Copyright 0 1990 by the Genetics Society of America

Genetic Differences in the Duration of the Lymphocyte Heat Shock Response in Mice

Virginia K. Mohl,* Gregory D. Bennettt and Richard H. Finnell*’T *Program in Genetics and Cell Biology and tDepartment of Veterinary and Comparative Anatomy, Pharmacology and Physiology,

College of Veterinary Medicine, Washington State University, Pullman, Washington 99164-6520 Manuscript received September 19, 1989

Accepted for publication December 22, 1989

ABSTRACT Lymphocytes from adult mice bearing a known difference in genetic susceptibility to teratogen-

induced exencephaly (SWV/SD, and DBA/2J) were evaluated for changes in protein synthesis following an in vivo heat treatment. Particular attention was paid to changes indicative of the heat shock response, a highly conserved response to environmental insult consisting of induction of a few, highly conserved proteins with simultaneous decreases in normal protein synthesis. The duration of heat shock protein induction in lymphocytes was found to be increased by 1 hr in the teratogen- sensitive SWV/SD strain as compared to the resistant DBA/2J strain. Densitometric analysis revealed a significant decrease in the relative synthesis of at least two non-heat shock proteins (36 kD and 45 kD) in the SWV/SD lymphocytes as compared to DBA/2J cells. The increased sensitivity of protein synthesis to hyperthermia in the SWV/SD lymphocytes were lost in the FI progeny of reciprocal crosses between SWV/SD and DBA/2J mouse strains. Sensitivity to hyperthermia-induced exence- phaly is recessive to resistance in these crosses. The relationship between altered protein synthesis ~,

and teratogen susceptibility is discussed.

I NVESTIGATION of the heat shock response be- gan with the discovery of a unique set of puffs in

the heat-pulsed salivary gland chromosomes of Dro- sophila busckii which were later associated with the production of specific proteins (RITOSSA 1962; TIS- SIERES, MITCHELL and TRACY 1974). Subsequent studies revealed the transient induction of a similar set of proteins known variously as heat shock or stress proteins, to be common to both eukaryotic and pro- karyotic heat-treated cells (SCHLESINGER, ASHBURNER and TISSIERES 1982; LINDQUIST 1986). T h e highly conserved nature of the heat shock protein genes (MORAN et al. 1982; LOWE, FULFORD and MORAN 1983; HUNT and MORIMOTO 1985), and their unique regulation during periods of cellular stress (TISSIERES, MITCHELL and TRACY 1974), led to the exploitation of the heat shock response as a model system with which to probe mechanisms of gene induction and regulation (NEIDHARDT, VANBOGELEL and VAUGHN 1984; BIENZ 1985). Heat shock proteins have also been implicated in the acquisition of thermotolerance (YAMAMORI and YURA 1982; LI and WERB 1982; LI and MAK 1985; BOSCH et al. 1988), and in the recovery of normal cellular processes following the application of a stress (YOST and LINDQUIST 1986). These proteins have been correlated with developmental changes in organisms as diverse as Drosophila melanogaster

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(MITCHELL and LIPPS 1978; MITCHELL et al. 1979), Naegleria gruberi (WALSH 1980) and yeast (KURTZ et al. 1986). Despite this scrutiny, the precise role of these proteins in resistance to cellular stress and their importance during development remains unclear (SCIANDRA and SUBJECK 1984; PETKO and LINDQUIST 1986; PRIMMETT et al. 1989).

A common approach utilized in the elucidation of the function of a gene or gene product is the use of mutations, three types of which currently are being actively applied in heat shock research. Mutant Esch- erichia coli strains that are defective in the positive regulation of the heat shock response (COOPER and RUETTINGER 1985; GROSSMAN, ERICKSON and GROSS 1984) have been used to demonstrate the importance of these proteins in cell growth at elevated tempera- tures (YAMAMORI and YURA 1982). A strain of Dic- tyostelium which fails to produce several specific heat shock proteins has a decreased ability to grow at elevated temperatures (LOOMIS and WHEELER 1982) while in some hydra strains, synthesis of a heat shock protein correlated with thermotolerance (BOSCH et al. 1988). In an attempt to specifically define the func- tions of a low molecular mass heat shock protein, PETKO and LINDQUIST (1986) created deletion and disruption mutations in the 26 kD heat shock protein gene (hsp 26) of yeast. They demonstrated that hsp 26 was not required for several different cellular functions including thermotolerance, germination, and growth at elevated temperatures (PETKO and

950 V. K. Mohl. G. D. Bennett and R. H. Finnell

LINDQUIST 1986). Unfortunately, similar mutations have yet be described in higher eukaryotes.

Lacking specific known mutations, the use of orga- nisms with known differences in their biological re- sponses to heat stress may provide insight into any possible homeostatic roles of these proteins. It has recently been demonstrated by FINNELL and col- leagues (1986) that a strain dependent hierarchy of susceptibility to a hyperthermia-induced neural tube defect (exencephaly) exists among inbred strains of mice. While the molecular basis of this strain variation is presently unknown, the response is clearly under genetic control. When susceptible (SWV/SD) animals are outcrossed to more resistant strains (LM/Bc, C576L/6J and DBA/ZJ; FINNELL et al. 1986; V. K. MOHL and R. H. FINNELL, unpublished data), the F1 hybrid progeny were resistant to hyperthermia in- duced exencephaly. The degree of resistance de- pended upon the outcross strain, yet all three outcross strains demonstrated that sensitivity was recessive to resistance to heat-induced exencephaly. Reciprocal cross experiments indicate that the genotype of the embryo, rather than the physiological response of the mother, is the major factor in determining suscepti- bility to heat-induced exencephaly (FINNELL et al. 1986). T h e discovery of one highly susceptible strain (SWV/SD), several moderately susceptible (LM/Bc, SWR/J) and resistant strains (DBA/ZJ, C57BL/6J) provides a unique model in which to investigate the importance of the heat shock response to embryonic development in a mammalian system.

Our working hypothesis has been that an environ- mental insult capable of disrupting normal mamma- lian development should have a demonstrable effect on maternal tissues. Previous results from our labo- ratory supported this hypothesis by demonstrating induction of heat shock proteins in both embryonic tissues and maternal lymphocytes (BENNETT, MOHL and FINNELL 1990). While investigations of the early embryo provide direct evidence for the teratogenic effects of the heat treatment, the severely limited amount of available tissue and its rapid differentiation complicate biochemical analysis of cell function. The lymphocyte assay complements the embryonic tissue studies by providing a sample tissue that is readily isolated with little trauma to the cells and which has well defined culture characteristics and functional ac- tivity assays.

In the studies reported here, the lymphocyte heat shock assay was expanded to examine the kinetics of protein synthesis following in vivo hyperthermia. Cells from adult mice bearing a known genetic susceptibility to heat-induced exencephaly were evaluated for changes in protein synthesis following a single in vivo heat treatment. In the kinetic study described herein, a heat treatment previously shown to induce devel-

opmental abnormalities (FINNELL et al. 1986) pro- duced differences in the heat shock response between highly inbred strains of mice susceptible (SWV/SD) and (DBA/ZJ) resistant to heat-induced exencephaly.

MATERIALS AND METHODS

Two inbred mouse strains (SWV/SD, and DBA/2J) were maintained on a 12-hr light cycle in the College Hall Vivar- ium at Washington State University, Pullman. Reciprocal cross experiments were performed using the FI generation progeny of matings between the highly susceptible SWV/ SD and the resistant DBA/2J strains. Healthy, pathogen- free mice were housed five per polycarbonate cage and allowed free access to Purina rodent chow and tap water. Animals 60 to 120 days of age were randomly assigned to treatment groups. All animal procedures were designed to minimize any stress and discomfort to the experimental animals.

Treatment of the adult animals consisted of a single, 10- min incubation in either a 43" waterbath for heat stressed animals or a 38" waterbath for control animals. This treat- ment is described in detail elsewhere (FINNELL et al. 1986). Core temperatures were recorded at one minute intervals using a YSI telethermometer and small animal probe (Yel- low Springs Instruments, Yellow Springs, Ohio). By chang- ing the depth of the restraining chamber in the waterbath, it is possible to control closely the rate of temperature increase and thus insure uniformity of heat treatment among individual animals. Therefore, the core body tem- perature did not vary among the parental or hybrid strains. Heat stressed animals not sacrificed immediately following treatment were patted dry and placed into a 38" warm air incubator for twenty minutes to allow the animal's body temperature to gradually return to its normal basal level. N o age or strain related differences in the kinetics of the temperature curve were noted. For each strain, a minimum of five animals were evaluated at each time point.

Following treatment, the mice were sacrificed by cervical dislocation, the spleen aseptically removed and placed into cold Hank's balanced salt solution (HBSS, GIBCO, Grand Island, New York). The spleen was then gently teased apart with mouse-toothed forceps and the cell containing super- natant collected. The number of nucleated cells was deter- mined using a hemocytometer. Both the cells and the buffer were kept on ice throughout the procedure. Cell viability as determined by trypan blue exclusion (MISHELL and SHIICI 1980) ranged between 98% and 96% viable cells.

To determine changes in specific proteins synthesized following an in vivo heat treatment, spleen cells were har- vested at 0, 1, 2, and 3 hr following a 10-min treatment at either control (38") or heat shock (43") temperatures. A volume of cell suspension containing between 2 and 5 mil- lion cells was collected for labeling with ["S]methionine. Each sample was spun down in a Beckrnan microfuge, the supernatant discarded and the resultant pellet resuspended into 200 pl of cold HBSS. Twenty microcuries [JsS]methio- nine (NEN Research Products, Boston, Massachusetts) was added and the suspension was allowed to incubate for one hour in a 37" water bath. Following incubation, the cells were washed twice with cold HBSS to remove any unincor- porated methionine and to minimize the potential degra- dation of newly synthesized heat shock proteins (MITCHELL, PETERSON and BUZIN 1985). The cells were then lysed with 100 pI LAEMMELI'S (1970) sample buffer and boiled for 10 min. To determine the amount of radioactivity that was incorporated into the cells, duplicate aliquots of sample were

Lymphocyte Heat Shock Response 95 1

counted i n a Beckman scintillation counter with 10 ml Aquasol (Amersham, Arlington Heights, Illinois) as the scin- tillant. The remainder of the sample was either used im- mediately for gel electrophoresis or frozen at 70".

Cells were considered to express a heat shock response if induction or enhancement of selected protein bands of appropriate molecular weights were detected on autoradi- ograms of samples separated by SDS-PAGE. The time of harvesting was selected to cover the period of greatest change in protein synthesis based on visual inspection of autoradiograms. Preliminary experiments indicated no sig- nificant induction of heat shock proteins due to this lym- phocyte isolation and protein labeling procedure (V. K. MOHL and R. H. FINNELL, unpublished results).

Electrophoresis was accomplished using the mini gel ap- paratus (Idea Scientific, Corvallis, OR) after the manner of LAEMMELI (1 970). For each sample, equal amounts of radio- activity as determined by scintillation counting was loaded onto gradient SDS polyacrylamide gels (12-20% acrylam- ide). Gradient gels were poured seven at a time using a gradient gel pouring stand (Idea Scientific, Corvallis, Ore- gon) and gravity feed. After electrophoresis, gels were stained with Coomassie blue, dried, and exposed to X-ray film (Kodak MNl X-ray film, Rochester, NY) for three days. Molecular weights were estimated based on the migra- tion of standard markers (nonradioactive markers: Sigma Chemical Co.; St. Louis, Missouri; radioactive markers: NEN Research Products, Boston, Massachusetts). Autora- diograms were evaluated visually and then run on a densi- tometer (Bio-Rad, Richmond, California).

T o facilitate between strain comparisons and to minimize the problems inherent in gel to gel comparisons, heat- treated samples were compared to control samples from the same experiment run on the same gel. This was accom- plished by dividing the integrated area of the treated peaks of interest (36,45,68-70,97 and 1 10 kD) by that of control peaks of the same molecular mass.

Factorial analysis of variance was used to determine the effects of genotype and time after heat treatment on the various parameters measured. Significant differences be- tween the strains and among the time points were further analyzed using a Student-Newman-Keds comparison of sample means (SOKAL and ROLF 1969; STEEL and TORRIE 1980). Tests for significance were set at a = 0.05 unless otherwise noted.

RESULTS

Immediately following the heat shock treatment, new protein bands appeared in all mouse strains at 68, 70, 97, and 1 10 kD, with an occasional band also appearing at approximately 50 kD (Figure I , A-D). The greatest difference between the strains in heat shock protein synthesis visible by autoradiography was the duration of active synthesis of the 68 and 70 kD heat shock proteins. In the DBA/2J strain, synthesis of hsp 68 and 70 is limited in its duration to no more than 1 hr post treatment. In the susceptible SWV/SD strain, there continued to be evidence of active syn- thesis of hsp 68 and 70 until at least 2 hr post treat- ment in all experiments. Synthesis of the 97 kD pro- tein appeared enhanced in all strains throughout the experimental period. The 110 kD protein demon- strated the greatest variation in terms of its detectable

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FIGURE 1 .-Autoradiograms. Fach gel represents one experi- ment with a separate mouse treated for each time point. Lympho- cytes were isolated at the indicated time following treatlnent and incubated for 1 hr with ["S]~nethionine a s described in the methods sections. Cells were lysed and equal anlounts of radio;Ictivity run 011 each lane of an SDS polyacryl;lmide gel. The protein bmds of interest to this study are indicated on the left side of the autoradi- ogram. The approxinxlte molecular weights were estimated by the inclusion of marker proteins with known molecular weights. The first lane of each autoradiogram contains the 38" control smple to which the other lanes nlay be colnpred. A. SWV/SD. The smile four bands seen with the DBA/2.J ;Inim;lls are apparent in cells from SWV/SD ;Inim;lls. However. induction of the 68- and 70-kD band continues to be expressed in the 4 3 " + 2 hr samples in all experi- ments. This was a consistent finding of five repeated experiments. B, DBA/2J. Induction of at least four prominent bands (68. 70, 97 and I10 kD) is apparent at 43" + 0 hr. and a t 43" + I hr. By the 43" + 2 hr time point, synthesis of the 68- and 70-kD proteins has returned to control levels. Wlis w a s a consistent finding of five repeated experiments. C, The F, offspring of matings between Sb'V/SD females and DBA/2J mdcs. Intiuction of the four protein bands followed the pattern described for the DBAILJ aninlals. D, the FI offspring of matings between the DBA/2J females and SWV/ S D nlales. Results resembled Figure 2. A and C, in four of five experiments. I n one experiment, the 68- and 70-kD bands were present at 43" + 2 hr.

induction period, with no consistent difference noted between the strains.

When similar experiments were repeated on the FI generation of DBA/2J X SWV/SD reciprocal crosses, lymphocytes from these animals responded very much like the heat-resistant DBA/2J parental mice. In par-

V. K. Mohl, G. D. Bennett and R. H. Finnell 952

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FIGURE 2.-A densitometric analysis comparing synthesis of two taon-hr;~t sllock proteins among the strains. FJch bar represents the relative incorpor;ation of methionine by the heat-treated samples dividrtl I)! the control s;~mple and averaged for five separate exper- iments. Rars extending below the "1 ." less than 100% of control, indicate treated cells synthesized less protein of that molecular weight than did control cells. Bars extending above the " I ," greater th;ao 100% of control, represent a relative induction of proteins at that nlolecu1;ar III;ISS. St;atistic;llly significant depression of both the 3ci-kD ;and the 4.5-kD proteins was found in the SWV/SD samples at 2 hr post heat-treatment when compared to the similarly nor- nxalized rate of protein synthesis in the DBA/2J strain and the FI recipror;al crosses. A, The 36-kD non-heat shock protein; B, 45-kD non-heat shock protein. (*) Significant at a = 0.05, Student-New- III>IWKCUIS test (S.N.K.).

ticular, the enhanced synthesis of the 68- and 70-kD proteins is observed immediately following heat treat- ment and by 2 hrs has returned to control levels. In fact, active synthesis of 68- and 70-kD heat shock proteins at 2 hr was detected in only one of the ten reciprocal cross experiments performed.

Densitometry provided a method to quantify the changes in protein synthesis seen on autoradiograms. Figures 2 and 3 summarize the effects of hyperther- mia on the synthesis of heat shock and non-heat shock proteins relative to control levels of synthesis among the mouse strains. Factorial analysis of variance was

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FIGURE 3.-Densitometric analysis of four heat-shock proteins: 68-70 kD, 97 kl). and 1 IO kD. The same comparison described in Figure 2 is presented in this figure for the heat-shock proteins. A, Methionine incorporation into the 68-70-kD protein was signifi- cantly increased in the SWV/SD and F, (DBA/2J female X SWV/ SD male) relative to the DBA/2J and FI reciprocal cross (SWV/SD female X DBA/2J male) at 1 hr post heat-treatment. B. Synthesis of the 97-kD protein was significantly increased in the SWV/SD strain relative to the other genotypes at 3 hr post-treatment. C , N o significant differences were detected among the strains in synthesis ofthe 1 IO-kD protein. (*) Significant at a = 0.05, S.N.K.

Lymphocyte Heat Shock Response 953

performed for each of three heat shock protein peaks (1 10 kD, 97 kD and 68-70 kD) and two non-heat shock protein peaks (45 kD, 36 kD). The 68 and 70 kD protein peaks were pooled as our densitometry system did not allow for complete separation of such closely migrating peaks. The two non-heat shock peaks were chosen based on their consistent appear- ance in both strains and at all treatment time points.

Examination of the effects of hyperthermia on non- heat shock proteins (36 kD, 45 kD; Figure 2, A and B) indicates in vivo hyperthermia treatment decreased synthesis of these lymphocyte proteins. Immediately following treatment, synthesis of the 36-kD protein was decreased by 20% (DBA X SWV) to 40% (SWV/ SD) relative to controls. The 45-kD protein was also affected at this time point, with a 10% (DBA/2J) to 50% (SWV/SD) reduction of protein synthesis. Syn- thesis of both the 36-kD and 45-kD proteins was significantly lower in the SWV/SD lymphocytes rela- tive to lymphocytes from the DBA/2J or F1 offspring at 2 hr post heat treatment (Figure 2).

In contrast to the 36-kD and 45-kD proteins, syn- thesis of the 68, 70, 97, and 110 kD proteins was enhanced by the 43" exposure. Immediately after treatment, synthesis of the 68-70-kD proteins dou- bled the control levels as determined by densitometric analysis (Figure 3A) with average synthesis 180% (DBA/2J) to 260% (DBA X SWV) of 38" treated controls from the respective genotypes. Lymphocytes from the DBA/2J strain returned to control levels by 1 hr while at this time the SWV/SD cells still demon- strated at least a 2-fold increase in synthesis of the 68-70-kD proteins. In the reciprocal cross experi- ments, the SWV/SD (M) X DBA/2J (P) resembled the DBA/2J cells at 1 hr post treatment with synthesis of the 68-70-kD protein significantly less than that of the SWV/SD cells or the other F1 cross (DBA X SWV). This densitometry result is anomalous as it does not reflect the results found by visible inspection of the autoradiograms and there were no statistically signif- icant differences between the reciprocal cross experi- ments' densitometry results (factorial analysis of vari- ance).

Synthesis of the 97-kD protein was increased by about 25% immediately following treatment in all experiments, with synthesis peaking in the 1-2-hr samples and remaining slightly enhanced in lympho- cytes from all animals throughout the experimental period (Figure 3B). However, at the 3 hr time point, relative synthesis of the 97-kD protein was signifi- cantly increased in the SWV/SD lymphocytes as corn- pared to all other cells examined.

Densitometry analysis found synthesis of the 110- kD protein to be highly variable as was the case for its visible induction o n autoradiograms. No significant differences were noted among the experiments. Syn-

thesis of the 1 IO-kD protein peaked from 1 to 2 hr post heat treatment (Figure 3C).

Changes in cellular protein synthesis as visualized on autoradiograms and verified by densitometry were accompanied by a change in the amount of radiolabel uptake by the cells. Immediately following the heat treatment in all three inbred strains, the amount of cellular radioactivity was significantly less than that found following control treatments (Figure 4). Two hours post treatment, no significant differences be- tween control and heat shocked cells were detected in any of the strains. In one of the F1 crosses (Figure 4), methionine incorporation at 3 hr actually exceeded control levels. It was noted that the SWV/SD cells incorporated less radioactivity on average than did the SWV/SD cells. However, there was no significant difference in cellular methionine incorporation of the controls among the different lymphocytes studied.

DISCUSSION

These experiments demonstrate that a heat treat- ment sufficient to induce developmental abnormali- ties in murine embryos is also capable of altering protein synthesis in at least one differentiated cell type. The changes seen in lymphocyte protein synthe- sis include the induction or enhancement of proteins of at least four different molecular masses (68, 70, 97 and 110 kD) following in vivo treatment at 43" for 10 min. Synthesis of at two non-heat shock proteins (36 and 45 kD) was depressed by the heat treatment, as determined by densitometric tracings of autoradi- ograms. A generalized decrease in cellular [35S]methi- onine uptake immediately following hyperthermia ex- posure was also noted. These results are consistent with the heat shock response as previously described (SCHLESINCER, ALIPERTI and KELLEY 1982; SUBJECK and SHYY 1986).

Genetic variation among the inbred strains follow- ing hyperthermic shock included differences in the kinetics of their lymphocyte heat shock response. Vis- ual inspection of autoradiograms demonstrated a con- sistently longer period of induction of the 68 and 70 kD heat shock proteins in the SWV/SD as compared to the DBA/2J mouse strains. Densitometry con- firmed this difference between the inbred strains with SWV/SD having the greater induction relative to control levels in their lymphocytes, particularly at 1 hr post treatment. The offspring of reciprocal crosses between these two strains were found to behave most like the parental DBA/2J strain with visual inspection of the autoradiograms. However, the densitometry results for the F1 reciprocal cross experiments were inconclusive. The inconsistency between the visual inspection of the autoradiograms and the densitome- try results in the F1 experiments indicate that in- creased duration of heat shock protein synthesis alone

954 V. K. Mohl, G. D. Bennett and R. H. Finnell

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is insufficient for evaluating the genetically deter- mined differences in cellular response to hyperther- mia between these mouse strains.

The affect of hyperthermia on normal protein syn- thesis had a more consistent correlation with sensitiv- ity to teratogen-induced exencephaly in these experi- ments. Synthesis of both of the normal proteins inves- tigated was most depressed in the SWV/SD lympho- cytes, particularly at 2 hr post heat treatment. This continued depression of protein synthesis was not found in the F1 reciprocal experiments, indicating the more rapid recovery is a dominant trait within the SWV/SD and DBA/2J genotypes. Thus, the inbred mouse strain whose spleen cells seem least affected by hyperthermia (DBA/2J) is the strain with the greatest resistance to heat-induced exencephaly (FINNELL et al. 1986) and this resistance trait is also dominant (V. K. MOHL and R. H. FINNELL, unpublished results).

While the similarities in the segregation of the al- leles controlling the lymphocyte heat shock response differences and that of susceptibility to hyperthermia- induced neural tube defects are intriguing, it is uncer- tain at present whether the differences in lymphocyte protein synthesis reported here can be directly related to abnormal embryonic development. GERMAN (1 984) hypothesized that preferential synthesis of heat shock protein could inhibit the synthesis of other proteins that are essential for normal development. However, the mere synthesis of heat shock proteins alone does not appear to be sufficient to lead to developmental defects in mouse embryos (BENNETT, MOHL and FIN- NELL 1990). Induction of these proteins may, in fact, protect the embryo from a later, more severe environ- mental stress (WALSH et al. 1987; MIRKES 1987). The

FIGURE 4.-Quantification of to- tal methionine uptake by the cells. The total amount of radioactivity in- corporated by the cells from DBA/ 25, SWV/SD and the F I offspring of reciprocal crosses between these two strains was determined in lysates from both control (38" + 0 hr) and heat-treated (43" = 0, 1 , 2 and 3 hr) animals. Following treatment, cells were labeled with ["'SJmethionine for 1 hr. Labeled cells were washed with excess cold HBSS to remove unincorporated methionine. The to- tal amount of radioactivity present in the cells was determined using liquid scintillation as described in the nleth- ods section. Uptake of the radiolabel was depressed immediately following heat-treatment in all experiments. Acid precipitable radioactivity was approximately 25% of the total ra- dioactivity in the lysates. (*) Signifi- cant at a = 0.05, S.N.K.

beneficial effect of the heat shock proteins in protect- ing normal development is reminiscent of the pro- posed role of these proteins in cellular homeostasis (SCIANDRA and SUBJECK 1984; LI and WERB 1982), and may better explain the relationship between the cellular effects described here and susceptibility to hyperthermia-induced exencephaly. For example, ex- perimental work conducted in cell culture suggest that cell survival is positively correlated with the extent to which normal protein synthesis is inhibited, the du- ration of heat shock protein induction and the time required to return to normal protein synthesis (re- viewed in SUBJECK and SHYY 1986). The ability of the cells to retain normal cellular functions even during periods of stress appears to play a key role in prevent- ing cell death (WELCH and SUHAN 1985). Thus, eval- uating normal cellular functions such as protein syn- thesis as well as the induction of heat shock proteins would seem likely to provide the best estimate of the response of cells to environmental stress.

In summary, the lack of appropriate animal models has hindered our understanding of the role of heat shock proteins in mammalian homeostatic responses to hyperthermia. As part of a series of experiments utilizing a strain hierarchy of susceptibility to hyper- thermia-induced neural tube defects, we have discov- ered a difference in the heat shock response of lym- phocytes from two inbred mouse strains. Our results indicate that the relative synthesis of normal proteins is most depressed in the SWV/SD strain, the strain most susceptible to heat-induced exencephaly. To- gether with the increased duration of heat shock protein synthesis, this indicates that lymphocytes from the SWV/SD strain are responding in a more dramatic

Lymphocyte Heat Shock Response 955

manner to the hyperthermic insult than are those from the other strains used in this study. These effects are lost in the F1 hybrid progeny of susceptible SWV/ SD and the more resistant DBA/2J strain.

This work was supported i n part by a National Science Founda- tion predoctoral fellowship to V.K.M., and by research grant ES 04326 from the National InstitutesofHealth to R.H.F. Theauthors wish to express their appreciation to MICHAEL VAN WAES for his critical review of the manuscript, PATRICIA AGER for her technical assistance, and C . SMITH, P. PERRON and R. THOMPSON for editorial and clerical assistance.

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BIENz, M., 1985 Transient and developnlental activation of heat- shock genes. Trends Biol. Sci. 00: 157-161.

BOSCH, T . C . G., S. M. KRYLOW, H. R. BODE and R. E. STEELE, 1988 Therniotoletance and synthesis of heat shock proteins: These responses are present in Hydra attenuate but absent in Hydra oligactis. Proc. Natl. Acad. Sci. USA 85: 7927-7931.

COOPER, S., and T . RUETTINGER, 1975 A temperature sensitive nonsense mutation affecting the synthesis of a major protein ofEscherichia coli K 1 2 . Mol. Gen. Genet. 139: 167-176.

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KURT%, S., J . ROW, L. PETKO and LINDQUIST, 1986 An ancient developnlental induction: Heat-shock proteins induced i n spor- ulation and oogenesis. Science 231: 1 1 54-1 157.

LAEMMIJ, U . K., 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 227: 680- 685.

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Communicating editor: R. E:. GANSCHOW