critical variables controlling cell proliferation in primary cultures of rat tracheal epithelial...

In Vitro Cell. Dev. Biol. 27A:ll05-814, October 1991 © 1991 Tissue Culture Association 0883-8364/91 $01.50+0.00

CRITICAL VARIABLES CONTROLLING CELL PROLIFERATION IN PRIMARY C U L T U R E S O F RAT TRACHEAL EPITHELIAL C E L L S

THOMAS GRAY, JOYCE RUNDHAUG, AND PAUL NETTESHEIM 1

National Institute of Environmental Health Sciences, Laboratory of Pulmonary Pathobiology, P.O. Box 12233, Research Triangle Park, North Carolina 27709

(Received 25 January 1991; accepted 29 May 1991)

SUMMARY

The purpose of our experiments was to examine variables affecting early events in the establishment of rat tracheal epithelial (RTE) cultures as well as factors regulating long-term RTE cell growth. The experiments showed that when RTE cells were seeded into complete serum-free medium between 13 and 30% of the seeded cells attached. Of the seeded cells, only ~ 2 % entered into DNA synthesis and underwent repeated cell divisions to form colonies containing > 20 cells. Coating the dishes with extracellular matrix components had little effect on cell attachment or colony forming efficiency (CFE). However, coating the dishes with fetal bovine serum markedly increased CFE. The media components bovine serum albumin and bovine pituitary extract were shown to be important in promoting cell attachment as well as CFE. Cholera toxin on the other hand had no effect on cell attachment but significantly increased CFE. These and other studies showed that cell attachment and cell proliferation are independently regulated. Studies on long-term culture growth indicated that the number of progeny produced per colony forming unit (CFU) is inversely proportional to the number of CFUs seeded. Inasmuch as the cultures did not become confluent under any of the culture conditions tested and media obtained from high density cultures were shown to be growth inhibitory, these findings suggest that a diffusible growth restraining factor is being produced by the cultures limiting clonal expansion. Experiments showing growth inhibitory effects of media conditioned by high cell density cultures support this interpretation. The putative factor reaches critical concentrations earlier in cultures seeded with high numbers of CFU than in cultures seeded with low numbers of CFU. Because the cultures are known to produce transforming growth factor-beta, this growth regulator probably plays a role in controlling RTE cell proliferation. However, it is likely than other events, such as depletion of growth factors from the media, also are significant in regulating the growth of the cultures.

Key words: rat tracheal epithelial cells; colony forming unit; transforming growth factor beta; transforming growth factor alpha; bovine serum albumin; cholera toxin.

INTRODUCTION

In recent years, the number of studies exploring mechanisms regulating growth and differentiation of tracheobronchial epithelium as well as mechanims of pathologic disturbances of conducting airway epithelium has greatly increased. To carry out these investigations, a great variety of organ culture and cell culture systems have been developed using airway epithelium from different species including humans, primates, rabbits, ferrets, hamsters, and rats (for review see 41). Such tissue and cell culture models have greatly facilitated, for example, studies of cellular and molecular mechanisms of differentiation (17), the role of specific factors regulating squamous and mucous differentiation including retinoids, transforming growth factor-beta (TGF-beta), and extraceilular matrix (9,18,28). They have also been used to investigate the regulation of mucous production and secretion by airway epithelium and glands and various factors stimulating or modifying airway secretions (1,10,20,31). The availability of cell culture models also greatly

To whom correspondence should be addressed.

805

facilitated studies of carcinogenesis of tracheobronchial epithelium, concerned with carcinogen metabolism (2,13), DNA adduct formation and DNA repair (5,6,15,16), the role of single and cooperating oncogenes and tumor suppressor genes in different lung cancer phenotypes (29), as well the antitransforming effects of retinoids, protease inhibitors, antioxidants, and other compounds (34).

Our own laboratory has utilized rabbit and airway cell cultures in studies of tracheal-bronchial cell differentiation (14,27,30) and, particularly the rat tracheal cell cultures, for analyzing the multistage process of neoplastic transformation and the effects of tumor promoters and antipromoting agents (8,11,21,22,24-26).

The studies reported here were aimed at obtaining information on the following questions: a) Are the colony forming cells (=colony forming units = CFU), which are present in freshly harvested rat tracheal epithelial (RTE) cells, the only cells capable of replication and which factors can influence the expression of their proliferative potential? b) Which variables significantly affect the extent of proliferation in the cultures? Several studies have been published recently describing the effects of various media components on RTE cell growth (39,40); however, these studies were mostly concerned

806 GRAY ET AL.

with early phases of culture growth and gave no clues as to which factors are crucial in promoting or limiting proliferation in later stages of culture. This aspect of growth regulation is particularly important for investigations of RTE cell transformation because such studies require that primary cultures be maintained for 4 to 5 wk at which time the variant colonies emerge (11,39). Both questions are pertinent to two fundamental issues in any cell transformation system: a) Which cells are the potential targets for the transforming agent, only the CFUs or also other cells which may be capable of proliferation but not of colony formation. We had noticed, for example, that rabbit tracheal cells seeded on plastic tissue culture surfaces do not form discrete, tightly packed colonies because the ceils are very migratory (unpublished observation). Mea- suring CFU may therefore not be an accurate measure of the size of the proliferative cell pool. b) Several recent reports point to the importance of culture variables that influence transformation frequencies in RTE cultures by affecting cell proliferation (37,38). It therefore was essential to gain a better understanding of factors that can limit cell proliferation in long-term RTE cultures. In particular we wanted to examine the influence of cell density on sustained proliferation in long-term primary cultures. The results of these studies showed that a) the CFUs are the only proliferative units in primary tracheal cell isolates; b) that the CFU seeding density has a marked affect on the number of progeny produced per CFU; and c) that this effect is in part mediated by growth inhibitory factor(s), possibly TGF-beta, produced in late logarithmic growth when the cell density reaches "~2.5 X 10 g cells/cm 2 ( ~ 5 × 105 cells" 60 mm -l dish). Depletion of growth factors in cultures containing >2.5 X 10 ¢ cells" cm 2 probably also plays a role in slowing the growth rate of late primary cultures.

MATERIALS AND METHODS

Cells and cell culture. Methods to establish primary cultures of RTE cells in serum-free medium have been previously reported (39). Briefly, cells were obtained from tracheas of 10- to 15-wk- old, male, Fisher 344, specific pathogen-free rats. The epithelial lining was dissociated by an overnight enzymatic digestion with 1% pronase (type 14, Sigma, St. Louis, MO) solution carried out at 40 ° C. Epithelial cells were flushed from the tracheas (viability was greater than 95% by trypan blue exclusion test), washed and suspended in culture medium, counted visually in a hemacytometer, and the desired number of ceils was plated into 60-mm dishes. RTE cultures were maintained at 37 ° C in a humidified atmosphere of 5% CO2 in air.

Rat tracheal epithelial ceils were seeded at various cell densities in complete serum-free medium (CSFM) composed of Ham's F12 medium (GIBCO, Grand Island, NY) supplemented with insulin (10 #g/ml), hydrocortisone (0.3 mM), transferrin (5 #g/ml), cholera toxin (CT, 0.1 #g/ml), phosphoethanolamine and ethanolamine (80 #M each), bovine serum albumin (BSA, 3 mg/ml, essentially globu- lin free), HEPES (15 raM, pH 7.2), and CaCI 2 (0.8 mM) all purchased from Sigma. In addition, the culture medium contained epidermal growth factor (5.0 ng/ml, Collaborative Research, Inc., New Bedford, MA) and 1% vol/vol bovine pituitary extract (BPE) which was prepared from whole pituitaries (Pel Freeze, Rogers, AK) as previously described (4). Penicillin and streptomycin (1% vol/ vol, GIBCO) were added to all media. To examine the role that BPE, BSA, and CT play in affecting cell attachment and clonal

growth, medium containing various concentrations of these factors was used. Culture medium was changed every 3 to 4 days except as noted.

Effects of substratum. To examine the effects of substratum on the attachment and growth of RTE cells, 60-ram tissue culture dishes were used either uncoated or were pretreated in several ways. Fibronectin, type I and IV collagens, laminin and Matrigel (all purchased from Collaborative Research) and FAV (a 1 )< solution contains fibronectin, and albumin at 10 #g/ml and type I collagen at 2.5 tsg/ml) were dissolved in Ham's F 12 medium and 5 ml of the appropriate concentration was added to each dish. After incubation overnight at 37 ° C the substratum containing medium was completely removed and RTE cells suspended in CSFM were inoculated at 1 to 2 X 104 cells per dish.

Determination of cell attachment. Cell attachment was determined 24 h after seeding 60-mm dishes with 2 X 104 RTE cells. The medium was gently removed and the cultures were carefully washed with phosphate buffered saline (PBS), 1 ml of 0.15% tryp- sin-0.06% EDTA was added to each dish followed by 10 rain incubation at 37 ° C. Cells were dispersed by repeated pipetting, and aliquots of the cell suspension were counted visually using a hemacytometer. Cell attachment was calculated by dividing the cell number per dish by the number of cells plated per dish. Values present the mean +_ standard deviation determined from 3 to 10 replicates per group. Confidence limits were determined using a t distribution with the number of degrees of freedom ranging from 3 to 10.

Colony forming efficiency (CFE), growth curves (attached and exfoliated cell number), cross-linked envelope (CLE) formation, and collection of conditioned media. To determine the number of clonogenic cells present in freshly isolated RTE cells suspensions obtained by type 14 pronase digestion, CFE was determined by seeding 5 X 103 to 2 × 104 cells/dish, three to five rephcates per group. Cultures were fixed and stained after 7 days and the number of colonies counted (colonies containing >20 cells were scored). CFE was calculated by dividing the mean number of colonies per dish by the number of cells seeded per dish X 100. Statistical comparisons were made using t distribution.

Determination of attached and exfoliated cell number per dish (3 to 10 replicates per time point) was made at various times after seeding 2 X 104 RTE cells/dish. The culture medium was removed and the cells suspended in the medium (exfoliated cells) pelleted, resuspended, and the cell number determined by hemacytometer counts. The remaining attached cells were dissociated as described above and the cell number determined visually. The number of cells containing a CLE was determined according to methods described by Sun and Green (36). Conditioned media was collected over a 48-h period from cultures seeded with 123 to 787 CFU/dish at various times during logarithmic-growth and at plateau. Control medium was collected simultaneously from cultures without cells. The conditioned medium was stored frozen at - 2 0 ° C until used undi- luted in a CFE assay using primary RTE cells as previously de-

scribed. Autoradiography and DNA synthesis. To determine the number

of seeded RTE ceils that undergo DNA synthesis within 72 h after plating, uncoated 60-mm culture dishes were seeded with 2 X 104 RTE cells in CSFM containing 10 #Ci/ml [SH]thymidine (methyl- [3H]TdR; specific activity 5.0 Ci/mmol, Amersham Corp, Arlington Heights, IL). After exposure, the medium was removed and the cultures were washed 3 times with cold PBS containing 1 #M cold

IN VITRO REGULATION OF PROLIFERATION OF RTE CELLS 8 0 7

thymidine followed by three 1-min rinses with ice-cold 10% tri- chloracetic acid (TCA), washed with distilled water, and air dried. The wall of the culture dish was carefully trimmed away and the dish bottoms were dipped in photographic emulsion (NTB-2, East- man Kodak, Rochester, NY) and stored for 4 wk at 4 ° C. Dishes were developed (5 min, 18 ° C; Kodak D-19 developer), fixed (Ko- dak fixer, 5 rain, 18 ° C), airdried, and stained for 10 min with 10% aqueous Giemsa. Cell nuclei having > 1 0 grains were considered positive. Labeling indices were made by scoring 500 cells.

DNA synthesis was also determined on cultures at various times after plating. Cultures were exposed to [aH]thymidine (10 ttCi/ml) for 2 h, the medium removed, and the cultures washed with PBS and TCA as described above. The cells were lysed in 2 ml of 0.3 N NaOH plus 1% sodium dodecyl sulfate (SDS), and the entire lysate was suspended in 10 ml of scintillant (Ecolume, ICN Biochemicals, Inc., Irvine, CA) and counted. Replicate dishes were used to determine cell number per culture and the data were expressed as cpm/ lO s cells.

RESULTS

Early events in RTE cultures. The purpose of these studies was to examine several factors which may affect early events in primary RTE cultures, namely, cell attachment and proliferation leading to formation of colonies. First we wanted to determine under routine culture conditions what fraction of the seeded cells attached, what fraction entered into DNA synthesis, and what fraction of cells had sufficient proliferative activity to form colonies containing at least 20 cells on Day 7. The first part of the experiment is summarized in Fig. 1 A; it showed that approximately 13% of the seeded cells attached. This was rather low; in other experiments attachment frequencies as high as 30% were not uncommon (see Fig. 3). Of the seeded cells, approximately 2% entered DNA synthesis during the first 24 h of culture and 1.5% were able to form colonies. In the second part of the experiment cultures were incubated with [3H]TdR for 48 and 72 h because the possibility existed that some cells might enter a first round of cell replication later than 24 h. We scored labeled cell clusters (i.e. one or more labeled cells within a cluster of cells constitutes a labeled cell cluster) instead of labeled individual cells, and expressed the data as fraction of cell clusters containing one or more labeled cells. It was to be expected that if no new cells entered DNA synthesis later than 24 h after seeding, the percent of labeled cell clusters would remain constant over time. The data summarized in Fig. 1 B revealed that the percent of labeled cell clusters, i.e. cell clusters with one or more labeled cells combined, was slightly higher at 48 h (23%) and 72 h (22%) than at 24 h (17%). At 24 h virtually all cells, labeled and unlabeled, were single. At 48 h most labeled cells were still single but a significant number of labeled doublets and multicell clusters occurred (8%). At 72 h only few single-labeled cells remained (4%) and more than 17% of labeled cell clusters were doublets and muhicell clusters, indicating that colony formation was well underway. The data indicated that a few of the attached cells entered DNA synthesis for the first time between 24 and 48 h. Together with the CFE results, the data suggested that in this experiment approximately 77% of the cells entering cell cycle during the first 24 to 48 h after seeding continued to proliferate and formed scorable colonies. In a separate experiment, the estimate was near 100%. Thus we con- cluded that most if not all of the cells involved in the first wave of

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FIG. 1. Relationship between cell attachment, entry into DNA synthesis, and colony formation. 2 X 104 RTE cells were seeded into uncoated, 60-mm tissue culture dishes and maintained in CSFM. A, the number of attached cells was determined after 24 h; [SH]TdR incubation was for 24 h; colonies were scored on Day 7. Data are expressed as the fraction of cells seeded which attached, underwent DNA synthesis, or formed colonies. B, cultures were incubated with [SH]TdR for 24, 48, or 72 h, prepared for autoradiography, and were scored for labeled cell "clusters" composed of a single cell ([]); two cells ([]); more than two cells (m); single and multieell clusters combined (11). Data are expressed as the fraction of cell clusters that were labeled. Differences between the fraction of clonogenic cells and the fraction of DNA synthesizing cells were examined by Student's t test distribution with 4 degrees of freedom. Values were not significantly different (0.1 > P > 0.05).

DNA synthesis proliferated sufficiently to form colonies (0.1 > P

> 0.05). Next we examined the effect of several attachment substrata

hsted in Table 1 on attachment frequency and CFE. Our goal was to determine whether extracellular matrices affect cell attachment and colony formation. Fibronectin increased CFE but only at the highest concentration tested, without causing statistically significant changes in attachment frequency. Type I and type IV collagen, laminin, and FAV (a mixture of fibronectin, albumin, and type I collagen), had no significant effect on either cell attachment or CFE. Coating of the culture dishes with fetal bovine serum (FBS) did not dramatically increase cell attachment but increased CFE up to 2.6-

fold. We also studied the effects of some CSFM components listed in

Table 2 on cell attachment, CFE, and cell number per colony. The

8 0 8 GRAY ET AL.

TABLE 1

EFFECT OF SUBSTRATUM ON CELL ATTACHMENT AND COLONY FORMING EFFICIENCY (CFE) a

Changes as % of eontroP Attachment Range of Substratum Concentrations Cell Attachment CFE

Fibronectin 0.1-10" 94-140 109-162 a Type I Collagen 0.1-10 ~ 81-96 91-106 Type IV Collagen 0.1-10 ~ 80-124 93-104 Laminin 0.1-10 ~ 78-94 89-113 FAV lx-5x" 75-107 71-106 FBS 2.5-20% 125-135 160-260 ~

Primary RTE cells were grown in CSFM. b Control cultures were grown in uncoated dishes; in seven experiments

control attachment ranged from 16.2 to 31.5% and CFE from 1.1 to 6.3%. c Concentration expressed as gg/ml.

Indicates values that are statistically significant above controls based on t test distribution analysis.

A lx solution of FAV contains 10 gg/ml of fibronectin and BSA and 2.5 gg/ml of type I collagen.

medium components we studied had been previously described (40) to significantly affect the clonal growth of RTE cells, but it was unclear whether the supplements increased cell attachment or acted as mitogenic stimuli. BSA significantly affected both cell attachment

and CFE. Without BSA, colony formation was almost completely

TABLE 2

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ent experiments. Different numbers of cells were seeded into 60-mm, uncoated tissue culture dishes (5 to 10 replicates per point). Numbers on the right indicate the number of clonogenic ceils (CFU/dish in the cultures seeded with the highest cell seeding density. Data are expressed as CFE + SD.

THE EFFECT OF MEDIUM COMPONENTS ON CELL ATTACHMENT AND CFE a

Medium Component Relative Relative CeU Number

(Cone.) AttaehmenL % CFE, % Per Colony b

BSA, mg/ml 3.0 1.0 ~ 1.0 c 186 1.0 0.62 + 0.21 a 0.95 -+ 0.09 177 0.3 0.50 + 0.194 0.67 -+ 0.044 212 0.0 0.24 + 0.15 n 0.002 + 0.002 d 156

BPE, %, vol 1.0 1.0 e 1.0 e 173 0.5 1.0 + 0.2 0.14 ___ 0.002 a 89 0.25 1.16 + 0.02 0.04 _+ 0.01 n 48 0.0 0.74 + 0.22 0.14 + 0.03 d 31

CT, ng/ml 10.0 1.0 c 1.0 c 157

1.0 0.96 + 0.13 0.79 --- 0.044 148 0.1 1.0 + 0.16 0.62 --- 0.07 n 200 0.0 0.91 + 0.19 0.59 --- 0.14 114

° For details see Table 1. RTE cells were plated in uncoated culture dishes in CSFM containing indicated concentrations of the individual component tested. Attachment was determined 24 h after seeding, whereas CFE was determined after 7 days.

b Cell number per colony from Day 7 cultures was calculated by dividing cell number per dish by colony number per dish.

BSA concentration in control medium was 3 mg/ml, attachment frequency was 32.9 + 9% and CFE was 4.2 _ 2%.

4 Statistically significant compared to controls based on t test distribution. BPE concentration was 1.0% vol in control medium, attachment fre-

quency was 31.5 -+ 5.6% and CFE was 4.7 + .3%.

abolished and as little as 0.3 mg/ml dramatically increased cell

attachment and colony formation. The number of cells per colony (colony size) was approximately the same in all colonies formed, regardless of the BSA concentration. Removal of BPE from the media had only a slight effect on cell attachment but decreased colony formation and colony size markedly. Removal of CT from the culture media had no effect on cell attachment but significantly

reduced CFE. Colony size did not seem to be affected unless CT was completely removed from the medium. Previously we reported

that the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) markedly increased CFE in RTE cultures grown on irra-

diated 3T3 feeder cells in serum-containing media (12,22). We retested the effect of TPA under the current, serum-free culture conditions and found that it had no effect on cell attachment but increased CFE 3.7- to 4-fold (data not shown) suggesting that under standard culture conditions (i.e. CSFM) only a fraction of the CFUs

are expressed. We also tested the effects of cell seeding density on attachment

and colony formation. This was deemed to be of importance because, as described below, cell seeding density or more accurately seeding density of clonogenic cells (CFU), influenced growth of primary RTE cells in important ways. The data depicted in Fig. 2 summarize the results of four typical experiments carried out at different times over a 20-too. period illustrating the marked experiment-to-experiment variability in CFE of primary RTE cells ranging from 1 to over 5%. These experiments demonstrated that over the 10-fold range of cell seeding densities tested, there was no statistically significant effect on CFE (P > 0.05). This was true whether

IN VITRO REGULATION OF PROLIFERATION OF RTE CELLS 8 0 9

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Fro. 3. Relationship between cell attachment and CFE. Data are a summary of 14 experiments carried out over a 20-mo. period. 5 × 103 to 2 × 104 cells were plated per culture in CSFM. Data represent the mean determined from three to five replicates per point. Slope of the line is -0.024. a y-intercept of 4.44. and a correlation coefficient of 0.074.

the number of clonogenic cells per dish ranged from 22 to 220 CFU or from 91 to 915 CFU (the slight downward trend at 500 CFU per dish and above is thought to be a result of counting errors due to fusion of some colonies).

Because there was such a large interexperiment variation in CFE, we considered whether this was related to differences in attachment frequency from one batch of cells to another. Figure 3 summarizes the relevant data from 14 experiments. It can be seen that moderate (3.5%) to high (>7%) CFEs could be observed even when the attachment frequencies were below 20%; the reverse also occurred, suggesting that there was no close correlation between cell attachment and colony formation (even though attachment is of course a precondition for colony formation).

Factors affecting long-term growth of primary RTE cells in culture. Very little is known about variables affecting late stages of RTE culture growth. Of considerable interest to us was a recent report (37) which suggested that the amount of proliferation in late stage cultures was inversely related to the number of clonogenic cells seeded. We therefore investigated in detail the effects of cell seeding density on the long-term growth of RTE cultures. Figure 4 shows the growth curves of RTE cultures seeded at cell densities of 76, 276, and 640 clonogenic cells per culture, respectively (in these experiments the media were changed every 3 to 4 days). In examining the three growth curves, several features are particularly noteworthy: a) The slopes of the curves during the logarithmic growth phase were similar, suggesting that the cultures were growing at similar rates and more importantly; b) in all three sets of cultures, the peak or plateau of growth occurred at similar levels, namely 3 to 7 × 10 s cells per culture, but at different times, namely, at Days 11 to 12 in the cultures seeded with 276 and 640 CFU per dish and at Day 19 in the cultures seeded with 76 CFU per dish. Figure 5 A,B show that neither of the cultures was confluent at plateau: thus confluency cannot be the reason for cultures ceasing to grow at approximately the same cell density even though cultures were started with significantly different numbers of CFU. When estimating the number of progeny produced per clonogenic cell in the different cultures, we found that those cultures seeded with 76 CFU produced ~ 5 3 0 0 progeny per CFU and the cultures seeded

with 276 and 640 CFU, respectively, produced ~ 1 1 0 0 progeny per CFU (assuming there was no cell loss, see below).

To examine the relationship between clonogenic cells seeded and number of progeny produced in greater detail, another experiment was performed. In addition to counting the number of ceils attached to the culture dish, we also counted the number of cells exfoliated into the media to obtain an estimate of the rate of cell death and a more accurate estimate of the number of progeny per clonogenic cell seeded; in addition we measured DNA synthesis. Cultures were started with either 102 or 878 CFU/dish. To obtain the most accurate count possible of the exfoliated cells, we changed the media every other day instead of every 3 to 4 days, as was done in the previous experiment. The shapes of the growth curves (Fig. 6 A, insert) were similar to those in previous experiments; however, more importantly, growth plateaued at a significantly higher cell number per dish. The effect of the frequency of media change on the growth rate of the cultures seeded with similar numbers of CFUs is illus- trated in Fig. 5 B-D. The cumulative number of cells produced as a function of time by high and low density cultures, taking attached as well as exfoliated cells into account, showed less than a two-fold difference (Fig. 6 A) despite a >eight-fold difference in cell seeding

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FIG. 4. The effect of cell seeding density on the growth of RTE cells in culture. Different numbers of cells were seeded into 60-mm uncoated culture dishes yielding 76 (O), 276 (ZX), and 640 (e) CFU/dish as determined by colony forming assay. Medium was changed every 3 to 4 days. Cell counts were determined at indicated times from three to five replicate cultures and represent the mean -+ SD.

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8 1 0 GRAY ET AL.

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Fic. 5. Plateau phase cultures and the effect of frequency of media change schedule. RTE cells were seeded into uncoated, 60-ram dishes at 2 X 1 0 3 o r 2 )< 104 cells/dish. A, 640 CFU per culture (fixed and stained Day 17); B, 76 CFU per culture (fixed and stained Day 17). In both cultures medium was changed every 3 to 4 days. C, 102 CFU per culture, medium was changed every 2 days (fixed and stained Day 17). D, effect of the frequency of media change on the growth of cultures seeded with 76 or 102 CFU/dish from B and C a b o v e .

density. We measured DNA synthesis in the same cultures. The data summarized in Fig. 6 B indicated that DNA synthesis, expressed as [3H]CPM/10s cells, was maximal on Day 5, dechning steadily thereafter. This decrease in DNA synthesis per unit cell number occurred even though the cultures were still in logarithmic growth phase (Fig. 6 A). In a repeat experiment (data not shown) similar data were obtained, ahhough the rate of DNA synthesis did not begin to decrease until after Day 7. Statistically significant differences in DNA synthesis between high and low cell density cultures occurred between Days 9 and 15.

To estimate the rate of cell death in the cultures we determined the number of cells exfoliated into the media during 2-day intervals (i.e. between media changes). The number of exfohating cells increased rapidly in both sets of cultures between Day 5 and Day 13

from a few hundred cells per culture to 2 to 20 × 104 cells per culture (Fig. 6 C). Thereafter the rate of exfohation remained fairly constant, leveling off at about 105 cells - culture - t " 48-1h. The high cell density cultures always showed a greater rate of exfoliation per culture than the lower cell density cultures. More importantly, the number of exfohated cells as a percent of attached cells was significantly greater, particularly between Days 11 and 13 and to a lesser extent thereafter in high compared to low density cultures (Fig. 6 D). This was also reflected in the frequency of attached cells having a crossed-linked envelope: in cultures seeded at low cell density CLE formation increased from 0.5 to 1.8% from Day 4 to 21 whereas in cultures seeded at high cell density CLE formation increased from 1.3 to 5.1% over the same time span (data not shown).

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IN VITRO REGULATION OF PROLIFERATION OF RTE CELLS 811

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FIG. 6. Effect of cell seeding density on several growth parameters and the rate of cell death in RTE cultures. Uncoated, 60-mm petri dishes were seeded with 102 (O) or 878 (e) colony forming cells. Culture medium was changed every 2 days. At the indicated time points, the following measurements were made: tile number of cells attached to the dish, the number of cells exfoliated into the medium during a 2-day interval, and DNA synthesis measured by [3H]TdR incorporation. All values represent the mean -+ SD from three replicates except for the number of exfoliated cells which was obtained by pooling the medium from three dishes at the time of medium change and counting the number of cells and cell ghosts. DNA synthesis was determined at the indicated time points by incubating the cultures with [3H]TdR for 2 h (i.e. from 46 to 48 h) after medium change. A, attached plus exfoliated cells, insert shows attached cells only; B, DNA synthesis, asterisk indicates statistically significant differences between two sets of cultures; C, exfoliated ceils collected at 2-day intervals; D, ratio of exfoliated ceils expressed as percent of attached cells.

812 GRAY ET AL.

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FiG. 7. Proliferative expansion of CFU in high and low density cultures. Cumulative number of cells generated per CFU was calculated by adding the number of attached cells/per dish at a given time point and the cumulative number of cells having exfoliated into the medium up to that time point and dividing the sum by the number of CFU which were originally seeded per culture. (©) cultures seeded with 102 CFU/dish; (e) cultures seeded with 878 CFU/dish.

To obtain an estimate of the proliferative activity of the clonogenic cells under the conditions of high and low density cultures we calculated the total number of progeny produced per CFU in the two sets of cultures, taking attached as well as exfoliated cells into account (Fig. 7). It can be seen that from about Day 9 onward, the clonal expansion in the cultures seeded at high cell density fell steadily behind that in the cultures seeded at low density. At Day 21 of culture, the cumulative number of progeny produced in high density cultures was ~ 3.9 X 10 z cells per CFU compared to ~ 2.0 )< 104 cells per CFU in low density cultures.

Inasmuch as the above experiments as well as the experiments reported by Terzaghi-Howe (37) suggested that growth inhibitory factors may be produced when cultures reach a certain cell density, we collected media over 48-h periods from cultures plated with 100 to 250 CFUs per dish and with 500 to 800 CFUs per dish. These media were assayed for growth inhibitory effects in colony forming assays using normal primary RTE cells. We found that media obtained from cultures during mid- to late-logarithmic growth phase (1 to 4 × 105 cells per culture) had growth stimulatory activity, causing two- to five-fold increases in CFE. In contrast, media obtained from early to mid-plateau phase cultures (6 to 10 )< 105 cells per

culture) had growth inhibitory activity causing 50 to 90% inhibition of CFE.

DIscussioN

In the RTE cell system, as in many other culture systems, CFE is used as an important parameter to measure proliferative capacity. The assumption is that the prohferating cells migrate litde, grow clonally, and have sufficient repheative potential to produce multicell colonies. We felt that this assumption might not always be correct. We had noticed in previous studies (26) that the CFE of actively growing primary RTE cultures upon subeulturing was quite low (1 to 2%) in spite of a high cell attachment frequency of 30 to 60%. This was true even during mid-logarithmic growth phase when 70 to 90% of the ceils in the primary cultures were undergo- ing DNA synthesis. Thus the question arose about the relationship between the ability of RTE cells to attach, to initiate DNA synthesis, and to form discrete colonies. The experiments showed that in primary RTE cultures most if not all cells entering DNA synthesis during the first 48 h after plating had sufficient proliferative potential to form colonies (which requires a minimum of four to five rounds of cell division). From that it follows that of all the cells seeded to initiate RTE cultures only a small fraction, namely, about 2 to 5%, is important in estabhshing the cultures. The question why highly prohferative RTE cultures have such low CFE when they are subcuhured remains unresolved. It is conceivable that most of the dividing cells are committed and terminally differentiate after a few rounds of cell replication before they can form scorable colonies.

We also examined the relationship between cell attachment and colony formation, investigating the role of different attachment substrata and different factors contained in the serum-free medium. These experiments indicated that cell attachment and CFE, both of which were highly variable from experiment to experiment, were regulated independently of each other. This was evident from the fact that under standard conditions, i.e. CSFM and uncoated culture dishes, the CFE could be high even when attachment frequency was low, and vice versa. Furthermore we found that several factors, namely, FBS, BPE, and CT had little or no effect on cell attachment but gready increased CFE; BSA on the other hand greatly affected cell attachment and had an even greater effect on CFE. We were surprised to find that attachment substrata such as collagen I and IV, fibronectin, and laminin as well as mixtures of substrata such as FAV and Matrigel (data not shown), which are used to enhance cell attachment in many other culture systems (e.g., 3), did not improve cell attachment significantly. Fibronectin was the only substratum causing an increase in CFE, but it did so only when used at very high concentrations. Conceivably the BSA in CSFM so efficiendy promoted attachment as well as prohferation that effects of other factors were overshadowed.

The major question raised by the studies on long-term RTE culture growth is concerned with the nature of the mechanism limiting the expansion of RTE cultures. Several formal possibilities have to be considered: a) normal RTE cells may be programmed to undergo a fixed numer of cell divisions; b) the clonal cell density, i.e. the cell density within each colony, may reach levels high enough to cause contact inhibition of growth (it has to be remembered that in no experiment did the cultures reach confluency); c) diffusible growth inhihitors may be produced and attain inhibitory concentrations once a critical cell number is reached; or d) growth factors and

IN VITRO REGULATION OF PROLIFERATION OF RTE CELLS 813

nutrients present in the media may become limiting. Our data showed that the peak (or plateau) of growth was independent of the number of clonogenic cells seeded. As Fig. 5 A,B illustrate, the cultures grew clonally and never reached confluency regardless of whether 76 or 640 CFUs were plated per dish. Because the data indicated that the total number of progeny per clonogenic cell was much greater in the cultures seeded at low cell density than in the cultures seeded at high density, clonal size and clonal cell density cannot have been the significant factors limiting proliferation in the cultures seeded at high cell density. The same experiments also ruled out the possibility that the number of cell divisions per clonogenic cell is preprogrammed, because both the level of DNA synthesis and the number of progeny per clonogenic cell depended on cell seeding density (and on feeding schedule). The data shown in Fig. 7 suggested that a growth-restraining mechanism began to exert its effect in the high density cultures between Days 7 and 9, i.e. during the late logarithimic growth phase, and between Days 9 and 13 a disproportionate number of cells exfoliated (Fig. 6 D). In low density cultures the reduction in the rate of growth was not observed until Day 15. It is tempting to speculate that a growth inhibitor such as TGF-beta is produced by the cultures, which limits proliferation. However, the results with conditioned media obtained from cultures of high cell density (7.5 × 10 s cells/dish) is equally compatible with depletion of growth factors as a cause for the reduction of growth rate. The situation is likely to be highly complex. That late primary cultures produce TGF-beta was recently reported by Ter- zaghi-Howe (37) and our own laboratory (7,35). Whether this is the only reason for the reduction in growth rate of high density cultures (i.e. > 5 X l 0 t cell/dish) is not entirely clear. Preliminary studies with TGF-beta antibodies suggest that TGF-beta secreted by the cultures may indeed act as an autocrine growth inhibitor (Rundhaug and Nettesheim, 1991 unpublished). On the other hand recent studies with human bronchial cells (19) indicate that with increasing cell density, cultures rapidly loose TGF-beta responsiveness. This would tend to reduce the effectiveness of TGF-beta. Whether this also occurs in RTE cell cultures is presently under investigation. We also know that not only transformed RTE cells but also normal RTE cells produce TGF-alpha (7,32) which can act as an autocrine growth stimulus. Perhaps this is the reason why conditioned media obtained from low density cultures (<5 × l 0 s cells/dish) were growth stimulatory in the CFE assay. With TGF-alpha as well as TGF-beta being produced in the cultures it seems quite plausible that a delicate balance of endogenous growth stimulators and growth inhibitors exist in the cultures, superimposed by the effects of the exogenous growth factors added to the media by the investi- gator. How rapidly the latter are depleted by the cultured cells is not known.

The RTE ceil culture system has been extensively used to investigate mechanisms of neoplastic transformation (for review see 24), to test various compounds for transforming potential (23,33), and others for antineoplastic effects (8, 21, 34). Previous studies (38) suggested that the emergence of transformed RTE cell variants is closely linked to the number of cell divisions allowed to occur before selection is imposed. The effect of cell seeding density on the number of cell divisions per clonogenic cell and on the effect of frequency of medium change on proliferation most likely will signifi- candy impact on the expression of the transformed cell phenotype and probably have important implications for designing cell transformation experiments utilizing this ceil culture model. Studies ex-

ploring these variables and their effects on cell transformation are in progress.

Rrvramcm

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