells to alter their surrounding matrix, but conversely adherence to matrix induces cells to make cytokines and controls how cells respond to cytokines. In such a complex interplay the fate of two neighboring cells may be completely different (229). 4) There are mutations, the probability of which increases with the number of mitoses (9, 123, 253). A good example of such an evolution is shown by the heterogeneous mammary or pancreatic neoplasia induced by the single tar- geted overexpression of TGF-cu in transgenic mice (202, 282). Also heterogeneity soon appears in the stimulated thyroids of mice submitted to hemithyroidectomy or methimazole treatment (298) or in the thyroids of transgenic mice constitutively stimulated by an adenosine AZ-receptor (Ledent and Dumont, unpublished observations). This heterogeneity is limited within one follicle by the intercellular communication of small molecules allowed by the gap junctions (223).

To understand the pathogenesis of goiter, it is thus advisable to study, as early as possible, simple lesions originated from a single cause. The lessons learned on simple models should help to understand complex and multifunctional diseases, just as the study of a congenital defect enlightens our understanding of all its phenotypic counterparts. We therefore mainly review goiter

formation in simple diseases such as Graves’ disease, hyperfunctioning adenomas, and adenomas with congenital defects.

The role of the signals and cascades controlling thyroid cell proliferation and differentiation in pathology has been until now little studied. In a few cases thyroid pathology can be explained in a straight forward man- ner within the framework just presented.

B. Graves’ Disease

The disease in which goiter is easiest to explain is Graves’ disease. In this disease autoantibodies directed against the TSH receptor (TSAb) activate this receptor and consequently the whole CAMP cascade (148, 193, 376). At the highest concentrations reached in pathology, these TSAbs do not activate, as TSH does, the Ca2’- PI cascade (180). Thus hyperthyroidism in Graves’ disease appears to result from a chronic hyperstimulation of the CAMP cascade. As shown, the effects of this cascade on cultured thyroid cells are to enhance function and proliferation while maintaining differentiation, i.e., they represent the in vitro counterparts of what is observed in Graves’ disease thyroids in vivo. Indeed TSAbs induce human thyroid cell growth and c-fos expression in vitro (138). It is interesting to note that, as in in vitro or in vivo chronically stimulated thyroids, the growth of thyroid in Graves’ disease is generally limited, although hyperfunction and thus hyperstimulation persist. The permanency of the stimulation is explained by the low desensitization of TSH receptors and the absence of downregulation of TSH receptor mRNA (61, 65; Maen- haut and Dumont, unpublished observations). This apparently simple and unicausal disease may lead in time to heterogeneous goiter. Also, in these chronically stimulated thyroids in which proliferation and the increasing number of mitoses are bound to allow the fixation of more mutations, cancer incidence has been shown to increase (207), although this is controversial (7). Thus even though the CAMP cascade itself maintains differentiation while promoting proliferation, the greater number of mitoses may give a higher probability of oc- currence to the rare mutagenic events that lead to carcinogenesis. Moreover, in the V79 cell line, activation of the CAMP cascade increases the mutation frequency induced by mutagenic methylating agents (131).

C. Congenital Defects

The goiter resulting from congenital defects in iodine metabolism by the gland is also simply explained by classic concepts of thyroid regulation. Deficiency in thyroid hormone formation resulting from the defect relieves the thyroid hormone feedback on the hypophysis and leads to increased TSH secretion and stimulation of the thyroid. In addition, a deficiency in iodine metabolism, at the level of trapping or iodination, will relieve the intrathyroid negative feedback of iodide and

684 DUMONT,LAMY,ROGER,AND MAENHAUT Vohme 72

increase the sensitivity of the gland to the TSH growth- promoting effect. Impaired iodination due to a congenital defect or to inhibition by antithyroid drugs has been shown to relieve the inhibitory effect of iodide on CAMP accumulation (66, 67). Defects of iodotyrosine coupling and iodotyrosine deiodination that also lead to iodine depletion will in time have the same effect. It is interesting to note that defects in iodination, which most se- verely affect the iodide inhibitory pathway, lead to the severest goiters, sometimes to follicular carcinoma (2). It is also important to note that even in this case where a single identified cause of the disease and its goitrogenic consequences exists, prolonged stimulation of the thyroid will in time generate heterogeneity and its ultimate result, the multinodular goiter (163, 278, 301). Similar considerations apply to endemic goiter in which iodine deficiency and sometimes goitrogen intake phenotypi- tally reproduce the congenital defects. However, in this case variations of the stimulation in time, e.g., by re- feeding iodine to the animals, will lead earlier to macro- scopic heterogeneity (116). After iodine administration in iodide-depleted glands, the death of many cells, the burst of cell proliferation, and the consequent remod- eling of the thyroid may explain these phenomena (70, 275).

D. Thyroid Adenoma

Thyroid adenomas, which by definition are monoclonal, i.e., are constituted by the progeny of one mutated cell, also result from a single initial biochemical lesion. A lesion can be called monoclonal if all cells of the lesion are derived from one cell that initiated the lesion. These cells have some common hereditary properties different from those of normal cells. The primary cell that acquired these properties first is mutated. Although this concept is clear, the terms oligo- and polyclonal are fuzzy; a lesion is monoclonal or is not monoclonal.

Adenomas are well-encapsulated homogenous neoplasia, whereas nodules are benign neoplasia that do not fit this definition (323). In mice Thomas et al. (323) have demonstrated that adenomas constitute the progeny of one mutated cell, i.e., are monoclonal; nodules are non- clonal. This has been recently confirmed for human thyroid lesions (133, 226).

There is ample indirect and direct evidence that monoclonal adenomas of various types exist in humans. I) The demonstration of a common biochemical defect in the whole lesion can best be explained by a genetic defect by somatic mutati .on in the cell at the origin of a clone. Defects entirely an .alogous to congenital de fects have been demonstrated in some well-studied adenomas: iodide trapping defects with normal iodination and iodination defects with normal trapping (66, 67, loo), constitutive activation of the GTP-binding protein that positively controls adenylate cyclase (175), enhanced iodide trapping and lack of protein substrate of CAMP-dependent protein kinase in autonomous ade-

nomas (337), and point mutations of ras protooncogenes (184, 228). When the biochemical defect corresponds to one identified mutation, the demonstration of this somatic mutation i .n the entire neoplasm but no t in the rest of the tissue is the demonstration that all cell s of the neopl asm are derived from a cell in which the mutation took place, i.e., that the neoplasm is monoclonal. 2) There is the demonstration by isoenzyme patterns of monoclonality in encapsulated experimental adenomas in rats. 3) There is also the demonstration by glucose-6-phosphate dehydrogenase isoenzymes and DNA methylation patterns of monoclonality in the few human thyroid adenomas studied (133).

The application of newly developed DNA recombinant-based techniques (DNA fingerprinting, methylation of X-linked restriction fragment length polymor- phism or RFLP) should soon allow for the extensive testing of this conclusion (340).

Of course the phenotype of such adenomas may be reproduced by any biochemical lesion that will inacti- vate or activate the systems involved in the genetic defects. However, just as a congenital defect of iodination is a good model for all congenital or pharmacological (goitrogens) iodination impairments, the adenomas represent simple genotypic models of a much larger group of similar phenotypic lesions, the “nodules.” For instance, patches of tissue of multinodular goiters exhibit the same defects in iodide trapping and iodide organification (290) that are expressed homogenously in adenomas (66, 67, 100). The model is simple because the pri .mary effect is unique and well defined.

Autonomous adenomas are well-encaps ulated thyroid neoplasms that take up and secrete thyroid hormones in the absence of a TSH stimulus. As the secreted hormones inhibit the pituitary thyrotrophs, TSH plasma levels decrease and the normal tissue becomes quiescent. By scintigraphy autonomous nodules appear as hot (taking up radiodide) lesions surrounded by cold (not incorporating iodide) tissue. Administration of exogenous thyroid hormones does not inhibit radioiodide uptake in the nodules, whereas administration of TSH restores this uptake in the contralateral quiescent tissue (285). If sufficiently large and with a diet sufficient in iodine, autonomous nodules cause hyperthyroidism (95). The defect in autonomous nodules is in the cells themselves and is not caused by exogenous growth factors: indeed xenotransplanted cells of such nodules retain their activity contrary to Graves’ disease thyroids (152).

A thyroid adenoma is called autonomous when it no longer requires its normal stimulant TSH to function and grow. This concept is relative. I) In a quantitative sense, a tumor may be more or less autonomous, requir- ing less TSH than normal tissue for its activity or no TSH at all (alteration in affinity). This has been well examplified in Cushing’s syndrome for the adrenal gland (169). In this first case the thyroid adenoma is suppressible by thyroid hormone treatment and therefore not truly au tonomous. It woul .d nevertheless appear as a hot nodule on a thyroid scintigram.

July m%? REGULATION OF THYROID CELL PROLIFERATION AND DIFFERENTIATION 685

In autonomous adenomas the function without TSH must be higher than in normal tissue without TSH (otherwise the “tumor” would behave normally) but can be set at different levels. If it is assumed th at a given cell has a maximal level of activi ty [in terms of enzyme kinetics, its maximum uptake rate ( Vmax)], autonomy can be defined as the ratio between basal activity without TSH (V,) and V,,,. It varies from a minimal value of Vo, corresponding to the basal activity of the normal tissue, to 1, where v0 = Vmax and VJV,,, = 1. Defined in this way autonomy i s the inverse of the stimulatory ratio. A tumor may lose activity, but if it lo osens its con trol and becomes autonomous, it can only do so by increasing v,/ Vmax and thus its basal activity V,. Fetal tissue is relatively autonomous for growth with regard to TSH (245).

2) Autonomy is also relative in a qualitative sense. The w hole biology (func tion and growth) can be autonomous: one would expect this from a constitutive activation (mu tation) upstream in the common steps i n the hormone receptor cascade ( g e. ., receptors, G pro teins, cyclase). On the other hand, some elements of the activating cascade may become autonomous (e.g., function or one step of the metabolism, such as iodide trapping, iodi nation, or growth). These latter mechanisms would not lead to adenomas as an increase i n function on ly and wou Id not confer any grow ,th advantage.

The s implest exa mple of a somatic m utation leading to autonomous hyperfunctioning adenomas has been demonstrated by Bourne’s group in the hypophysis of acromegalic rats. In the rat somatotrophs, as in dog and human thyroid cells, the activating hormone, growth hormone-releasing hormone, acts by activating adenylate cyclase and the CAMP cascade, which leads tional activatio n and growth. In hyperfu nction

to func- ing, au-

tonomous adenomas of the somatotrophs (175) they demonstrated a mutation in G, that causes constitutive activation of this transducing protein and consequently of the whole CAMP cascade. A systematic search for similar lesions in other tumors has allowed the demonstration of sever al identical mutations in the G, of thyroid adeno mas ( 194). Of course, any som atic mutation leading to the constitutive activation of the first elements of the CAMP cascade (TSH or other stimulating receptor, cyclase, protein kinase) or to the inactivation of a negative controlling element (inhibiting receptor, Gi, and a postulated iodine inhibitor XI) could result in a similar phenotype of constitutive activation of the CAMP cascade and consequently of the function and growth of the affected cells, i.e., in a autonomous thyroid adenoma. Similarly the expression in the thyroids of transgenic mice of a constitutively activated adenosine A,-receptor, w hich stimulates cyclase, induces hyperfunction, (Ledent and

growth, and autonomy of the whole thyroid Dumont, un *published observations). Thus

although available data on the CAMP system man autonomous thyroid adenoma suggest

1 n the hu- that the

constitutive G, or adenosine AZ-receptors will only explain a minority of cases, they provide a useful paradigm for the study of other mechanisms of autonomy.

Autonomy in these adenomas corresponds to an uncoupling of thyroid function from the level of activation by TSH but not to a total loss of control: the adenomas still contain TSH receptor mRNA (G. Brabant and J. E. Du- mont, unpublished observations) and respond to TSH, but the basal level of iodide trapping and thyroid secretion is high and the stimulation ratio much decreased (337). The synthesis of DNA is also high but can be stimulated by TSH (221, 222). Activating mutations of G, have also been found in differentiated thyroid tumors, suggesting that they contributed to the tumorigenic phenotype (310). As pointed out earlier, not all autonomous nodules observed in vivo are monoclonal adenomas (227). The autonomy of some nodules has been shown to be reversible, which would be difficult to explain in a lesion caused by a mutation (295). Moreover, areas of autonomy may be spread in the whole tissue of a multinodular goiter (210).

Similar mutations inducing constitutive activation in the elements (genes or proteins) of the Ca2’-PIP, cascade of cells secreting by an exocytic process could explain autonomous functional adenomas of such exocrine or endocrine organs. It will be of interest to look for such defects in calcitonin-secreting cell tumors.

Well-defined mutations of two of the ras genes have been demonstrated in some adenomas (228). These mutations induce a constitutive activation of the Ras proteins, i.e., of GTP-binding transducing proteins. In fact the hydrolysis of GTP by the protein that abolishes its activity is no longer possible. Although the effector system(s) of the Ras proteins has not been identified yet, its permanent activation leads to a proliferation program and to transformation. Again the autonomy of growth corresponds to a loss of control. The type of ras mutation encountered depends on the cause of the tumor, lesions of Ha-ras in carcinogen-induced tumors and mutation of Ki-ras in radiation-induced tumors (184). A similar pattern is found in human tumors (358).

The isolated defects in iodide trapping and of iodination in a few well-studied “cold” adenomas could perhaps also explain the growth of these adenomas (66,67, 294). In the absence of iodine trapping or oxidation, the negative control of iodide is relieved, which might confer a selective advantage to the affected cells, favor- ing the appearance of new mutations and of tumorigen- em.

Finally, response to heterotypic hormones (e.g., ACTH) would confer autonomy to the affected cells. Such heterotypic responses have been observed in adrenal tumors (203) and with platelet-derived growth factor receptors in an anaplastic thyroid carcinoma (127). However, in thyroid autonomous adenomas, only an increased response to P-adrenergic agents has been found in some cases (337). Undoubtedly, these mechanisms do not account for all thyroid adenomas, and other causes will be found. It should be noted that even in such well- defined clonal lesions as these adenomas the histology may be or become very heterogeneous: local factors such as blood supply and new mutations generate heteroge- nei ty.


E. Sporadic Goiter

It is striking that the cause and mechanisms of the most common form of goiter in developed countries, sporadic goiter, are still completely unknown. The great heterogeneity of such goiters, especially late in their evolution (multinodular goiter), does not exclude a single initial homogeneous cause for each. Indeed, investi- gation of a series of patients with sporadic goiter suggests a continuous growth and increasing nodularity in the natural history of the disease (20). Moreover, we have seen that the evolution of goiters due to simple causes, such as a defect in iodine metabolism or iodine deficiency, ends up in a similar heterogeneity. Relative iodine deficiency and heterozygocity of congenital defects have long been suspected to play a role in sporadic goiters (5, 103). The introduction of a simple gene for TGF-CY with a mammary tissue-specific promoter is sufficient to induce very complex and heterogeneous tumors in the affected transgenic mice (202, 282). In the mechanisms generating this heterogeneity the first is certainly that fixation of mutations requires cell division and thus that the mutation load depends on the number of past mitoses. Any mutation causing a loss of control, i.e., a degree of autonomy, will give a selective advantage to the affected cell. This explains why the permanent stimulation by TSH of the CAMP cascade in human thyroids, i.e., of proliferation and differentiation, eventually leads to an increase in the frequency of thyroid cancers. In this regard secretion of autocrine or paracrine growth factors or modulation of the number of receptors to those factors per cell will, as in many tumors, play a large role. It is also probable that local factors, such as irrigation, or its decrease by sclerosis or autoimmune reactions will greatly modify the tissue biology from one locus to another. It has been well shown in endothelial cells that local factors, such as cell ten- sion and shape, that are controlled by the extracellular matrix greatly influence the proliferation of the cells and their responses to growth factors (141-144). In human thyroid cells a set of changes in protein synthesis induced by TSH and CAMP is reproduced by just cultur- ing the cells in dense aggregates (50). Conversely, treatment of dog cells with TSH or phorbol esters but not with EGF induces a disorganization of the actin network (268) and a switch in the expression of tropomyosin isoforms.

VIII. HYPOTHETICAL AND EXISTING EXAMPLES OF

CONSTITUTIVE ACTIVATION OF ADENOSINE 3’,5’- CYCLIC MONOPHOSPHATE CASCADE

As shown, among the mitogenic cascades the CAMP pathway is the only pathway that in some cell types induces both proliferation and differentiation and also activates function (84). In the thyroid and hypophysis its permanent stimulation leads to hyperfunctioning hypertrophied lesions. These involve the whole organ in

A B C H-,+ R P I I

4 + * 1 Gs I % Gs I I I

i

I I

+ 1 A.CYCLASE

+ $

A.CYCLASE A.CY&ASE I I I I I I

1 + 1 + i +

CAMP CAMP CAMP A A A

+J’ ‘\ + +J’ ‘\ + +/ ‘\ + FUNCT GROWTH FUNCT GROWTH FUNCT GROWTH

FIG. 5. CAMP cascade as a possible oncogenic pathway. Models of constitutive activation of CAMP cascade in systems in which cascade is mitogenic. A: normal; causal relations between hormone receptor interaction, CAMP generation, and stimulation of function and growth. B: mutated G, in hyperfunctioning hypophysial adenoma; relations when G, is constitutively activated. C: relations when receptor activating G, is constitutively activated. A cyclase, adenylate cyclase; funct, function; H, hormone; R, receptor; G,, GTP-binding protein that activates adenylate cyclase; *, constitutive activation.

the case of Graves’ disease, where the whole thyroid is constantly stimulated by thyroid-stimulating immuno- globins, or an adenoma in cases where a somatic mutation causes constitutive activation of one element of the cascade in the cell of origin. Many possible somatic mutations could conceivably cause constitutive activation of the CAMP cascade and hyperfunctioning adenomas. Any permanent enhancement of a positive control or suppression of a negative control could give a selective advantage to the affected cells. This advantage would be expressed by a higher mitogenic rate and therefore a higher probability of mutations and of further progression of the cells to autonomy and tumorigenesis. How- ever, only constitutive enhancement of a positive control could lead to autonomy from the normal regulatory feedback control (such as the thyroid pituitary feedback). Similar somatic mutations in cells in which CAMP is a negative signal for growth would of course have the opposite effect; they would select against the affected cells. In the thyrocytes, the following models can be proposed (Fig. 5).

A. Adenylate Cyclase-Coupled Receptors

There are several ways by which receptors could activate adenylate cyclase chronically. Overexpression of the receptor could lead to permanent stimulation if the unoccupied receptor had some basal activity or at least could render the thyrocyte more sensitive to TSH and thus give it a selective advantage. Mutation of the receptor could induce or increase its constitutive activity as has been shown for the EGF receptor in v-e&B oncogene transformation (339). Heterotypic expression of a nonthyroid receptor (such as the ,&adrenergic re-


ceptor) would make the cells independent of the normal thyroid pituitary feedback. Heterotypic expression of receptors coupled to the PI cascade [serotonin 5-HT,, (154)], a putative angiotensin receptor, i.e., the KW,S oncogene (147), leads to fibroblast transformation in vitro. Heterotypic expression of platelet-derived growth factor receptors has been demonstrated in anaplasic thyroid carcinoma (127). Similarly, expression of a receptor to a metabolite (e.g., adenosine) or a signal (e.g., a prostaglandin) that is constantly produced by the cell would also cause apparently constitutive activation. Such physiologically constitutive activation has been conferred in culture on various types of cells by the expression of the high-affinity adenosine AZ-receptor (198). Expression of this gene in the thyroid of transgenic mice leads to hyperfunction and thus hyperthyroidism and increased proliferation and thus goiter (Ledent and Dumont, unpublished observations). These causes of chronic activation would of course have to override the normal desensitization mechanisms, but these are weak in the thyroid (65; Maenhaut and Dumont, unpublished observations). On the contrary, low levels of thyroid stimulation increase the sensitivity of thyroid cells to TSH (315).

B. Cyclase-Activating Guanosine Triphosphate-Binding Protein

Constitutive activation by mutational impairment of the GTPase activity of G, or its subunit cy, would, as in the inhibition of this activity by cholera toxin, lead to permanent constitutive activation of adenylate cyclase. This type of somatic mutation, discovered by Landis et al. (175) in pituitary adenomas, has been described earlier. An inactivating mutation of the inhibitory subunit p of G, would have the same effect. Overexpression of G, could possibly induce a permanent activation of adenylate cyclase.

C. Adenosine 3’,5’-Cyclic Monophosphate-Dependent Protein Kinase

Inactivating mutations of the inhibitory regulatory subunit of CAMP protein kinase lead to constitutive activation of the enzyme. Such mutants have been produced in yeast (185).

D. Negative Controls of Adenosine 3’,5’-Cyclic Monophosphate Cascade

Mutations in any negative control element could lead to activation of the affected cell and would confer it a selective advantage, for example, I) negative feed- backs in CAMP action (phosphorylated protein inhibitor as postulated in autonomous nodules) (337), R) the receptor-Gi pathway, and 3) the iodide inhibitory pathway (at the level of its action on adenylate cyclase iodide transport, oxidation, or at the level of the synthesis of

the inhibitory iodinated derivative XI) (48). It is interesting in this regard that adenoma with defects in some steps in the latter pathway have been demonstrated (66, 67, 100).

IX. CONCLUSIONS

Apart from the few well-defined pathologies described, our expanding knowledge on the secretion in the thyroid of growth factors and local hormones and on the effects on thyrocytes of such factors, of cytokines, and of hormones has not been really translated in the study of disease until now. To investigate the role of such factors it will be necessary to ascertain, in primary cultures, which growth factors, local hormones, and neu- rotransmitters act on human thyroid cells. The recent separations and cloning of these factors and of their receptors now give the tools necessary to investigate, by immunohistochemistry and in situ hybridization on in- dividual cells in thyroid sections, the local pathogenetic process involved in such common diseases as simple goiter and thyroiditis. This will allow researchers to cor- relate directly the various histological aspects with glimpses of the biological situations of the cells that these tools will provide. Also it will be necessary to investigate by double-labeling methods whether the pic- tures that histology and autoradiography provide correspond to a more or less permanent situation of a cell or to a snapshot at one point in time of the dynamic evolution of these cells. Concretely, will the follicle that takes up iodide now become quiescent later and vice versa; will the cell that synthezises DNA now become quiescent for long time or will it keep dividing? The next review on the subject will certainly involve a great deal of such information.

The authors thank Dr. G. Vassart for advice and sugges- tions.

This work was supported by the Minis&e de la Politique Scientifique, Fonds National de la Recherche Scientifique, Fonds de la Recherche Scientifique Medicale, Caisse Generale d’Epargne et de Retraite, Televie, Loterie Nationale, and Asso- ciation contre le Cancer.

REFERENCES

1.

2.

3.

4.

5.

ABRAMOWICZ, M., G. VASSART, AND D. CHRISTOPHE. Thy- roid peroxidase gene promoter confers TSH responsiveness to heterologous reporter genes in transfection experiments. Bio- them. Biophys. Res. Commun. 166: 12%1264,199O. ABS, R., J. VERHELST, E. SCHOOFS, AND E. DE SOMMER. Hyperfunctioning metastatic follicular thyroid carcinoma in Pendred’s syndrome. Cancer 67: 2191-2193,199l. ADAIR, T. H., W. J. GAY, AND J. P. MONTANI. Growth regulation of the vascular system: evidence for a metabolic hypothesis. Am. J. Physiol. 259 (Regulatory Integrative Comp. Physiol. 28): R393-R404,1990. ADAMS, D. D. Thyroid-stimulating autoantibodies. Vitam. Horm. 38: 120-147, 1980. AGERBAEK, H. Non-toxic goitre. Acta Endocrinol. 76: 74-82, 1974.


6. AHREN, B. Atria1 natriuretic factor (ANF) inhibits thyroid hormone secretion in the mouse. Life Sci. 47: 1973-19771990.

7. AHUJA, S., AND H. ERNST. Hyperthyroidism and thyroid carcinoma. Acta EndocrinoZ. 124: 146-151,199l.

8. AMBESI-IMPIOMBATO, F. S., L. A. M. PARKS, AND H. G. COON. Culture of hormone-dependent functional epithelial cells from rat thyroids. Proc. N&Z. Acad. Sci. USA 77: 3455-3459,198O.

9. AMES, B. N., AND L. S. GOLD. Too many rodent carcinogens: mitogenesis increases mutagenesis. Science Wash. DC 249: 970- 971,199o.

27. BONE, E., L. D. KOHN, AND P. CHOMCZYNSKI. Thyroglobulin gene activation by thyrotropin and CAMP in hormonally depleted FRTL-5 thyroid cells. Biochem. Biophys. Res. Commun. 141: 1261- 1266,1986.

10. AOUANI, A., S. HOVSEPIAN, AND G. FAYET. Multihormonal regulation of thyroglobulin production by the OVNIS 6H thyroid cell line. Harm. Metab. Res. 20: 91-95, 1988.

11. ASHIZAWA, K., S. YAMASHITA, Y. NAGAYAMA, H. KI- MURA, H. HIRAYU, M. IZUMI, AND S. NAGATAKI. Interferon- y inhibits thyrotropin-induced thyroidal peroxidase gene expression in cultured human thyrocytes. J. CZin. Endocrinol. Metub. 69: 475-477,1986.

28. BijSWALD, M., S. HARASIM, AND B. MAURER-SCHULTZE. Tracer dose and availability time of thymidine and bromodeoxyuridine: application of bromodeoxyuridine in cell kinetic studies. CeZL Tissue Kinet. 23: 169-181, 1990.

29. BRANDI, M. L., C. M. ROTELLA, C. MAVILIA, F. FRANCES- CHELLI, A. TANINI, AND R. TOCCAFONDI. Insulin stimulates cell growth of a new strain of differentiated rat thyroid cells. MoZ. Cell. Endocrinol. 54: 91-103, 1987.

30. BRAY, G. A. Increased sensitivity of the thyroid in iodine-depleted rats to the goitrogenic effects of thyrotropin. J. Clin. In- vest. 47:1640-1647, 1968.

12. ATKINSON, S., AND P. KENDALL-TAYLOR. Effect of thyrotropin on epidermal growth factor receptors in monolayer cultures of porcine thyroid cells. J. EndocrinoZ. 114: 179-184,1987.

13. AVVEDIMENTO, V. E., A. M. MUSTI, M. UEFFING, S. OBICI, A. GALLO, M. SANCHEZ, D. BEBRASI, AND M. E. GOTTES- MAN. Reversible inhibition of a thyroid-specific trans-acting factor by ras. Genes Dev. 5: 22-28,199l.

14. BACHRACH, L. K., M. C. EGGO, R. L. HINTZ, AND G. N. BURROW. Insulin-like growth factors in sheep thyroid cells: action, receptors and production. Biochem. Biophys. Res. Commun. 154: 861-867,1988.

31. BRENNER-GATI, L., K. A. BERG, AND M. C. GERSHENGORN. Insulin-like growth factor-I potentiates thyrotropin stimulation of adenylyl cyclase in FRTL-5 cells. Endocrinology 125: 1315- 1320, 1989.

32. BRETON, M. F., P. P. ROGER, B. OMRI, J. E. DUMONT, AND M. PAVLOVIC-HOURNAC. Thyrotropin but not epidermal growth factor down-regulates the isozyme I (PKa I) of cyclic AMP-dependent protein kinases in dog thyroid cells in primary cultures. Mol. Cell. Endocrinol. 61: 49-55, 1989.

15. BACHRACH, L. K., M. C. EGGO, W. W. MAK, AND G. N. BURROW. Phorbol esters stimulate growth and inhibit differentiation in cultured thyroid cells. Endocrinology 116: 1603-1609, 1985.

16. BASERGA, R. The Biology of Cell Reproduction. Cambridge, MA: Harvard University Press, 1985, p. 251.

17. BASERGA, R. Growth regulation of the PCNA gene. J. CeZZ Sci. 98:433-436,199l.

33. BROOKS, R. F., F. N. RICHMOND, P. N. RIDDLE, AND K. M. V. RICHMOND. Apparent heterogeneity in the response of quiescent Swiss 3T3 cells to serum growth factors: implications for the transition probability model and parallel with “cellular senescence” and “competence.” J. Cell. Physiol. 121: 341-350, 1984.

34. BROWN, R. S., P. KEATING, AND E. MITCHELL. Maternal thyroid-blocking immunoglobulins in congenital hypothyroidism. J. Clin. EndocrinoZ. Metub. 70: 1341-1346, 1990.

35. BURTON, F. H., K. W. HASEL, F. E. BLOOM, AND J. G. SUT- CLIFFE. Pituitary hyperplasia and gigantism in mice caused by a cholera toxin transgene. Nature Lond. 350: 74-77,199l.

36. CARPENTER, G., AND S. COHEN. Epidermal growth factor. J. Biol. Chem. 265: 7709-7712,199O.

18. BECKS, G. P., M. C. EGGO, AND G. N. BURROW. Organic iodide inhibits deoxyribonucleic acid synthesis and growth in FRTL-5 cells. Endocrinology 123: 545-550, 1988.

19. BELLUR, S., K. TAHARA, M. SAJI, E. F. GROLLMAN, AND L. D. KOHN. Repeatedly passed FRTL-5 rat thyroid cells can develop insulin and insulin-like growth factor-I-sensitive cy- clooxygenase and prostaglandin E, isomerase-like activities to- gether with altered basal and thyrotropin-responsive thymidine incorporation into DNA. Endocrinology 127: 1526-1539,199O.

20. BERGHOUT, A., W. M. WIERSINGA, N. J. SMITS, AND J. L. TOUBER. Interrelationships between age, thyroid volume, thyroid nodularity, and thyroid function in patients with sporadic nontoxic goiter. Am. J. Med. 89: 602-608, 1990.

21. BERLINGIERI, M. T., A. M. MUSTI, V. E. AVVEDIMENTO, R. DI LAURO, P. P. DI FIORE, AND A. FUSCO. The block of thyroglobulin synthesis, which occurs upon transformation of rat thyroid epithelial cells, is at the transcriptional level and it is associated with methylation of the 5’ flanking region of the gene. Exp. CeZl Res. 183: 277-283, 1989.

37. CHABAUD, O., M. CHAMBARD, N. GAUDRY, AND J. MAU- CHAMP. Thyrotropin and cyclic AMP regulation of thyroglobulin gene expression in cultured porcine thyroid cells. J. Endocr. 116:25-33,1987.

38. CHAMBARD, J. C., S. PARIS, G. L’ALLEMAIN, AND J. POUYSEGUR. Two growth factor signalling pathways in fibroblasts distinguished by pertussis toxin. Nature Lond. 326: 800- 803,1987.

39. CHAZENBALK, G., R. P. MAGNUSSON, AND B. RAPOPORT. Thyrotropin stimulation of cultured thyroid cells increases steady state levels of the messenger ribonucleic acid for thyroid peroxidase. Mol. Endocrinol. 1: 913-917, 1987.

40. CHOI, P. M., K. M. TCHOU-WONG, AND I. B. WEINSTEIN. Overexpression of protein kinase C in HT29 colon cancer cells causes growth inhibition and tumor suppression. MoZ. CeZl. BioZ. 10: 4650-4657,199O.

22. BERLINGIERI, M. T., G. PORTELLA, M. GRIECO, M. SAN- TORO, AND A. FUSCO. Cooperation between the polyomavirus middle-T-antigen gene and the human c-myc oncogene in a rat thyroid epithelial differentiated cell line: model of in vitro progression. Mol. CeZI Biol. 8: 2261-2266, 1988.

23. BIDEY, S. P. Control of thyroid cell and follicle growth. Trends EndocrinoZ. Metub. 1: 174-180, 1990.

24. BILLAH, M. M., AND J. C. ANTHES. The regulation and cellular functions of phosphatidylcholine hydrolysis. Biochem. J. 269: 281-291,199O.

41. CHOW, C. C., D. I. W. PHILLIPS, J. H. LAZARUS, AND A. B. PARKES. Effect of low dose iodide supplementation on thyroid function in potentially susceptible subjects: are dietary iodide levels in Britain acceptable? Clin. Endocrinol. 34: 413-416, 1991.

42. CHRISTOPHE, D., C. GERARD, C. HANSEN, C. CHRIS- TOPHE-HOBERTUS, G. JUVENAL, F. LIBERT, P. ROGER, J. E. DUMONT, AND G. VASSART. Control of thyroglobulin gene expression. Horm. CeZZ Regul. 153: 205-213, 1987.

43. CHRISTOPHE, D., C. GERARD, G. JUVENAL, A. BACOLLA, E. TEUGELS, C. LEDENT, C. CHRISTOPHE-HOBERTUS, J. E. DUMONT, AND G. VASSART. Identification of a CAMP-responsive region in thyroglobulin gene promoter. MoZ. Cell. Endocrinol. 64:5-18,1989.

25. BLACK, E. G., A. LOGAN, J. R. E. DAVIS, AND M. C. SHEP- 44. CHRISTOV, K. Cell population kinetics and DNA content during PARD. Basic fibroblast growth factor affects DNA synthesis and thyroid carcinogenesis. Cell Tissue Kinet. 18: 119-131, 1985. cell function and activates multiple signalling pathways in rat 45. CHRISTOV-TZELKOV, K., AND G. GRASCHEV. Cell population thyroid FRTL-5 and pituitary GM3 cells. J. Endocrinol. 127: 39- kinetics of thyroid follicular cells in infant rats. CeLZ Tissue Kinet. 46, 1990. 15:275-284,1982.

26. BLAU, H. M., AND D. BALTIMORE. Differentiation requires continuous regulation. J. CelZ Biol 112: 781-783, 1991.

46. CIVITAREALE, D., R. LONIGRO, A. J. SINCLAIR, AND R. DI LAURO. A thyroid-specific nuclear protein essential for tissue-


47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

specific expression of the thyroglobulin promoter. EM’0 J. 8: 2537-2542,1989. CLARK, 0. H., R. R. CAVALIERI, C. MOSER, AND S. H. ING- BAR. Iodide-induced hypothyroidism in patients after thyroid resection. Eur. J. Clin. Invest. 20: 573-580, 1990. COCHAUX, P., J. VAN SANDE, S. SWILLENS, AND J. E. DU- MONT. Iodide-induced inhibition of adenylate cyclase activity in horse and dog thyroid. Eur. J Biochem. 170: 435-442,1987. COCLET, J., F. FOUREAU, P. KETELBANT, P. GALAND, AND J. E. DUMONT. Cell population kinetics in dog and human adult thyroid. CZin. EndocrinoZ. 31: 655-665, 1989. COCLET, J., F. LAMY, F. RICKAERT, J. E. DUMONT, AND P. P. ROGER. Intermediate filaments in normal thyrocytes: modulation of vimentin expression in primary cultures. Mol. CeZZ. Endo- crinol. 76:135-148, 1991. COLLETTA, G., A. M. CIRAFICI, AND G. VECCHIO. Induction of the c-fos oncogene by thyrotropic hormone in rat thyroid cells in culture. Science Wash. DC 233: 458-460,1986. COMBEST, W. L., AND D. H. RUSSELL. Alteration in cyclic AMP-dependent protein kinases and polyamine biosynthetic en- zymes during hypertrophy and hyperplasia of the thyroid in the rat. Mol. Pharmacol. 23: 641-647,1983. CONNORS, J. M., L. J. HUFFMAN, AND G. A. HEDGE. Effects of thyrotropin on the vascular conductance of the thyroid gland. Endocrinology 122: 921-929,1988. CONTOR, L., F. LAMY, R. LECOCQ, P. P. ROGER, AND J. E. DUMONT. Differential protein phosphorylation in induction of thyroid cell proliferation by thyrotropin, epidermal growth factor, or phorbol ester. 1MoZ. CeZZ. BioZ. 8: 2494-2503, 1988. COOPER, E., AND S. W. SPAULDING. HMG (high-mobility- group)-14/17-like proteins in calf thyroid. Biochem. J. 215: 643- 649,1983. COOPER, E., AND S. W. SPAULDING. Hormonal control of the phosphorylation of histones, HMG proteins and other nuclear proteins. Mol. CeU. Endocrinol. 39: l-20, 1985. CORCORAN, J. M., M. J. WATERS, C. J. EASTMAN, AND G. JORGENSEN. Epidermal growth factor: effect on circulating thyroid hormone levels in sheep. Endocrinology 119: 214-217, 1986. CZECH, M. P. Signal transmission by the insulin-like growth factor. CeZZ 59: 235-238, 1989. DAMANTE, G., F. COX, AND B. RAPOPORT. IGF-I increases c-fos expression in FRTL-5 rat thyroid cells by activating the c-fos promoter. Biochem. Biophys. Res. Commun. 151: 1194-1199, 1988. DAMANTE, G., AND B. RAPOPORT. TSH stimulates the activity of the c-fos promoter in FRTL-5 rat thyroid cells. Mol. Cell. Endo- crinol. 58: 279-282, 1988. DAVIES, T. F. Positive regulation of the guinea pig thyrotropin receptor. EndocrinoZogy 117: 201-207,1985. DAVIES, T. F., AND M. PLATZER. hCG-induced TSH receptor activation and growth acceleration in FRTL-5 thyroid cells. Endo- crinology 118: 2149-2151,1986. DAVIES, T. F., C. YANG, AND M. PLATZER. Cloning the fisher rat thyroid cell line (FRTL-5): variability in clonal growth and 3’,5’-cyclic adenosine monophosphate response to thyrotropin. En- docrinoZogy 121: 78-83,1987. DEGROOT, L. J., A. NAKAI, A. SAKURAI, AND E. MACCHIA. The molecular basis of thyroid hormone action. J. Endocrinol. Invest. 12: 843-861,1989. DELBEKE, D., J. VAN SANDE, S. SWILLENS, C. ERNEUX, AND J. E. DUMONT. Cooling enhances adenosine 3’5’ monophosphate accumulation in thyrotropin stimulated dog thyroid slices. Metabolism 31: 797-804, 1982. DEMEESTER-MIRKINE, N., J. VAN SANDE, J. CORVILAIN, AND J. E. DUMONT. Benign thyroid nodule with normal iodide trap and defective organification. J. Clin. Endocrinol. 41: 1169- 1171,1975. DEMEESTER-MIRKINE, N., J. VAN SANDE, P. DOR, R. HEI- MANN, P. COCHAUX, AND J. E. DUMONT. Iodide organification defect in a cold thyroid nodule: absense of iodide effect on cyclic AMP accumulation. CZin. EndocrinoZ. 20: 473-479, 1984. DENEF, J. F., A. C. CORDIER, S. HAUMONT, AND C.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

87.

88.

BECKERS. The influence of thyrotropin and growth hormone on the thyroid gland in the hereditary dwarf mouse: a morphometric study. Endocrinology 107: 1249-1257, 1980. DENEF, J. F., S. HAUMONT, C. CORNETTE, AND C. BECKERS. Correlated functional and morphometric study of thyroid hyperplasia induced by iodine deficiency. Endocrinology 108: 2352- 2358,198l. DENEF, J. F., C. OVAERT, AND M. C. MANY. La goitrogenese experimentale. Ann. Endocrinol. 50: l-15, 1989. DERE, W. H., H. HIRAYU, AND B. RAPOPORT. TSH and CAMP enhance expression of the myc proto-oncogene in cultured thyroid cells. Endocrinology 117: 2249-2251, 1985. DERE, W. H., H. HIRAYU, AND B. RAPOPORT. Thyrotropin and cyclic AMP regulation of ras proto-oncogene expression in cultured thyroid cells. FEBS Lett. 196: 305-308, 1986. DERE, W. H., AND B. RAPOPORT. Control of growth in cultured rat thyroid cells. Mol. CeZZ. EndocrinoZ. 44: 195-199, 1986. DICICCO-BLOOM, E., E. TOWNES-ANDERSON, AND I. B. BLACK. Neuroblast mitosis in dissociated culture: regulation and relationship to differentiation. J. CelZ Biol. 110: 2073-2086, 1990. DINSART, C., R. LECOCQ, J. E. DUMONT, AND G. VASSART. Control by TSH of protein turnover in thyroid subcellular fractions. Horm. Metab. Res. 8: 140-145, 1976. DOMANN, F. E., J. M. MITCHEN, AND K. H. CLIFTON. Restora- tion of thyroid function after total thyroidectomy and quantitative thyroid cell transplantation. Endocrinology 127: 2673-2678, 1990. DONIACH, I. Types of thyroid growth. Br. Med. BUZZ. 16: 99-104, 1960. DOW, C. J., J. E. DUMONT, AND P. KETELBANT. Etude du pourcentage de cellules epitheliales, fibroblastes et cellules en- dotheliales dans les thyroi’des de chien. C. R. Seances Sot. BioZ. Fil. 180: 629-632,1986. DREXHAGE, H. A., G. F. BOTTAZZO, D. DONIACH, L. BI- TENSKY, AND J. CHAYEN. Evidence for thyroid-growth-stimulating immunoglobulins in some goitrous thyroid diseases. Lan- cet 9: 287-292,198O. DREXHAGE, H. A., L. J. HAMMOND, L. BITENSKY, J. CHAYEN, G. F. BOTTAZZO, AND D. DONIACH. The involvement of the pentose shunt in thyroid metabolism after stimulation with TSH or with immunoglobulins from patients with thyroid disease. 1. The generation of NADPH in relation to stimulation of thyroid growth. Clin. Endocrinol. 16: 46-56, 1982. DUGRILLON, A., G. BECHTNER, W. M. UEDELHOVEN, P. C. WEBER, AND R. GARTNER. Evidence that an iodolactone mediates the inhibitory effect of iodide on thyroid cell proliferation but not on adenosine 3’,5’-monophosphate formation. EndocrinoZ- ogyl27:337-343,199O. DUMONT, J. E. The action of thyrotropin on thyroid metabolism. Vitam. Harm. 29: 287-412, 1971. DUMONT, J. E., J. M. BOEYNAEMS, C. DECOSTER, C. ER- NEUX, F. LAMY, R. LECOCQ, J. MOCKEL, J. UNGER, AND J. VAN SANDE. Biochemical mechanisms in the control of thyroid function and growth. Adv. Cyclic Nucleotide Res. 9: 723-734,1978. DUMONT, J. E., J. C. JAUNIAUX, AND P. P. ROGER. The cyclic AMP-mediated stimulation of cell proliferation. Trends Bio- them. Sci. 14: 67-71,1989. DUMONT, J. E., AND P. ROCMANS. In vivo effects of thyrotropin on the metabolism of the thyroid gland. J Physiol. Lond. 174: 26-45,1964. DUMONT, J. E., P. ROGER, B. VAN HEUVERSWYN, C. ER- NEUX, AND G. VASSART. Control of growth and differentiation by known intracellular signal molecules in endocrine tissues: the example of the thyroid gland. Adv. Cyclic NucZeotide Protein Phosphorylation Res. 17: 337-342, 1984. DUMONT, J. E., P. P. ROGER, AND M. LUDGATE. Assays for the thyroid growth immunoglobulins and their clinical implications: methods, concepts and misconceptions. Endocr. Rev. 8: 448- 452,1987. DUMONT, J. E., G. VASSART, AND C. REFETOFF. Thyroid dis- orders. In: The Metabolic Basis of Inherited Disease, edited by


89.

90.

91.

92.

93.

94.

95.

96.

97.

98.

99.

100.

101.

102.

103.

104.

105.

106.

107.

108.

C. R. Striver, A. L. Beaudet, W. S. Sly, and D. Valle. New York: McGraw-Hill, 1989, p. 1843-1879. EALEY, P. A., C. A. AHENE, J. M. EMMERSON, AND N. J. MARSHALL. Forskolin and thyrotropin stimulation of rat FRTL-5 thyroid cells growth: the role of cyclic AMP. J. Endo- crinol. 114: 199-205, 1987. EGGO, M. C., L. R. BACHRACH, G. FAYET, J. ERRICK, J. E. KUDLOW, M. F. COHEN, AND G. N. BURROW. The effects of growth factors and serum on DNA synthesis and differentiation in thyroid cells in culture. Mol. Cell Endocrinol. 38: 141-150,1984. EGGO, M. C., W. W. MAK, L. K. BACHRACH, J. E. ERRICK, AND G. N. BURROW. Cultured thyroids. Is immortality the an- swer? Thyroglobulin-the prothyroid hormone. Prog. Endocr. Res. Ther. 2: 201-210, 1985. EMOTO, N., 0. ISOZAKI, M. ARAI, H. MURAKAMI, K. SHI- ZUME, A. BAIRD, T. TSUSHIMA, AND H. DEMURA. Identifica- tion and characterization of basic fibroblast growth factor in porcine thyroids. EndocrinoZogy 128: 58-64, 1991. ENDO, T., H. SHIMURA, T. SAITO, AND T. ONAYA. Cloning of malignantly transformed rat thyroid (FRTL) cells with thyrotropin receptors and their growth inhibition by 3’,5’-cyclic adenosine monophosphate. Endocrinology 126: 1492-1497,199O. ENOMOTO, T., H. SUGAWA, S. KOSUGI, D. INOUE, T. MORI, AND H. IMURA. Prolonged effects of recombinant human interleukin-lcu on mouse thyroid function. Endocrinology 127: 2322- 2327,199O. ERMANS, A. M., AND M. CAMUS. Modifications of thyroid function induced by chronic administration of iodide in the presence of “autonomous” thyroid tissue. Acta EndocrinoZ. 70: 463-475, 1972. ERRICK, J. E., K. W. A. ING, M. C. EGGO, AND G. N. BURROW. Growth and differentiation in cultured human thyroid cells: effects of epidermal growth factor and thyrotropin. In Vitro 22: 28-36,1986. FAYET, G., AND S. HOVSEPIAN. Demonstration of growth in porcine thyroid cell culture, Biochimie 61: 923-930, 1979. FAYET, G., AND S. HOVSEPIAN. Strategy of thyroid cell culture in defined media and the isolation of the ovnis and porthos cell strains. In: Thyroglobulin. The Prothyroid Hormone, edited by M. C. Eggo and E. G. Burrow. New York: Raven, 1985, p. 211- 224. FAYET, G., M. MICHEL-BECHET, AND S. LISSITZKY. Thyro- tropin-induced aggregation and reorganization into follicles of isolated porcine thyroid cells in culture. Eur. J. Biochem. 24: lOO- 111,197l. FIELD, J. B., R. LARSEN, K. YAMASHITA, K. MASHITER, AND A. DEKKER. Demonstration of iodide transport defect but normal iodide organification in nonfunctioning nodules of human thyroid glands. J. C&n. Invest. 52: 2404-2410, 1973. FOLKMAN, J., AND M. KLAGSBURN. Angiogenic factors. Science Wash. DC 235: 442-447,1987. FOTI, D., G. DAMANTE, AND B. RAPOPORT. Studies on the role of c-fos in TSH-stimulated thyroid cell proliferation. CeZZ. MO,!. Biol. 36: 363-373, 1990. FRASER, G. R. Some genetical determinants in the pathogenesis of goiter. J. Genet. Hum. 11: 240-250, 1962. GARBI, C., G. COLLETTA., A. M. CIRAFICI, P. C, MARCHISIO, AND L. NITSCH. Transforming growth factor-beta induces cytoskeleton and extracellular matrix modifications in FRTL-5 thyroid epithelial cells. Eur. J. CeZl BioZ. 53: 281-289, 1990. GARTNER, R., G. BECHTNER, D. STUBNER, AND W. GREIL. Growth regulation of porcine thyroid follicles in vitro by growth factors. Harm. Metab. Res. 23, Suppl.: 61-67, 1990.

GARTNER, R., W. GREIL, P. DEMHARTER, AND K. HORN. Involvement of cyclic AMP, iodine and metabolites of arachidonic acid in the regulation of cell proliferation of isolated porcine thyroid follicles. MoC Cell. Endocrinol. 42: 145-155, 1985. GEELHOED-DUIJVESTIJN, P. H. L. M., J. K. BUSSEMAKER, AND F. ROELFSEMA. Changes in basal and stimulated TSH and other parameters of thyroid function in acromegaly after transsphenoidal surgery. Acta EndocrinoZ. 121: 207-215,1989. GERARD, C., A. LEFORT, D. CHRISTOPHE, F. LIBERT, J. VAN SANDE, J. E. DUMONT, AND G. VASSART. Control of

109.

110.

111.

112.

113.

114.

115.

116.

117.

118.

119.

120.

121.

122.

123.

124.

125.

126.

127.

128. HELDIN, N. E., F. A. KARLSSON, AND B. WESTERMARK. A

thyroperoxidase and thyroglobulin transcription by CAMP: evidence for distinct regulatory mechanisms. Mol. Endocrinol. 3: 2110-2118, 1989. GERARD, C., A. LEFORT, F. LIBERT, D. CHRISTOPHE, J. E. DUMONT, AND G. VASSART. Transcriptional regulation of the thyroperoxidase gene by thyrotropin and forskolin. MO,!. CeZC En- docrinol. 60: 239-242, 1988. GERARD, C. M., P. P. ROGER, AND J. E. DUMONT. Thyroglobu- lin gene expression as a differentiation marker in primary cultures of calf thyroid cells. Mol. CeU. Endocrinol. 61: 23-35, 1989. GETZENBERG, R. H., K. J. PIENTA, AND D. S. COFFEY. The tissue matrix: cell dynamics and hormone action. Endocr. Rev. 11: 399-417,199o. GILL, G. N., P. J. HORNSBY, AND M. H. SIMONIAN. Hormonal regulation of the adrenocortical cell. J. Supramol. Struct. 14: 353- 369,199O. GILLET, C., J. CORVILAIN, J. MATTE-HIRIART, D. WIL- LEMS, AND P. BERGMANN. Effect of acute hypercalcemia on thyrotropin (TSH) and triiodothyronine responses to TSH-releasing hormone in man. J. CZin. Endocrinol. Metab. 71: 516-519, 1990. GOLDSTEIN, S. Replicative senescence: the human fibroblast comes of age. Science Wash. DC 249: 1129-1132,199O. GOODMAN, A. L., AND J. D. RONE. Thyroid angiogenesis: en- dotheliotropic chemoattractant activity from rat thyroid cells in culture. EndocrinoZogy 121: 2131-2140,1987. GREER, M. A., H. STUDER, AND J. W. KENDALL. Studies on the pathogenesis of colloid goiter. Endocrinology 81: 623-632, 1967. GREIL, W., M. RAFFERZEDER, G. BRECHTNER, AND R. GARTNER. Release of an endothelial cell growth factor from cultured porcine thyroid follicles. MoZ. Endocrinol. 3: 858-865, 1989. GRENIER, G., J. VAN SANDE, C. WILLEMS, P. NEVE, AND J. E. DUMONT. Effects of microtubule inhibitors and cytochala- sin B on thyroid metabolism in vitro. Biochimie 57: 337-341,1975. GRIEP, A. E., AND H. WESTPHAL. Antisense Myc sequences induce differentiation of F9 cells. Proc. Natl. Acad. Sci. USA 85: 6806-6810,1988. GRUBECK-LOEBENSTEIN, B., G. BUCHAN, R. SADEGHI, M. KISSONERGHIS, M. LONDEI, M. TURNER, K: PIRICH, R. ROKA, B. NIEDERLE, H. KASSAL, W. WALDHAUSL, AND M. FELDMAN. Transforming growth factor beta regulates thyroid growth. J. Clin. Invest. 83: 764-770, 1989. HAASKJOLD, E., S. B. REFSUM, R. BJERKNES, AND T. 0. PAULSEN. The labelling index is not always reliable. Discrepan- cies between the labelling index and the mitotic rate in the rat cornea1 epithelium after intraperitoneal and topical administration of tritiated thymidine and colcemid. CeZZ Tissue Kinet. 21: 389-394,1988. HANSEN, C., C. GERARD, G. VASSART, P. STORDEUR, AND D. CHRISTOPHE. Thyroid-specific and CAMP-dependent hyper- sensitive regions in thyroglobulin gene chromatin. Eur. J. Bio- them. 178: 387-393,1988. HARTWELL, L. H., AND T. A. WEINERT. Checkpoints: controls that ensure the order of cell cycle events. Science Wash. DC246: 629-634,1989. HAY, N., M. TAKIMOTO, AND J. M. BISHOP. A fos protein is present in a complex that binds a negative regulator of myc. Genes Dev. 3: 293-303,1989. HAYE, B., J. L. AUBLIN, S. CHAMPION, B. LAMBERT, AND C. JACQUEMIN. Tetradecanoyl phorbol-13-acetate counteracts the responsiveness of cultured thyroid cells to thyrotropin. Biochem. Pharmacol. 34: 3795-3802,1985. HEIKKILA, R., G. SCHWAB, S. WICKSTROM, S. LOONG LOKE, D. V. PLUZNIK, R. WATT, AND L. M. NECKERS. A c- myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from GO to Gl. Nature Land. 328: 445-449,1987. HELDIN, N. E., B. GUSTAVSSON, L. CLAESSON-WELSH, A. HAMMACHER, J. MARK, C. H. HELDIN, AND B. WESTER- MARK. Aberrant expression of receptors for platelet-derived growth factor in an anaplastic thyroid carcinoma cell line. Proc. Natl. Acad. Sci. USA 85: 9302-9306, 1988.


growth stimulatory effect of iodide is suggested by its effects on c-myc messenger ribonucleic acid levels, rH]thymidine incorporation, and mitotic activity of porcine follicle cells suspension culture. Endocrinology 121: 757-764, 1987.

129. HELDIN, N. E., Y. PAULSSON, K. FORSBERG, C. H. HELDIN, AND B. WESTERMARK. Induction of cyclic AMP synthesis by forskolin is followed by a reduction in the expression of c-myc messenger RNA and inhibition of 3H-thymidine incorporation in human fibroblasts. J. Cell. Physioh 138: 17-23, 1989.

130. HELDIN, N. E., AND B. WESTERMARK. Epidermal growth factor, but not thyrotropin, stimulates the expression of c-fos and c-myc messenger ribonucleic acid in porcine thyroid follicle cells in primary culture. Endocrinology 122: 1042-1046,1988.

131. HEPBURN, A., AND J. E. DUMONT. Effect of adenosine 3’5’ monophosphate on the mutation frequency induced by N- methyl-N’-nitro-N-nitrosoguanidine. Mutat. Res. 199: 221-228, 1988.

132. HERSHMAN, J. M., H. Y. LEE, M. SUGAWARA, C. J. MIRELL, X. P. PANG, M. YANAGISAWA, AND A. E. PEKARY. Human chorionic gonadotropin stimulates iodide uptake, adenylate cyclase, and deoxyribonucleic acid synthesis in cultured rat thyroid cells. J. Clin. Endocrinol. Metab. 67: 74-79, 1988.

133. HICKS, D. G., V. A. LIVOLSI, J. A. NEIDICH, J. M. PUCK, AND J. A. KANT. Clonal analysis of solitary follicular nodules in the thyroid. Am. J. Pathol. 137: 553-562, 1990.

134. HILFER, S. R., AND R. L. SEARL. Differentiation in the thyroid in the hypophysectomized chick embryo. Dev. BioL. 79: 107-118, 1980.

135. HIRAYU, H., W. H. DERE, AND B. RAPOPORT. Initiation of normal thyroid cells in primary culture is associated with enhanced c-myc messenger ribonucleic acid levels. Endocrinology 120: 924-928,1987.

136. HORNSBY, P. J. Regulation of adrenocortical cell proliferation in culture. Endocr. Res. 10: 259-281, 1985.

137. HUBER, G. K., AND T. F. DAVIES. Human fetal thyroid cell growth in vitro: system characterization and cytokine inhibition. Endocrinology 126: 869-875, 1990.

138. HUBER, G. K., R. SAFIRSTEIN, D. NEUFELD, AND T. F. DA- VIES. Thyrotropin receptor autoantibodies induce human thyroid cell growth and c-fos activation. J. Clin. Endocrinol. Metab. 72: 1142-1148, 1991.

139. HUME, W. J. A reproducible technique combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine for double labelling studies of cell proliferation in par- affin sections of tissues. CeZZ Tissue Kinet. 23: 161-168, 1990.

140. IMADA, M., M. KUROSUMI, AND H. FUJITA. Three-dimen- sional aspects of blood vessels in thyroids from normal, low iodine diet-treated, TSH-treated, and PTU-treated rats. Cell Tissue Res. 245: 291-296,1986.

141. INGBER, D. E. Fibronectin controls capillary endothelial cell growth by modulating cell shape. Proc. N&Z. Acad. Sci. USA 87: 3579-3583,199O.

142. INGBER, D. E., AND J. FOLKMAN. How does extracellular matrix control capillary morphogenesis? CeZZ 58: 803-805, 1989.

143. INGBER, D. E., J. A. MADRI, AND J. FOLKMAN. Endothelial growth factors and extracellular matrix regulate DNA synthesis through modulation of cell and nuclear expansion. In Vitro CeZl. Dev. Biol. 5: 387-394, 1987.

144. INGBER, D. E., D. PRUSTY, J. V. FRANGIONI, E. J. CRAGOE, JR., C. LECHENE, AND M. A. SCHWARTZ. Control of intracellular pH and growth by fibronectin in capillary endothelial cells. J. Cell Biol. 110: 1803-1811, 1990.

145. ISLER, M. Loss of mitotic response of the thyroid gland to TSH in hypophysectomized rats and its partial restoration by anterior and posterior pituitary hormones. Anat. Rec. 180: 369-376,1974.

146. ISOZAKI, O., L. D. HOHN, C. A. KOZAK, AND S. KIMURA. Thy- roid peroxidase: rat cDNA sequence, chromosomal localization in mouse, and regulation of gene expression by comparison to thyroglobulin in rat FRTL-5 cells. Mol. Endocrinol. 3: 1681-1692, 1989.

147. JACKSON, T. R., L. A. C. BLAIR, J. MARSHALL, M. GOEDERT, AND M. R. HANLEY. The mas oncogene encodes an angiotensin receptor. Nature Lond. 335: 437-440,1988.

148. JIN, S., F. J. HORNICEK, D. NEYLAN, M. ZAKARIJA, AND J. M.

MCKENZIE. Evidence that adenosine 3’,5’-monophosphate mediates stimulation of thyroid growth in FRTL-5 cells. Endocrinol- ogy 119: 802-810,1986.

149. JOLIN, T., G. MORREALE DE ESCOBAR, AND F. ESCOBAR DEL REY. Differential effects in the rat of thyroidectomy, propylthiouracil and other goitrogens on plasma insulin and thyroid weight. Endocrinology 87: 99-110, 1970.

150. JOLIN, T., M. J. TARIN, AND M. D. GARCIA. Effects of adrenal- ectomy and corticosterone on thyroid weight of goitrogen- treated rats. Role of adrenal-corticoids in the insulin increase of goitre weight. Acta Endocrinol. 75: 734-747, 1974.

151. JONCKHEER, M. H., Y. MICHOTTE, A. C. VAN STEIR- TEGHEM, AND F. DECONINCK. The adaptation of the human thyroid gland to a physiological regimen of iodide intake: evidence for a transitory inhibition of thyroid hormone secretion modulated by the intrathyroidal iodine stores. J. Endocrinol. In- vest. 6: 267-272, 1983.

152. JijRTSij, E., J. MijLNE, B. BOERYD, L. E. ERICSON, H. HJELM, V. JOHANSSON, J. PERSLIDEN, L. TEGLER, AND S. SMEDS. Effects of thyroid stimulating immunoglobulin on function and morphology of xenotransplanted toxic diffuse, toxic nodular and normal thyroid tissue. J. Endocrinol. Invest. 10: 435-442, 1987.

153. JOST, A. Sur le developpement de thyroi’de chez le foetus de lapin ditcapite. Arch. Anat. Microsc. Morphol. Exp. 42: 168-183, 1953.

154. JULIUS, D., T. J. LIVELLI, T. M. JESSELL, AND R. AXEL. Ec- topic expression of the serotonin lc receptor and the triggering of malignant transformation. Science Wash. DC 244: 1057-1062, 1989.

155. KACZMAREK, L. Protooncogene expression during the cell cycle. Lab. Invest. 54: 365-376, 1986.

156. KAISSLING, B., AND 0. BUCHER. Alterations of the mitotic index of C cells and follicular cells of the rat thyroid during the circadian rhythm and after exposure to cold. 2. ZelZfbrsch. Mikrosk. Anat. 146: 417-423,1973.

157. KALISNIK, M., R. ZORC-PLESKOVIC, Z. PAJER, AND K. PAV- LIN. The effect of chronic hypercalcemia or hypocalcemia on the follicular and parafollicular cells in rat thyroid gland. Am. J. Anat. 189: 201-206,199O.

158. KARSENTY, G., C. ALQUIER, C. JELSEMA, AND B. D. WEIN- TRAUB. Thyrotropin induces growth and iodothyronine production in a human thyroid cell line without affecting adenosine 3’,5’-monophosphate production. Endocrinology 123: 1977-1983, 1988.

159. KASAGI, K., A. HIDAKA, H. HATABU, Y. TOKUDA, T. MI- SAKI, Y. IIDA, M. YOSHIDA, S. FUJII, H. TAII, AND J. KONI- SHI. Stimulation of cyclic AMP production in FRTL-5 cells by crude immunoglobulin fractions of serum from pregnant women. Clin. Endocrinol. 31: 267-275, 1989.

160. KASAI, K., M. HIRAIWA, Y. SUZUKI, T. EMOTO, N. BANDA, T. NAKAMURA, AND S. I. SHIMODA. Presence of epidermal growth factor receptors on human thyroid membranes. Acta En- docrinol. 114: 396-401,1987.

161. KASAI, K., T. OHMORI, N. KOIZUMI, T. HOSOYA, M. HI- RAIWA, T. EMOTO, Y. HATTORI, AND S. I. SHIMODA. Regula- tion of thyroid peroxidase activity by thyrotropin, epidermal growth factor and phorbol ester in porcine thyroid follicles cultured in suspension. Life Sci. 45: 1451-1459,1989.

162. KAWABE, Y., K. EGUCHI, C. SHIMOMURA, M. MINE, T. OT- SUBO, Y. UEKI, H. TEZUKA, H. NAKAO, A. KAWAKAMI, K. MIGITA, S. YAMASHITA, M. MATSUNAGA, N. ISHIKAWA, K. ITO, AND S. NAGATAKI. Interleukin-1 production and action in thyroid tissue. J. CZin. Endocrinol. Metab. 68: 1174-1183, 1989.

163. KENNEDY, J. S. The pathology of dyshormonogenetic goitre. J. Pathol. 99: 251-264, 1969.

164. KERKOF, P. R., P. J. LONG, AND I. L. CHAIKOFF. In vitro effects of thyrotropin hormone. On the pattern of organization of monolayer cultures of isolated sheep thyroid gland. Endocrinol- ogy 74: 170-179,1964.

165. KRAIEM, Z., 0. SADEH, AND E. SOBEL. Thyrotropin, acting at least partially via adenosine 3’,5’-monophosphate, exerts both mitogenic and antimitogenic effects in cultured human thyroid cells. J. CZin. Endocrinol. Metab. 70: 497-502, 1990.

692 DUMONT, LAMY, ROGER, AND MAENHAUT Volume 7.2

166. KRAIEM, Z., E. SOBEL, 0. SADEH, A. KINARTY, AND N. LA- HAT. Effects of y-interferon on DR antigen expression, growth, 3,5,3’-triiodothyronine secretion, iodide uptake, and cyclic adenosine 3’,5’-monophosphate accumulation in cultured human thyroid cells. J. Clin. Endocrinol. Metab. 71: 817-824, 1990.

167. KUNG, A. W. C., AND K. S. LAU. Interferon-y inhibits thyrotropin-induced thyroglobulin gene transcription in cultured human thyrocytes. J Clin. Endocrinol. Metab. 70: 15121517, 1990.

168. LAMBERG, B. A., R. PELKONEN, A. ARO, AND B. GRAHNE. Thyroid function in acromegaly before and after transsphenoidal hypophysectomy followed by cryoapplication. Acta EndocrinoZ. 82: 254-266, 1976.

J. J. M. DE VIJLDER. Methimazole increases thyroid-specific mRNA concentration in human thyroid cells and FRTL-5 cells. Mol. Cell. Endocrinol. 78: 221-228, 1991.

184. LEMOINE, N. R., E. S. MAYALL, E. D. WILLIAMS, V. THUR- STON, AND D. WYNFORD-THOMAS. Agent-specific ras oncogene activation in rat thyroid tumours. Oncogene 3: 541-544,1988.

185. LEVIN, L. R., J. KURET, K. E. JOHNSON, S. POWERS, S. CA- MERON, T. MICHAELI, M. WIGLER, AND M. J. ZOLLER. A mutation in the catalytic subunit of CAMP-dependent protein kinase that disrupts regulation. Science Wash. DC 240: 68-70, 1988.

169. LAMBERTS, S. W. J., J. ZUIDERWIJK, P. UITTERLINDEN, J. J. BLIJD, H. A. BRUINING, AND F. H. DE JONG. Characteriza- tion of adrenal autonomy in Cushing’s syndrome: a comparison between in vivo and in vitro responsiveness of the adrenal gland. J. Clin. Endocrinol. Metab. 70: 192-199, 1990.

169a.LAMY, F., R. LECOCQ, AND J. E. DUMONT. Thyrotropin stimulation of the phosphorylation of serine in the N-terminal region of thyroid Hl histones. Eur. J. Biochem. 73: 529-535, 1977.

170. LAMY, F., P. P. ROGER, L. CONTOR, S. REUSE, E. RASP& J. VAN SANDE, AND J. E. DUMONT. Control of thyroid cell proliferation: the example of the dog thyrocyte. Horm. CeZZ Regul. 153: 169-180,1987.

171. LAMY, F., P. P. ROGER, R. LECOCQ, AND J. E. DUMONT. Dif- ferential protein synthesis in the induction of thyroid cell proliferation by thyrotropin, epidermal growth factor or serum. Eur. J. Biochem. 155: 265-272, 1986.

186. LEVITZKI, A. Tyrphostins-potential antiproliferative agents and novel molecular tools. Biochem. PharmacoZ.40: 913-918,199O.

187. LEWINSKI, A. Effect of dibutyryl cyclic adenosine 3’,5’-monophosphate on mitotic activity in the thyroid of hypophysectomized rats. CeZL Tissue Res. 211: 349-352, 1980.

188. LEWINSKI, A. Pituitary is not required for the compensatory thyroid hyperplasia. Harm. Res. Basell5: 189-197, 1981.

189. LEWINSKI, A., A. BARTKE, AND N. K. R. SMITH. Compensa- tory thyroid hyperplasia in hemithyroidectomized snell dwarf mice. Endocrinology 113: 2317-2318,1983.

190. LEWINSKI, A., A. ESQUIFINO, AND A. BARTKE. Influence of thyrotropin, growth hormone and prolactin on mitotic activity of the thyroid in normal and hereditary dwarf mice. Neuroendo- crinol. Lett. 3: 177-182, 1984.

172. LAMY, F., P. P. ROGER, R. LECOCQ, AND J. E. DUMONT. Pro- tein synthesis during induction of DNA replication in thyroid epithelial cells: evidence for late markers of distinct mitogenic pathways. J. Cell. Physiol. 138: 568-578, 1989.

173. LAMY, F., M. TATON, J. E. DUMONT, AND P. P. ROGER. Con- trol of protein synthesis by thyrotropin and epidermal growth factor in human thyrocytes: role of morphological changes. Mol. Cell. EjIdocrinol. 73: 195-209, 1990.

174. LAMY, F., C. WILLEMS, R. LECOCQ, C. DELCROIX, AND J. E. DUMONT. Stimulation by thyrotropin in vitro of uridine incorporation into the RNA of thyroid slices. Harm. Metab. Rex 3: 414- 422, 1971.

191. LOMBARDI, A., B. M. VENEZIANI, D. TRAMONTANO, AND S. H. INGBAR. Independent and interactive effects of tetradecanoyl phorbol acetate on growth and differentiated functions of FRTL-5 cells. Endocrinology 123: 1544-1552, 1988.

192. LU, C., K. KASAGI, A. HIDAKA, H. HATABU, Y. IIDA, AND J. KONISHI. Simultaneous measurement of TSH-binding inhibitor immunoglobulin and thyroid stimulating autoantibody activities using cultured FRTL-5 cells in patients with untreated Graves’ disease. Acta Endocrinol. 123: 282-290, 1990.

193. LUDGATE, M., J. PERRET, M. PARMENTIER, C. GERARD, F. LIBERT, J. E. DUMONT, AND G. VASSART. Use of the recombinant human thyrotropin receptor (TSH-R) expressed in mammalian cell lines to assay TSH-R autoantibodies. Mol. Cell. Endo- crinol. 73: R13-R18, 1990.

175. LANDIS, C. A., S. B. MASTERS, A. SPADA, A. M. PACE, H. R. BOURNE, AND L. VALLAR. GTPase inhibiting mutations activate the cy chain of G, and stimulate adenylate cyclase in human pituitary tumours. Nature Lond. 340: 692-696, 1989.

176. LANOTTE, M., J. B. RIVIERE, S. HERMOUET, G. HOUGE, 0. K. VINTERMYR, B. T. GJERTSEN, AND S. 0. DOSKELAND. Programmed cell death (apoptosis) is induced by rapidly and with positive cooperativity by activation of cyclic adenosine monophosphate-kinase I in a myeloid leukemia cell line. J. CeZZ. Ph ysiol. 146: 73-80, 1991.

194. LYONS, J., C. A. LANDIS, G. HARSH, L. VALLAR, K. GRUNEWALD, H. FEICHTINGER, Q. Y. DUH, 0. H. CLARK, E. KAWASAKI, H. R. BOURNE, AND F. MCCORMICK. Two G protein oncogenes in human endocrine tumors. Science Wash. DC 249: 655-659, 1990.

195. LYONS, R. M., AND H. L. MOSES. Transforming growth factors and the regulation of cell proliferation. Eur. J. Biochem. 187: 467-473,199O.

177. LARSEN, P. R. Thyroid-pituitary interaction. Feedback regulation of thyrotropin secretion by thyroid hormones. N. Engl. J. Med. 306: 23-32, 1982.

178. LAURENT, E., J. MOCKEL, K. TAKAZAWA, C. ERNEUX, AND J. E. DUMONT. Stimulation of generation of inositol phosphates by carbamylcholine and its inhibition by phorbol esters and iodide in dog thyroid cells. Biochem. J. 263: 795-801, 1989.

179. LAURENT, E., J. MOCKEL, J. VAN SANDE, I. GRAFF, AND J. E. DUMONT. Dual activation by thyrotropin of the phospholipase C and CAMP cascades in human thyroid. MoZ. CeZZ. Endo- crinol. 52: 273-278, 1987.

180. LAURENT, E., J. VAN SANDE, M. LUDGATE, B. CORVILAIN, P. ROCMANS, J. E. DUMONT, AND J. MOCKEL. Unlike thyrotropin, thyroid-stimulating antibodies do not activate phospholipase C in human thyroid slices. J. Clin. Invest. 87: l-9, 1991.

181. LECOCQ, R., F. LAMY, AND J. E. DUMONT. Pattern of protein phosphorylation in intact stimulated cells: thyrotropin and dog thyroid. Eur. J. Biochem. 102: 147-152, 1979.

182. LECOCQ, R., F. LAMY, AND J. E. DUMONT. Use of two-dimen- sional gel electrophoresis and autoradiography as a tool in cell biology: the example of the thyroid and the liver. Electrophoresis 11: 200-212,199l.

196. MACIEL, R. M. B., A. C. MORES, G. VILLONE, D. TRAMON- TANO, AND S. H. INGBAR. Demonstration of the production and physiological role of insulin-like growth factor II in rat thyroid follicular cells in culture. J. Clin. Invest. 82: 1546-1553, 1988.

197. MAENHAUT, C., A. LEFORT, F. LIBERT, M. PARMENTIER, E. RASP& P. ROGER, B. CORVILAIN, E. LAURENT, S. REUSE, J. MOCKEL, F. LAMY, J. VAN SANDE, AND J. E. DU- MONT. Function, proliferation and differentiation of the dog and human thyrocyte. Horm. Metab. Res. 23: 51-61,199O.

198. MAENHAUT, C., J. VAN SANDE, F. LIBERT, M. ABRAMO- WICZ, M. PARMENTIER, J. J. VANDERHAEGEN, J. E. DU- MONT, G. VASSART, AND S. SCHIFFMANN. RDC8 codes for an adenosine A, receptor with physiological constitutive activity. Biochem. Biophys. Res. Commun. 173: 1169-1178,199O.

199. MAK, W. N., M. C. EGGO, AND G. N. BURROW. Thyrotropin regulation of plasminogen activator activity in primary cultures of ovine thyroid cells. Biochem. Biophys. Res. Commun. 123: 633- 640,1984.

200. MANY, M. C., J. F. DENEF, P. GATHY, AND S. HAUMONT. Morphological and functional changes during thyroid hyperplasia and involution in C3H mice: evidence for folliculoneogenesis during involution. Endocrinology 112: 1292-1302,1987.

201. MANY, M. C., J. F. DENEF, AND S. HAUMONT. Precocity of the endothelial proliferation during a course of rapid goitrogenesis. Acta Endocrinol. 105: 487-491, 1984.

183. LEER, L. M., M. CAMMENGA, E. R. VAN DER VORM, AND 202. MATSUI, Y., S. A. HALTER, J. T. HOLT, B. L. M. HOGAN, AND

Jz& l&9;;! REGULATION OF THYROID CELL PROLIFERATION AND DIFFERENTIATION 693

R. J. COFFEY. Development of mammary hyperplasia and neoplasia in MMTV-TGF~u transgenic mice. CeZZ 61: 1147-1155,199O.

203. MATSUKURA, S., T. KAKITA, S. SUEOKA, H. YOSHIMI, Y. HIRATA, M. YOKOTA, AND T. FUJITA. Multiple hormone receptors in the adenylate cyclase of human adrenocortical tumors. Ca.ncer Rex 40: 3768-3771, 1980.

204. MATSUZAKI, S., T. KAKEGAWA, M. SUZUKI, AND K. HA- MANA. Thyroid function and polyamines. III. Changes in ornithine decarboxylase activity and polyamine contents in the rat thyroid during hyperplasia and involution. EndocrinoZ. Jpn. 25: 129-139, 1978.

205. MATSUZAKI, S., AND M. SUZUKI. Thyroid function and polyamines. II. Thyrotropin stimulation of polyamine biosynthesis in the rat thyroid. Endocrinol. Jpn. 22: 339-345,1975.

206. MAUCHAMP, J., B. CHARRIER, N. TAKASU, A. MARGOTAT, M. CHAMBARD, AND M. DUMAS. Modulation by thyrotropin, prostaglandin E, and catecholamines of sensitivity to acute stimulation in cultured thyroid cells. Horm. Cell. Regul. 3: 51-68,1979.

207. MAZZAFERRI, E. L. Thyroid cancer and Graves’ disease. J. CZin. Endocrinol. Metab. 70: 826-829, 1990.

208. MESTDAGH, C., M. C. MANY, S. HALPERN, C. BRIANCON, P. FRAGU, AND J. F. DENEF. Correlated autoradiographic and ion-microscopic study of the role of iodine in the formation of “cold” follicles in young and old mice. Cell Tissue Res. 260: 449- 457,199o.

209. MILLER, H., C. ASSELIN, D. DUFORT, J. Q. YANG, K. GUPTA, K. B. MARCU, AND A. NEPVEU. A cis-acting element in the promoter region of the murine c-myc gene is necessary for transcriptional block. Mol. CeLZ. Biol. 9: 5340-5349, 1989.

210. MILLER, J. M., R. C. HORN, AND M. A. BLOCK. The evolution of toxic nodular goiter. Arch. Int. Med. 113: 72-88, 1964.

211. MINE, M., D. TRAMONTANO, W. W. CHIN, AND S. H. INGBAR. Interleukin-1 stimulates thyroid cell growth and increases the concentration of the c-myc proto-oncogene mRNA in thyroid follicular cells in culture. Endocrinology 120: 1212-1214, 1987.

212. MIYAKAWA, M., M. SAJI, T. TSUSHIMA, K. WAKAI, AND K. SHIZUME. Thyroid volume and serum thyroglobulin levels in patients with acromegaly: correlation with plasma insulin-like growth factor I levels. J. Clin. Endocrinol. Metab. 67: 973-978, 1988.

213. MOCKEL, J., G. DECAUX, J. UNGER, AND J. E. DUMONT. In vitro regulation of ornithine decarboxylase in dog thyroid slices. Endocrinology 107: 2069-2075,198O.

214. MOCKEL, J., J. VAN SANDE, C. DECOSTER, AND J. E. DU- MONT. Tumor promoters as probes of protein kinase C in dog thyroid cell: inhibition of the primary effects of carbamylcholine and reproduction of some distal effects. Metabolism 36: 137-143, 1987.

215. MOLNE, S., E. JORTSO, S. SMEDS, AND L. E. ERICSON. Vascu- larization of normal human tissue transplanted to nude mice. Exp. Cell BioL. 55: 104-110, 1987.

216. MONCADA, S., R. M. J. PALMER, AND E. A. HIGGS. Biosynthe- sis of nitric oxide from L-arginine: a pathway for the regulation of cell function and communication. Biochem. Pharmacol. 38: 1709-1715,1989.

217. MORIS, J. C., III, G. RANGANATHAN, I. D. HAY, R. E. NEL- SON, AND N. S. JIANG. The effects of transforming growth factor-@ on growth and differentiation of the continuous rat thyroid follicular cell line, FRTL-5. Endocrinology 123: 1385-1394, 1988.

218. MOSES, A. C., D. TRAMONTANO, B. M. VENEZIANI, AND A. G. FRAUMAN. Adenosine has divergent effects on deoxyribonucleic acid synthesis in FRTL-5 cells: inhibition of thyrotropin- stimulated and potentiation of insulin-like growth factor-I stimulated thymidine incorporation. Endocrinology 125: 2758-2765, 1989.

219. MULCAHI, R. T., D. P. ROSE, J. M. MITEKEN, AND K. M. CLIF- TON. Hormonal effects on the quantitative transplantation of monodispersed rat thyroid cells. Endocrinology 106: 1769-1775, 1980.

220. MULLER, R. Cellular and viral fos genes: structure, regulation of expression and biological properties of their encoded products. Bi@im. Biojphys. Acta 823: 207-225, 1986.

221. MULLER-GARTNER, H. W., H. BAISCH, M. GARN, AND J. DE

LA ROCHE. Individually different proliferation responses of differentiated thyroid carcinomas to thyrotropin. In: Growth Reg- ulation of Thyroid Gland and Thyroid Tumors, edited by P. E. Goretzki and H. D. Roher. Basel: Karger, 1989, vol. 18, p. 137-151. (Front. Horm. Res.)

222. MULLER-GARTNER, H. W., H. BAISCH, T. WIEGEL, B. KREMER, AND C. SCHNEIDER. Increased deoxyribonucleic acid synthesis of autonomously functioning thyroid adenomas: independent of but further stimulable by thyrotropin. J. Clin. Endocrinob Metab. 68: 39-45, 1989.

223. MUNARI-SILEM, Y., M. MESNIL, S. SELMI, F. BERNIER-VA- LENTIN, R. RABILLOUD, AND B. ROUSSET. Cell-cell interactions in the process of differentiation of thyroid epithelial cells into follicles: a study by microinjection and fluorescence micros- copy on in vitro reconstituted thyroid follicles. J. CeZZ. Physiol. 145: 414-427,199O.

224. NAGAYAMA, Y., S. YAMASHITA, H. HIRAYU, M. IZUMI, T. UGA, N. ISHIKAWA, K. ITO, AND S. NAGATAKI. Regulation of thyroid peroxidase and thyroglobulin gene expression by thyrotropin in cultured human thyroid cells. J. CZin. Endocrinol. Metab. 68: 1155-1159,1989.

225. NAITO, K., S. SKOG, B. TRIBUKAIT, L. ANDERSSON, AND H. HISAZUMI. Cell cycle related [3H]thymidine uptake and its significance for the incorporation into DNA. CeZL Tissue Kinet. 20: 447-457,1987.

226. NAMBA, H., K. MATSUO, AND J. A. FAGIN. Clonal composition of benign and malignant human thyroid tumors. J. Clin. Invest. 86: 120-125,199O.

227. NAMBA, H., J. L. ROSS, D. GOODMAN, AND J. A. FAGIN. Soli- tary polyclonal autonomous thyroid nodule: a rare cause of child- hood hyperthyroidism. J. Clin. Endocrinol. Metab. 72: 1108-1112, 1991.

228. NAMBA, H., S. A. RUBIN, AND J. A. FAGIN. Point mutations of ras oncogenes are an early event in thyroid tumorigenesis. Mol. Endocrinol. 4: 147’4-1479,199O.

229. NATHAN, C., AND M. SPORN. Cytokines in context. J. CeLZ Biol. 113: 981-986, 1991.

230. NICOLSON, G. L. Tumor cell instability, diversification, and progression to the metastatic phenotype: from oncogene to oncofetal expression. Cancer Res. 47: 1473-1487,1987.

231. NIELSEN, T. B., M. S. FERDOWS, B. R. BRINKLEY, AND J. B. FIELD. Morphological and biochemical responses of cultured thyroid cells to thyrotropin. Endocrinology 116: 788-797, 1985.

232. NITSCH, L., AND S. H. WOLLMAN. Thyrotropin preparations are mitogenic for thyroid epithelial cells in follicles in suspension culture. Proc. Natl. Acad. Sci. USA 77: 2743-2748,198O.

233. NURSE, P. Universal control mechanism regulating onset of M- phase. Nature Lond. 344: 503-508, 1990.

234. OHTA, K., T. ENDO, AND T. ONAYA. The mRNA levels of thyrotropin receptor, thyroglobulin and thyroid peroxidase in neoplas- tic human thyroid tissues. Biochem. Biophys. Res. Commun. 174: 1148-1153, 1991.

235. OLLIS, C. A., R. DAVIES, D. S. MUNRO, AND S. TOMLINSON. Relationship between growth and function of human thyroid cells in culture. J. Endocrinol 108: 393-398, 1986.

236. OLLIS, C. A., D. J. HILL, AND D. S. MUNRO. A role for insulin- like growth factor-I in the regulation of human thyroid cell growth by thyrotropin. J. Clin. Endocrinol. 123: 495-500,1989.

237. OZAWA, S., L. G. SHEFLIN, AND S. W. SPAULDING. Thyroxine increases epidermal growth factor levels in the mouse thyroid in vivo. Endocrinology 128: 1396-1403, 1991.

238. OZAWA, S., AND S. W. SPAULDING. Epidermal growth factor inhibits radioiodine uptake but stimulates deoxyribonucleic acid synthesis in newborn rat thyroids grown in nude mice. Endocri- nology 127: 604-612, 1990.

239. PARDEE, A. B. Gl events and regulation of cell proliferation. Science Wash. DC 246: 603-608,1989.

240. PAYET, N., J. G. LEHOUX, AND H. ISLER. Effect of ACTH on the proliferative and secretory activities of the adrenal glomeru- losa. Acta Endocrinol. 93: 365-374, 1980.

241. PEKONEN, F., H. ALFTHAN, U. H. STENMAN, AND 0. YLI- KORKALA. Human chorionic gonadotropin (hCG) and thyroid function in early human pregnancy: circadian variation and evi-

694 DUMONT, LAMY, ROGER, AND MAENHAUT Volume Z?

242. 260.

243. 261.

dog primary thyrocytes: a positive and negative control by cyclic AMP on c-myc expression. Exp. Cell. Res. 189: 33-40, 1990. ROESLER, W. J., G. R. VANDENBARK, AND R. W. HANSON. Cyclic AMP and the induction of eukaryotic gene transcription. J. Biol. Chem. 263: 9063-9066,1988. ROGER, P. P., AND J. E. DUMONT. Epidermal growth factor controls the proliferation and the expression of differentiation in canine thyroid cells in primary culture. FEBS Lett. 144: 209-212, 1982.

244. 262.

245. 263.

246. 264.

247.

ROGER, P. P., AND J. E. DUMONT. Thyrotropin and the differential expression of proliferation and differentiation in dog thyroid cells in primary culture. J. Endocrinol. 96: 241-249, 1983. ROGER, P. P., AND J. E. DUMONT. Factors controlling proliferation and differentiation of canine thyroid cells cultured in reduced serum conditions: effects of thyrotropin, cyclic AMP and growth factors. Mol. Cell. Endocrinol. 36: 79-93, 1984. ROGER, P. P., J. E. DUMONT, AND J. M. BOEYNAEMS. Lack of prostaglandin involvement in the mitogenic effect of TSH on canine thyroid cells in primary culture. FEBS Lett. 166: 136-140, 1984.

265.

248.

249.

266.

267.

250.

251.

dence for intrinsic thyrotropic activity of hCG. J. Clin. Endo- crinol. Metab. 66: 853-856, 1988. PENN, L. J. Z., M. W. BROOKS, E. M. LAUFER, AND H. LAND. Negative autoregulation of c-myc transcription. EMBO J. 9: 1113-1121,199O. PEREIRA, A., J. C. BRAEKMAN, J. E. DUMONT, AND J. M. BOEYNAEMS. Identification of a major iodolipid from the horse thyroid gland as 2-iodohexadecanal. J. Biol. Chem. 265: 17018- 17025,199O. PETER, M. J., M. GERBER, H. STUDER, AND S. SMEDS. Patho- genesis of heterogeneity in human multinodular goiter. J. Clin. Invest. 76: 1992-2002,1985. PETER, M. J., M. STUDER, AND P. GROSCURTH. Autonomous growth, but not autonomous function, in embryonic human thyroids: a clue to understanding autonomous goiter growth. J. Clin. Endocrinol. Metab. 66: 968-973, 1988. PHILP, J. R., J. CROOKS, A. G. MACGREGOR, AND J. A. R. MCINTOSH. The growth curve of the rat thyroid under a goitro- genie stimulus. Br. J. Cancer 23: 515-523,1969. PIC, P., M. MICHEL-BECHET, F. EL ATIQ, AND A. M. ATHOUEL-HAON. Forskolin stimulates CAMP production and the onset of the functional differentiation in the fetal rat thyroid in vitro. Biol. Cell 57: 231-238, 1986. PISAREV, M., L. J. DEGROOT, AND J. F. WILBER. Cyclic-AMP production of goiter. Endocrinology 87: 339-342, 1970. PISAREV, M. A., G. D. CHAZENBALK, R. M. VALSECCHI, G. BURTON, L. KRAWIEC, E. MONTEAGUDO, G. J. JUVENAL, R. J. BOADO, AND H. A. CHESTER. Thyroid autoregulation. In- hibition of goiter growth and of cyclic AMP formation in rat thyroid by iodinated derivatives of arachidonic acid. J. Endo- crinol. Invest. 11: 669-687, 1988. POHL, V., P. P. ROGER, D. CHRISTOPHE, G. PATTYN, G. VASSART, AND J. E. DUMONT. Differentiation expression during proliferative activity induced through different pathways: in situ hybridization study of thyroglobulin gene expression in thyroid epithelial cells. J. Cell Biol. 111: 663-672, 1990. POLYCHRONAKOS, C., H. J. GUYDA, B. PATEL, AND B. L. POSNER. Increase in the number of type II insulin-like growth factor receptors during propylthiouracil-induced hyperplasia in the rat thyroid. Endocrinology 119: 1204-1209,1986. PRATT, M. A. C., M. C. EGGO, L. K. BACHRACH, P. CARAYON, AND G. N. BURROW. Regulation of thyroperoxidase, thyroglobulin and iodide levels in sheep thyroid cells by TSH, tumor promoters and epidermal growth factor. Biochimie 71: 227-235, 1989. PRESTON-MARTIN, S., M. C. PIKE, R. K. ROSS, P. A. JONES, AND B. E. HENDERSON. Increased cell division as a cause of human cancer. Cczncer Res. 50: 7415-7421, 1990. PROCHOWNIK, E. V., J. KUKOWSKA, AND C. RODGERS. c- myc antisense transcripts accelerate differentiation and inhibit Gl progression in murine erythroleukemia cells. Mol. Cell. Biol. 8: 3683-3695,1988. RAPOPORT, B. Dog thyroid cells in monolayer tissue culture: adenosine 3’-5’-cyclic monophosphate response to thyrotropic hormone. Endocrinology 98: 1189-1197, 1976. RASMUSSEN, A. K., L. KAYSER, K. BECH, U. FELDT-RAS- MUSSEN, H. PERRILD, AND K. BENDTZEN. Differential effects of interleukin lcu and lp on cultured human and rat thyroid epithelial cells. Acta Endocrinol. 122: 520-526, 1990. RASPE, E., S. REUSE, C. MAENHAUT, P. P. ROGER, B. COR- VILAIN, E. LAURENT, J. MOCKEL, F. LAMY, J. VAN SANDE, AND J. E. DUMONT. Importance and variability of transducing systems in the control of thyroid cell function, proliferation and differentiation. In: Growth Regulation of Thyroid Gland and Thy- roid Tumors, edited by P. E. Goretzki and H. D. Riiher. Basel: Karger, 1989, vol. 18, p. l-13. (Front. Horm. Res. Ser.) REDMOND, O., AND A. R. TUFFERY. Mitotic rate of the rat’s thyroid gland during hypertrophy induced by an antithyroid agent (carbimazole). J. Anat. 127: 353-362, 1978. REUSE, S., C. MAENHAUT, AND J. E. DUMONT. Regulation of protooncogenes c-fos and c-myc expressions by protein tyrosine kinase, protein kinase C, and cyclic AMP mitogenic pathwavs in

268.

269.

252.

270.

253. 271.

254. 272.

255. 273

256.

274.

257. 275.

276.

258. 277.

259.

ROGER, P. P., A. HOTIMSKY, C. MOREAU, AND J. E. DU- MONT. Stimulation by thyrotropin, cholera toxin and dibutyryl cyclic AMP of the multiplication of differentiated thyroid cells in vitro. Mol. Cell. Endocrinol. 26: 165-176, 1982. ROGER, P. P., S. REUSE, P. SERVAIS, B. VAN HEUVERS- WYN, AND J. E. DUMONT. Stimulation of cell proliferation and inhibition of differentiation expression by tumor-promoting phorbol esters in dog thyroid cells in primary culture. Cancer Res. 46: 898-906, 1986. ROGER, P. P., F. RICKAERT, G. HUEZ, M. AUTHELET, F. HOFMANN, AND J. E. DUMONT. Microinjection of catalytic subunit of cyclic AMP-dependent protein kinases triggers acute morphological changes in thyroid epithelial cells. FEBS Lett. 232: 409-413,1988. ROGER, P. P., F. RICKAERT, F. LAMY, M. AUTHELET, AND J. E. DUMONT. Actin stress filter disruption and tropomyosin isoform switching in normal thyroid epithelial cells stimulated by thyrotropin and phorbol esters. Exp. Cell. Res. 182: l-13,1989. ROGER, P. P., P. SERVAIS, AND J. E. DUMONT. Stimulation by thyrotropin and cyclic AMP of the proliferation of quiescent canine thyroid cells cultured in a defined medium containing insulin. FEBS Lett. 157: 323-329, 1983. ROGER, P. P., P. SERVAIS, AND J. E. DUMONT. Induction of DNA synthesis in dog thyrocytes in primary culture: synergistic effects of thyrotropin and cyclic AMP with epidermal growth factor and insulin. J. Cell. Physiol. 130: 58-67, 1987. ROGER, P. P., P. SERVAIS, AND J. E. DUMONT. Regulation of dog thyroid epithelial cell cycle by forskolin, an adenylate cyclase activator. Exp. Cell. Res. 172: 282-292, 1987. ROGER, P. P., M. TATON, J. VAN SANDE, AND J. E. DUMONT. Mitogenic effects of thyrotropin and cyclic AMP in differentiated human thyroid cells in vitro. J. Clin. Endocrinol. Metab. 66: 1158- 1165, 1988. ROGER, P. P., B. VAN HEUVERSWYN, C. LAMBERT, S. REUSE, G. VASSART, AND J. E. DUMONT. Antagonistic effects of thyrotropin and epidermal growth factor on thyroglobulin mRNA level in cultured thyroid cells. Eur. J Biochem. 152: 239- 245,1985. ROGNOGNI, J. B., C. PENEL, AND F. DUCRET. Vascularization and iodide transport down regulation in rat goitre. Acta Endo- crinol. 105: 40-48, 1984. ROGNONI, J. B., C. PENEL, J. GOSTEIN, P. GALAND, AND J. E. DUMONT. Cell kinetics of thyroid epithelial cells during hyper- plastic goitre involution. J. Endocrinol. 114: 483-490, 1987. ROSSOMONDO, A. J., M. J. WEBER, AND T. W. STURGILL. Evidence that pp42, a major tyrosine kinase target protein, is MAP kinase, a mitogen-activated serine/threonine protein kinase. Proc. N&l. Acad. Sci. USA 86: 6940-6943, 1989. ROTELLA, C. M., C. MAVILIA, U. FREDIANI, AND R. TOCCA- FONDI. Calf serum modifies the mitogenic activity of epidermal growth factor in WRT thyroid cells. Mol. Cell. Endocrinol. 65: 63-74,1989. SACREZ. R.. J. G. JUIF. J. MANDRY. C. KELLERSHORN. R. 278.

July 199.z REGULATION OF THYROID CELL PROLIFERATION AND DIFFERENTIATION 695

279.

280.

281.

282.

283.

284.

285.

286.

287.

288.

289.

290.

291. 292.

293.

294

295

296.

297.

298.

299.

RIVIERE, AND D. COMAR. Le syndrome de Pendred. I. Etude critique, clinique et genetique. Ann. Pediatr. Paris 1: 5-15, 1967. SAJI, M., 0. ISOZAKI, T. TSUSHIMA, M. ARAI, M. MIYA- KAWA, Y. OHBA, Y. TSUCHIYA, T. SANO, AND K. SHIZUME. The inhibitory effect of iodide on growth of rat thyroid (FRTL-5) cells. Acta EndocrinoL 119: 145-151, 1988. SAJI, M., AND L. D. KOHN. Effect of hydrocortisone on the ability of thyrotropin to increase deoxyribonucleic acid synthesis and iodide uptake in FRTL-5 rat thyroid cells: opposite regulation of adenosine 3’,5’-monophosphate signal action. EndocrinoL- ogy 127: 1867-1876,199O. SAJI, M., T. TSUSHIMA, 0. ISOZAKI, H. MURAKAMI, Y. OHBA, K. SATO, M. ARAI, A. MARIKO, AND K. SHIZUME. In- teraction of insulin-like growth factor I with porcine thyroid cells cultured in monolayer. Endocrinology 121: 749-756, 1987. SANDGREN, E. P., N. C. LUETTEKE, R. D. PALMITER, R. L. BRINSTER, AND D. C. LEE. Overexpression of TGFcw in trans- genie mice: induction of epithelial hyperplasia, pancreatic meta- plasia, and carcinoma of the breast. CeZl61: 1121-1135,199O. SANTISTEBAN, P., L. D. KOHN, AND R. DI LAURO. Thyroglob- ulin gene expression is regulated by insulin and insulin-like growth factor I, as well as thyrotropin, in FRTL-5 thyroid cells. J. Biol. Chem. 262: 4048-4052,1987. SAUNIER, B., K. DIB, B. DELEMER, C. JACQUEMIN, AND C. CORREZE. Cyclic AMP regulation of G, protein. J. BioZ. Chem. 265: 19942-19946,199O. SAVOIE, J. C. Etude clinique et biologique de quarante-trois cas d’adenome toxique thyroidien. Rev. Fr. Etud. Clin. Biol. 6: 263- 275,196l. SCHEINMAN, S. J., AND G. N. BURROW. In vitro stimulation of thyroid ornithine decarboxylase activity and polyamines by thyrotropin. Endocrinology 101: 1088-1094,1977. SCHERBERG, N. H. Isolation of thyroglobulin messenger RNA from rats: increased yield in propylthiouracil-induced hyperplasia. Biochem. Biophys. Res. Commun. 85: 1415-1423, 1978. SCHERBERG, N. H., G. VASSART, R. LECOCQ, J. E. DUMONT, AND S. REFETOFF. Modulation of thyroglobulin messenger RNA accumulation in the rat thyroid. EndocrinoZogy 109: 1650- 1656,198l. SCHRADER, J., A. DEUSSEN, AND R. T. SMOLENSKI. Adeno- sine is a sensitive oxygen sensor in the heart. Experientia BaseZ 46: 1172-1174,199O. SCHURCH, M., H. J. PETER, H. GERBER, AND H. STUDER. Cold follicles in a multinodular human goiter arise partly from a failing iodide pump and partly from deficient iodine organification. J. CZin. Endocrinol. Metab. 71: 1224-1229, 1990. SELL, S. Is there a liver stem cell? Cancer Res. 1: 3811-3815,199O. SELLITTI, D. F., AND C. HUGHES. Immunoreactive atria1 natriuretic peptide in the thyroid gland. Regul. Pept. 27: 285-298,199O. SEUWEN, K., AND J. POUYSSEGUR. Serotonin as a growth factor. Biochem. PharmacoZ. 39: 985-990,199O. SHIROOZU, A., K. INOUE, T. NAKASHIMA, K. OKAMURA, M. YOSHINARI, H. NISHITANI, AND T. OMAE. Defective iodide transport and normal organification of iodide in cold nodules of the thyroid. CZin. Endocrinol. 15: 411-416, 1981. SILVERSTEIN, G. E., G. BURKE, AND R. COGAN. The natural history of the autonomous hyperfunctioning thyroid nodule. Ann. Intern. Med. 67: 539-548,1967. SINCLAIR, A. J., R. LONIGRO, D. CIVITAREALE, L. GHI- BELLI, AND R. DI LAURO. The tissue-specific expression of the thyroglobulin gene requires interaction between thyroid-specific and ubiquitous factors. Eur. J. Biochem. 193: 311-318, 1990. SISSON, J. C., W. H. BEIERWALTES, G. PATTON, AND S. SER- FONTEIN. Effect of thyroidectomy with and without thyroxine replacement on transplantable thyroid tumors in rats. Endocri- nology 74: 925-929,1964. SMEDS, S., H. J. PETER, E. JijRTSij, H. GERBER, AND H. STUDER. Naturally occurring clones of cells with high intrinsic proliferation potential within the follicular epithelium of mouse thyroids. Cancer Res. 47: 1646-1651,1987. SMEDS, S., AND S. H. WOLLMAN. 3H-thymidine labeling of endothelial cells in thyroid arteries, veins, and lymphatics during thvroid stimulation. Lab. Invest. 48: 285-291. 1983.

300.

301.

302.

303.

304.

305.

306.

307.

308.

309.

310.

311.

312.

313.

314.

315.

316.

317.

318.

319.

SMETS, L. A. Discrepancies between precursor uptake and DNA synthesis in mammalian cells. J. CeZZ. Physiol. 74: 63-68, 1969. SMITH, J. F. The pathology of the thyroid in the syndrome of sporadic goitre and congenital deafness. Q. J. Med. 114: 297-303, 1960. SMITH, P., E. D. WILLIAMS, AND D. WYNFORD-THOMAS. In vitro demonstration of a TSH-specific growth desensitising mechanism in rat thyroid epithelium. MoZ. CeZZ. Endocrinol. 51: 51-58,1987. SMITH, P., D. WYNFORD-THOMAS, B. M. J. STRINGER, AND E. D. WILLIAMS. Growth factor control of rat thyroid follicular cell proliferation. Endocrinology 119: 1439-1445, 1986. SPAULDING, S. W., N. W. FUCILE, D. P. BOFINGER, AND L. G. SHEFLIN. Cyclic adenosine 3’,5’-monophosphate-dependent phosphorylation of HMG 14 inhibits its interactions with nucleosomes. Mob EndocrinoZ. 5: 42-50, 1991. SPEIGHT, J. W., W. I. BABA, AND G. M. WILSON. The effect of propylthiouracil and thyroid-stimulating hormone on the sur- vival of rat thyroid cells in vivo and in vitro. J. Endocr. 41: 577- 591,1968. STRANICKY, K., AND B. MESS. Effect of TSH on the rate of DNA and protein synthesis in embryonic chicken thyroid cells, investigated with 3H-thymidine and 3H-methionine autoradiography. Acta Biol. Hung. 18: 221-229, 1967. STRINGER, B. M. J., D. WYNFORD-THOMAS, H. R. HARACH, AND E. D. WILLIAMS. Reparative growth in the rat thyroid. CeZZ Tissue Kinet. 16: 571-576, 1983. STRINGER, B. M. J., D. WYNFORD-THOMAS, AND E. D. WIL- LIAMS. In vitro evidence for an intracellular mechanism limit- ing the thyroid follicular cell growth response to thyrotropin. Endocrinology 116: 611-615,1985. STUBNER, D., R. GARTNER, W. GREIL, K. GROPPER, G. BRABANT, W. PERMANETTER, K. HORN, AND C. R. PICK- ARDT. Hypertrophy and hyperplasia during goitre growth and involution in rats-separate bioeffects of TSH and iodine. Acta EndocrinoZ. 116: 537-548,1987. SUAREZ, H. G., J. A. DU VILLARD, B. CAILLOU, M. SCHLUM- BERGER, C. PARMENTIER, AND R. MONIER. gsp mutations in human thyroid tumours. Oncogene 6: 677-679,199l. SUAREZ NUNEZ, J. M. Effect of removal of submaxillary glands on the thyroid gland. J. Dent. Res. 49: 454, 1970. TAHARA, K., E. F. GROLLMAN, M. SAJI, AND L. D. KOHN. Regulation of prostaglandin synthesis by thyrotropin, insulin or insulin-like growth factor-I, and serum in FRTL-5 rat thyroid cells. J. Biol. Chem. 266: 440-448, 1991. TAKADA, K., N. AMINO, H. TADA, AND K. MIYAI. Relation- ship between proliferation and cell cycle-dependent Ca2’ influx induced by a combination of thyrotropin and insulin-like growth factor-I in rat thyroid cells. J. C&n. Invest. 86: 1548-1555, 1990. TAKAHASHI, S. I., M. CONTI, AND J. J. VAN WIJK. Thyrotro- pin potentiation of insulin-like growth factor-I dependent deoxyribonucleic acid synthesis in FRTL-5 cells: mediation by an autocrine amplification factor(s). Endocrinology 126: 736-745, 1990. TAKASU, N., B. CHARRIER, J. MAUCHAMP, AND S. LIS- SITZKY. Positive and negative regulation by thyrotropin of thyroid cyclic AMP response to thyrotropin in porcine thyroid cells. FEBS Lett. 84: 191-194,1977. TAKASU, N., I. KOMIYA, Y. NAGASAWA, T. ASAWA, Y. SHI- MIZU, AND T. YAMADA. Cytoplasmic pH in the action of insulin-like growth factor-I in cultured porcine thyroid cells. J. Endo- crinol. 127: 305-309, 1990. TAKASU, N., I. KOMIYA, Y. NAGASAWA, T. ASAWA, T. SHINODA, T. YAMADA, AND Y. SHIMIZU. Stimulation of porcine thyroid cell alkalinization and growth by EGF, phorbol ester, and diacylglycerol. Am. J. Physiol. 258 (Endocrinol. Metab. 21): E445-E450,1990. TAKASU, N., Y. NAGASAWA, I. KOMIYA, T. YAMADA, AND Y. SHIMIZU. Cytoplasmic pH in the action of epidermal growth factor (EGF) in cultured porcine thyroid cells. Biochem. Biophys. Res. Commun. 157: 346-349,1988. TAKASU, N., Y. SHIMIZU, AND T. YAMADA. Tumour promoter 12-0-tetradecanoylphorbol13-acetate and epidermal growth fac-


320.

321.

322.

323.

324.

325.

326.

327.

328.

329.

330.

331.

332.

333.

335.

336.

tor stimulate proliferation and inhibit differentiation of porcine thyroid cells in primary culture. J. EndocrinoZ. 113: 485-487,1987. TAKASU, N., M. TAKASU, I. KOMIYA, Y. NAGASAWA, T. ASAWA, Y. SHIMIZU, AND T. YAMADA. Insulin-like growth factor I stimulates inositol phosphate accumulation, a rise in cytoplasmic free calcium, and proliferation in cultured porcine thyroid cells. J. BioC Chem. 264: 18485-18488, 1989. TAKASU, N., M. TAKASU, T. YAMADA, ANDY. SHIMIZU. Epi- dermal growth factor (EGF) produces inositol phosphates and increases cytoplasmic free calcium in cultured porcine thyroid cells. Biochem. Biophys. Res. Cowzmun. 151: 530-534, 1988. TAKIMOTO, M., J. P. QUINN, A. R. FARINA, L. M. STAUDT, AND D. LEVENS. Fos/jun and octamer-binding protein interact with a common site in a negative element of the human c-myc gene. J. Biol. Chem. 264: 8992-8999,1989. THOMAS, G. A., D. WILLIAMS, AND E. D. WILLIAMS. The clonal origin of thyroid nodules and adenomas. Am. J. Pathol. 134: 141-151,1989. THORBURN, G. D., M. J. WATERS, I. R. YOUNG, D. BUNTINE, AND P. S. HOPKINS. Epidermal growth factor: a critical factor in fetal maturation. In: The Fetus and Independent Life. London: Pitman, 1981, p. 172-198. (CIBA Found. Symp. 86) TRAMONTANO, D., W. W. CHIN, A. C. MOSES, AND S. H. ING- BAR. Thyrotropin and dibutyryl cyclic AMP increase levels of c-myc and c-fos mRNAs in cultured rat thyroid cells. J. Biol. Chem. 261: 3919-3922,1986. TRAMONTANO, D., G. CUSHING, A. C. MOSES, AND S. H. ING- BAR. Insulin-like growth factor-I stimulates the growth of rat thyroid cells in culture and synergizes the stimulation of DNA synthesis induced by TSH and Graves’ IgG. EndocrinoZogy 119: 940-942,1986. TRAMONTANO, D., A. C. MOSES, AND S. H. INGBAR. The role of adenosine 3’,5’-monophosphate in the regulation of receptors for thyrotropin and insulin-like growth factor I in the FRTL-5 rat thyroid follicular cell. Endocrinology 122: 133-136, 1988. TRAMONTANO, D., A. C. MOSES, B. M. VENEZIANI, AND S. H. INGBAR. Adenosine 3’,5’-monophosphate mediates both the mitogenic effect of thyrotropin and its ability to amplify the response to insulin-like growth factor I in FRTL-5 cells. EndocrinoZ- ogy122:127-132,1988. TRAMONTANO, D., C. M. ROTELLA, R. TOCCAFONDI, AND S. F. AMBESI-IMPIOMBATO. Thyrotropin-independent mutant clones from FRTL-5 rat thyroid cells: hormonal control mechanisms in differentiated cells. Endocrinology 118: 862-868, 1986. TSUSHIMA, T., M. ARAI, M. SAJI, Y. OHBA, H. MURAKAMI, E. OHMURA, K. SATO, AND K. SHIZUME. Effects of transforming growth factor-p on deoxyribonucleic acid synthesis and iodine metabolism in porcine thyroid cells in culture. Endocrinol- ogy 123: 1187-1194,1988. VALENTE, W. A., P. VITTI, L. D. KOHN, M. L. BRANDI, C. M. ROTELLA, R. TOCCAFONDI, D. TRAMONTANO, S. M. ALOJ, AND F. S. AMBESI-IMPIOMBATO. The relationship of growth and adenylate cyclase activity in cultured thyroid cells. Separate bioeffects of thyrotropin. Endocrinology 112: 71-79,1983. VALENTE, W. A., P. VITTI, C. M. ROTELLA, M. M. VAUGHAN, S. M. ALOJ, E. F. GROLLMAN, F. S. AMBESI-IMPIOMBATO, AND L. D. KOHN. Antibodies that promote thyroid growth. A distinct population of thyroid-stimulating autoantibodies. N. Engl. J. Med. 309: 1028-1034,1983. VAN HEUVERSWYN, B., A. LERICHE, J. VAN SANDE, J. E. DUMONT, AND G. VASSART. Transcriptional control of thyroglobulin gene expression by cyclic AMP. FEBS Lett. 188: 192-196, 1985. VAN HEUVERSWYN, B., C. STREYDIO, H. BROCAS, S. REFE- TOFF, J. E. DUMONT, AND G. VASSART. Thyrotropin controls transcription of the thyroglobulin gene. Proc. Nut,!. Acad. Sci. USA 81: 5941-5945,1984. VAN SANDE, J., G. GRENIER, C. WILLEMS, AND J. E. DU- MONT. Inhibition by iodide of the activation of the thyroid cyclic 3’,5’-AMP system. Endocrinology 96: 781-786, 1975.

337.

338.

339.

340.

341.

342.

343.

344.

345.

346.

347.

348.

349.

350.

351.

352.

353.

354.

355.

356.

VAN SANDE, J., F. LAMY, R. LECOCQ, N. MIRKINE, P. ROC- MANS, P. COCHAUX, J. MOCKEL, AND J. E. DUMONT. Patho- genesis of autonomous thyroid nodules: in vitro study of iodine and adenosine 3’,5’-monophosphate metabolism. J. Gin. Endo- crinol. Metab. 66: 570-579, 1988. VAN SANDE, J., A. LEFORT, S. BEEBE, P. ROGER, J. PERRET, J. CORBIN, AND J. E. DUMONT. Pairs of cyclic AMP analogs, that are specifically synergistic for type I and type II CAMP-dependent protein kinases, mimic thyrotropin effects on the function, differentiation expression and mitogenesis of dog thyroid cells. Eur. J. Biochem. 183: 699-708, 1989. VELU, T. J. Structure, function and transforming potential of the epidermal growth factor receptor. Mol. Cell EndocrinoZ. 70: 205-216,199O. WAINSCOAT, J. S., AND M. F. FEY. Assessment of clonality in human tumors: a review. Cancer Res. 50: 1355-1360,199O. WATANABE, H., M. N. GOULD, P. A. MAHLER, R. T. MUL- CAHY, AND K. H. CLIFTON. The influence of donor and recipient age and sex on the quantitative transplantation of monodispersed rat thyroid cells. Endocrinology 112: 172-177, 1983. WATANABE, Y., N. AMINO, H. TAMAKI, Y. IWATANI, AND K. MIYAI. Bovine thyrotropin inhibits DNA synthesis inversely with stimulation of cyclic AMP production in cultured porcine thyroid follicles. EndocrinoZ. Jpn. 32: 81-88, 1985. WATERS, M. J., R. C. TWEEDALE, T. A. WHIP, G. SHAW, S. W. MANLEY, AND J. R. BOURKE. Dedifferentiation of cultured thyroid cells by epidermal growth factor: some insights into the mechanism. Mol. CeU. Endocrinol. 49: 109-117,1987. WATT, F. M. Cell culture models of differentiation. FASEB J 5: 287-293,1991. WEETMAN, A. P., R. BRIGHT-THOMAS, AND M. FREEMAN. Regulation of interleukin-6 release by human thyrocytes. J. En- docrinol. 127:357-361,199O. WESTERMARK, B., F. A. KARLSSON, AND 0. WALINDER. Thyrotropin is not a growth factor for human thyroid cells in culture. Proc. N&Z. Acad. Sci. USA 76: 2022-2026,1979. WESTERMARK, K., F. A. KARLSSON, AND B. WESTERMARK. Epidermal growth factor modulates thyroid growth and function in culture. Endocrinology 112: 1680-1686,1983. WESTERMARK, K., F. A. KARLSSON, AND B. WESTERMARK. Thyrotropin modulates EGF receptor function in porcine thyroid follicle cells. MoZ. CeZZ. EndocrinoZ. 40: 17-23, 1985. WESTERMARK, K., AND B. WESTERMARK. Mitogenic effect of epidermal growth factor on sheep thyroid cells in culture. Exp. Cell. Res. 138: 47-55, 1982. WESTERMARK, K., B. WESTERMARK, F. A. KARLSSON, AND L. E. ERICSON. Location of epidermal growth factor receptors on porcine thyroid follicle cells and receptor regulation by thyrotropin. Endocrinology 118: 1040-1046,1986. WILDERS-TRUSCHNIG, M. M., H. A. DREXHAGE, G. LEB, 0. EBER, H. P. BREZINSCHEK, G. DOHR, G. LANZER, AND G. J. KREJS. Chromatographically purified immunoglobulin G of endemic and sporadic goiter patients stimulates FRTL-5 cell growth in a mitotic arrest assay. J. Clin. EndocrinoZ. Metab. 70: 444-452,199O. WILLIAMS, D. W., D. WYNFORD-THOMAS, AND E. D. WIL- LIAMS. Control of human thyroid follicular cell proliferation in suspension and monolayer culture. MoZ. Cell. Endocrinol. 51: 33- 40, 1987. WILLIAMS, D. W., E. D. WILLIAMS, AND D. WYNFORD- THOMAS. Loss of dependence on IGF-1 for proliferation of human thyroid adenoma cells. Br. J. Cancer 57: 535-539,1988. WILLIAMS, D. W., E. D. WILLIAMS, AND D. WYNFORD- THOMAS. Evidence for autocrine production of IGF-1 in human thyroid adenomas. Mol. CeZZ. Endocrinol. 61: 139-147, 1989. WOLFF, J., AND S. VARRONE. The methyl xanthines-a new class of goitrogens. Endocrinology 85: 410-414,1969. WOLLMAN, S. H., AND J. P. HERVEG. Thyroid capsule changes during the development of thyroid hyperplasia in the rat. Am. J. Pathol. 93: 639-654,1978.

357. WOLLMAN, S. H., J. P. HERVEG, J. D. ZELIGS, AND L. E. ER-

Jz4ly 1992 REGULATION OF THYROID CELL PROLIFERATION AND DIFFERENTIATION 697

358.

359.

360.

361.

36.2.

363.

364.

365.

366.

367.

368.

ICSON. Blood capillary enlargement during the development of thyroid hyperplasia in the rat. Endocrinology 103: 2306-2314, 1978. WRIGHT, P. A., E. D. WILLIAMS, N. R. LEMOINE, AND D. WYNFORD-THOMAS. Radiation-associated and “spontaneous” human thyroid carcinomas show a different pattern of ras oncogene mutation. Oncogene 6: 471-473, 1991. WUSTER, C., G. STEGER, A. SCHMELZLE, J. GOTTS- WINTER, H. W. MINNE, AND R. ZIEGLER. Increased incidence of euthyroid and hyperthyroid goiters independently of thyrotropin in patients with acromegaly. Harm. Metab. Res. 23: 131-134, 1991. WYLLIE, F. S., T. DAWSON, J. A. BOND, P. GORETZKI, S. GAME, S. PRIME, AND D. WYNFORD-THOMAS. Correlated abnormalities of transforming growth factor-@1 response and p53 expression in thyroid epithelial cell tansformation. 2MoL Cell. En- docrinol. 76: 13-21, 1991. WYNFORD-THOMAS, D., P. SMITH, AND E. D. WILLIAMS. Proliferative response to cyclic AMP elevation of thyroid epithelium in suspension culture. Mol. Cell Endocrinol. 51: 163-166, 1987. WYNFORD-THOMAS, D., B. M. J. STRINGER, H. R. HARACH, AND E. D. WILLIAMS. Mitotic response to wounding in goitrous and normal rat thyroid: implications for thyroid growth control. Cell Tissue Kinet. 18: 467-473, 1985. WYNFORD-THOMAS, D., B. M. J. STRINGER, AND E. D. WIL- LIAMS. Circadian rhythm of mitotic activity in normal and hy- perplastic rat thyroid. Acta Endocrinol. 100: 46-50, 1982. WYNFORD-THOMAS, D., B. M. J. STRINGER, AND E. D. WIL- LIAMS. Desensitization of rat thyroid to the growth-stimulating action of TSH during prolonged goitrogen administration. Per- sistence of refractoriness following withdrawal of stimulation. Acta EndocrinoZ. 101: 562-569, 1982. WYNFORD-THOMAS, D., B. M. J. STRINGER, AND E. D. WIL- LIAMS. Dissociation of growth and function in the rat thyroid during prolonged goitrogen administration. Acta Endocrinol. 101: 210-216,1982. WYNFORD-THOMAS, D., B. M. J. STRINGER, AND E. D. WIL- LIAMS. Goitrogen-induced thyroid growth in the rat: a quantitative morphometric study. J. Endocr. 94: 131-140,1982. WYNFORD-THOMAS, D., B. M. J. STRINGER, AND E. D. WIL- LIAMS. Control of growth in the rat thyroid-an example of specific desensitization to trophic hormone stimulation. Exgoer- ientia BaseZ 39: 421-423, 1983. YAMAMOTO, M., T. TAKIZAWA, K. ARISHIMA, AND Y. EGU- CHI. Differentiation and thyroid-stimulating hormone (TSH) sensitivity of the fetal rat thyroid in organ culture. Anat. Rec. 215: 361-364.1986.

369.

371.

372.

373.

374

375.

376.

377.

378.

379.

380.

381.

YAMASHITA, S., H. KIMURA, K. ASHIZAWA, Y. NAGA- YAMA, H. HIRAYU, M. IZUMI, AND S. NAGATAKI. Interleu- kin-l inhibits thyrotrophin-induced human thyroglobulin gene expression. J. EndocrinoZ. 122: 177-183,1989. YAP, A. S., J. R. BOURKE, AND S. W. MANLEY. Role of cell-cell contact in the preservation of differentiation and response to thyrotropin in cultured porcine thyroid cells. J. EndocrinoZ. 113: 223-229,1987. YOSHIKAWA, N., M. NISHIKAWA, M. HORIMOTO, M. YO- SHIMURA, S. SAWARAGI, Y. HORIKOSHI, I. SAWARAGI, AND M. INADA. Thyroid-stimulating activity in sera of normal pregnant women. J. CZin. EndocrinoZ. Metab. 69: 891-895, 1989. YOSHIKAWA, N., M. NISHIKAWA, M. HORIMOTO, M. YO- SHIMURA, N. TOYODA, AND M. INADA. Human chorionic gonadotropin promotes thyroid growth via thyrotropin receptors in FRTL-5 cells. Endocrinol. Jpn. 37: 639-648, 1990. YOSHIMURA, M., M. NISHIKAWA, M. HORIMOTO, N. YO- SHIKAWA, S. SAWARAGI, Y. HORIKOSHI, I. SAWARAGI, AND M. INADA. Thyroid-stimulating activity of human chorionic gonadotropin in sera of normal pregnant women. Acta Endo- crinol. 123: 277-281, 1990. YOSHIMURA, M., M. NISHIKAWA, N. YOSHIKAWA, M. HO- RIMOTO, N. TOYODA, I. SAWARAGI, AND M. INADA. Mecha- nism of thyroid stimulation by human chorionic gonadotropin in sera of normal pregnant women. Acta EndocrinoZ. 124: 173-178, 1991. ZAKARIJA, M., F. J. HORNICEK, S. LEVIS, AND J. M. McKEN- ZIE. Effects of y-interferon and tumor necrosis factor cy on thyroid cells: induction of class II antigen and inhibition of growth stimulation. MoZ. Cell. EndocrinoC 58: 129-136, 1988. ZAKARIJA, M., S. JIN, AND J. M. MCKENZIE. Evidence supporting the identity in Graves’s disease of thyroid-stimulating antibody and thyroid growth-promoting immunoglobulin G as as- sayed in FRTL5 cells. J. CZin. Invest. 81: 879-884, 1988. ZAKARIJA, M., AND J. M. MCKENZIE. Influence of cytokines on growth and differentiated function of FRTL-5 cells. Endocrinol- ogy 125: 1260-1265, 1989. ZAKARIJA, M., AND J. M. MCKENZIE. Variations in the culture medium for FRTL5 cells: effects on growth and iodide uptake. Endocrinology 125: 1253-1259,1989. ZAKARIJA, M., AND J. M. MCKENZIE. Do thyroid growth-promoting immunoglobulins exist? J. CZin. Endocrinol. Metab. 70: 308-310,199O. ZARRILLI, R., S. FORMISANO, AND B. DI JESO. Hormonal regulation of thyroid peroxidase in normal and transformed rat thyroid cells. Mol. Endocrinol. 4: 39-45, 1990. ZOR, U. Role of cytoskeletal organization in the regulation of adenylate cyclase-cyclic adenosine monophosphate by hormones. Endocr. Rev. 4: l-21,1983.

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