Effects of cell density and cell growth alterations on cyclic nucleotide levels in cultured human diploid fibroblasts.

cGMP and cAMP concentrations were studied in cultures of two strains of normal human diploid lung fibroblasts, WI38 and KL-2, under various conditions which alter growth rate. Higher levels of cAMP were found in fibroblasts grown in medium with low (0.1 – 1.0%) serum concentration and thus exhibiting a decreased rate of growth. A rise in cAMP also preceded the decreased growth rate when medium was not changed for 4 days or longer (starvation). The reinitiation of cell growth by addition of fresh medium containing the standard 10% serum to either starved or serum-restricted cells was preceded by a rapid drop in cAMP level. Cellular cAMP levels increased to a moderate extent as sparse cultures first increased in density, but did not continue to rise as the culture approached saturation density. cGMP levels were inversely related to cell density: much higher cellular cGMP levels were found at low density than at higher cell density, whether cells were rapidly proliferating under standard growth conditions or had their growth arrested by omission of medium change or restriction of serum. Thus, under these conditions the steady state levels of cGMP appear to be related to cell density rather than rate of cell proliferation. However, a transient but appreciable increase in cGMP did occur upon the addition of fresh medium containing 10% serum to starved or serum-restricted cells, a condition leading to reinitiation of cell proliferation. Smaller but significant increases in cGMP were also evident following routine addition of fresh medium with serum to growing cells fed every other day and following mild EDTA-trypsin treatment of confluent WI38 fibroblasts. Thus, at least dual control mechanisms appear to be involved in the regulation of cGMP levels. Comparison of mid- and late-passage WI38 cells revealed no significant differences either in the levels of cGMP at sparse densities or in the density-dependent change in levels. These results suggest that levels of both cAMP and cGMP are influenced by cell density and also by conditions which alter the rate of cell proliferation.     

The growth of WI-38 cells in a serum-free,growth factor-free,medium with elevated calcium concentrations

Summary The growth of WI-38 cells in serum-free growth medium with and without hormone supplementation in the presence of elevated Ca²⁺ concentrations was investigated. At 5 mM CaCl₂, WI-38 cells seeded at low density without serum or hormone supplementation showed up to a 12-fold increased in cell number at saturation density over that obtained at day 1. Saturation densities were comparable when either 5 mM CaCl₂ or epidermal growth factor (1 mM CaCl₂) was used in the presence of insulin, dexamethasone and transferrin. Combining suboptimal doses of epidermal growth factor and CaCl₂ resulted in an additive effect on saturation density. Thus, nornal human diploid cells are capable of substantial growth in serum-free, hormone-free growth medium. In contrast, confluent cultures refed with the same medium are not responsive to elevated Ca²⁺ concentrations. In fact, elevated Ca²⁺ concentrations inhibited the proliferative response of confluent cultures to epidermal growth factor, but enhanced their response to the combined treatment of insulin, transferrin and dexamethasone. This work was supported by the United States Public Health Society grants T-32, CA09171 and AG-00378. Editor's Statement This paper rigorously dissects the interplay among external Ca²⁺ concentration, cell density and specific growth factors on fibroblast growth in defined medium. Wallace L. McKeehan     

Umasking large and persistent reductions in proliferation rate of aging cells

Summary We have reported that nontransformed sublines of NIH 3T3 cells that are incubated under the growth constraint of confluence for 10 d or longer exhibit heritable reductions of growth rate upon serial subculture at low density, which simulate the effects of aging in vivo on cell growth. There is also a marked increase in the likelihood of neoplastic transformation. After switching to a new batch of calf serum (CS), we found the reduced growth rate was no longer produced within the previously established timeframe. However, substitution of fetal bovine serum (FBS) for CS during the period of recovery from confluence or the following tests of growth rate resulted in profound inhibition of growth in cells serially subcultured from confluent cultures. In some cases, fewer than one in a thousand cells from subcultures of confluent cultures formed colonies in FBS although they cloned at relatively high efficiency in CS. The reduced growth in FBS was retained in the postconfluent subcultures after many generations of multiplication at low density in CS. Generally, similar results with individual variations were obtained with three other batches of FBS. The numbers of cells per 3-d colony initiated from subcultures of confluent cultures were lower than those of control cultures that had never been confluent. Supplementation of FBS-containing medium with CS fully restored the growth of the postconfluent subcultures to the rate in CS medium, indicating that there is a deficiency of growth factor(s) in FBS rather than the presence of an inhibitor. The results show that prolonged incubation at confluence induces a populationwide heritable increase in requirement for growth factor(s) in short supply in FBS. Because clonal studies have shown that the reduction in growth rate is irreversible and varies in degree from clone to clone, we propose it arises from damage to DNA at any of many different genetic loci or from chromosome aberrations. Such genetic damage is also consistent with the increased tendency for neoplastic transformation in subcultures from the long-term confluent cultures.     

Human epidermal growth factor and the proliferation of human fibroblasts

The effect of human epidermal growth factor (hEGF), a 5,400 molecular weight polypeptide isolated from human urine, on the growth of human foreskin fibroblasts (HF cells) was studied by measuring cell numbers and the incorporation of labeled thymidine. The addition of hEGF to HF cells growing in a medium containing 10% calf serum resulted in a 4-fold increase in the final density. The presence of hEGF also promoted the growth of HF cells in media containing either 1% calf serum or 10% gamma globulin-free serum. The addition of hEGF to quiescent confluent monolayers of HF cells, maintained in a medium with 1% calf serum for 48 hours, resulted in a 10- to 20-fold increase in the amount of ³H-thymidine incorporation after 20–24 hours. The stimulation of thymidine incorporation was maximal at an hEGF concentration of 2 ng/ml, was dependent on the presence of serum, and was enhanced by the addition of ascorbic acid. In confluent cultures of HF cells, subject to density dependent inhibition of growth, hEGF was able to stimulate DNA synthesis more effectively than fresh calf serum. Human EGF stimulated DNA synthesis in quiescent cultures, however, regardless of cell density. The addition of rabbit anti-hEGF inhibited all effects of this growth factor on HF cells.     

Growth requirements and long-term subcultivation of bovine granulosa cells cultured in a serum-free medium

Summary We have developed an improved serum-free medium to optimize the cell growth of bovine granulosa cells. The cells on collagen-coated culture plates proliferated extensively in a nutrient medium supplemented with insulin, heparin binding growth factor-2 (HBGF-2), lipoprotein, and bovine serum albumin (BSA). The cell doubling time at logarithmic phase and final cell density at confluent cultures were equal to those of cultures grown in the presence of medium supplemented with optimal concentration (10%) of fetal bovine serum (FBS). Whereas HBGF-2 or insulin alone had a small mitogenic effect of granulosa cells, lipoprotein or BSA did not. When lipoprotein, BSA, or insulin was added together with HBGF-2, synergistic cell proliferation was observed in all combinations. Insulin or lipoprotein had an additive mitogenic stimulation of these cells in the presence of BSA. After granulosa cells were subcultivated in a serum-containing medium until three generations [8.5 cumulative population doubling level (CPDL)], subsequent subcultivation of the cells in a complete serum-free medium could be achieved up to six generations (14.4 CPDL). These results demonstrate that this serum-free medium can support the optimal cell growth and long-term subcultivation of bovine granulosa cells.     

Modulation of WI-38 cell proliferation by elevated levels of CaCl2

Elevating the level of extracellular calcium (CaEx2+) increases the saturation density achieved by the normal human diploid cell line, WI-38, but does not change the growth rate. Day 7 cell yields remain unchanged when [CaEx2+] is between 0.5 mM and 3.0 mM, decrease when [CaEx2+] less than 0.5 mM, and increase when [CaEx2+] greater than 3.0 mM. Combining hydrocortisone with additional CaCl2 results in an additive effect on the saturation density relative to that obtained with each treatment separately. The stimulatory effect of elevated [CaCl2] is independent of serum concentration but is lost when WI-38 cells are grown in conditioned medium. Stimulation is recovered when conditioned medium is diluted with serum-free medium. In the case of young cultures grown in conditioned medium, stimulation can also be recovered when higher than usual levels of additional CaCl2 are used (2-3 mM). A glutamine supplementation to the conditioned medium potentiates cell response to elevated [CaCl2]. These results indicate that the loss of an enhanced saturation density when cells are grown in conditioned medium is not due to serum depletion but is more likely the effect of metabolites and/or nutrient depletion. When older or less vigorously growing cultures are grown in conditioned medium, additions of up to 3 mM CaCl2 only lead to inhibition, suggesting an age-related change in proliferative regulation. Elevated levels of CaEx2+ also enhance the proliferative response of quiescent monolayers to serum stimulation. This finding, along with the increase in saturation density of Ca2+-treated cultures, suggests that an elevated level of CaEx2+ affects cell entry into and exit from quiescence brought on by density-dependent inhibition.     

Role of serum components in density-dependent inhibition of growth of cells in culture. Platelet-derived growth factor is the major serum determinant of saturation density

The effects of platelet-derived growth factor and plasma components on saturation density in cultures of 3T3 cells were investigated. Both of these components of whole blood serum affect saturation density; however, when 3T3 cells become quiescent at high density in medium containing whole blood serum, only platelet-derived growth factor and fresh whole blood serum are capable of stimulating proliferation. Addition of fresh plasma- derived serum has little effect on cell growth. These results suggest that the platelet factor is the major determinant of saturation density in cultures of 3T3 cells maintained in medium supplemented with whole blood serum. Experiments were performed to investigate the mechanism by which platelet-derived growth factor regulates saturation density. We investigated the possibilities of inactivation of growth factors by proliferating cells, and the effects of cell density on the response of 3T3 cells to platelet-derived growth factor. The amount of platelet- derived growth factor required to initiated DNA synthesis increases with increasing cell density. Some inactivation of growth factors by growing cells was detected, but this depletion was only evident at high cell density. We propose that density-dependent inhibition in cultured 3T3 cells is the result both of an increased requirement for the platelet- derived growth factor as the cultures become more crowded and of inactivation of growth factor activity by growing cells.     

Effect of cultivation conditions on the growth properties of Swiss 3T3 cells

A possibility of using Swiss 3T3 cells, adapted to the growth in the Eagle basal medium and bovine serum, in studies of cell proliferation and quiescent state was shown on the basis of their growth characteristics. Proliferative activity of cultures was estimated by measuring the intensity of DNA synthesis (incorporation of labeled thymidine and flow cytofluorometric analysis), mitotic index and cell number counts. Growth rate and saturation density of the culture were analyzed depending on serum concentration, substrate quality and medium changes both in growth and quiescent states. In spite of repeated medium changes such adapted cells had saturation density within 4.10(4)--7.10(4) cells/cm2, standard for this line. Besides, a distinct inhibition of cell proliferation at confluence or after incubation with low serum (0.5%) and a possibility of the following stimulation of cell divisions by adding a fresh medium containing different concentrations of serum were demonstrated. The increased rate of adipose conversion was detected in resting confluent 3T3 cells cultivated in closed vessels, as compared to cells growing in tissue culture dishes in the CO2 incubator.     

Interaction of estradiol and high density lipoproteins on proliferation of the human breast cancer cell line MCF-7 adapted to grow in serum free conditions

The responsiveness of the human mammary carcinoma cell line MCF-7 to estradiol and tamoxifen treatment has been studied in different culture conditions. Cells from exponentially growing cultures were compared with cells in their initial cycles after replating from confluent cultures ("confluent-log" cells). It has been observed that estradiol stimulation of tritiated thymidine incorporation decreases with cell density and that "confluent-log" cells are estrogen unresponsive for a period of four cell cycles in serum-free medium conditions. On the other hand, growth of cells replated from exponentially growing, as well as from confluent cultures, can be inhibited by tamoxifen or a combined treatment with tamoxifen and the progestin levonorgestrel. This growth inhibitory effect can be rescued by estradiol when cells are replated from exponentially growing cultures. The growth inhibitory effect cannot be rescued by estradiol alone (10(-10) to 10(-8) M) when cells are replated from confluent cultures. In this condition, the addition of steroid depleted serum is necessary to reverse the state of estradiol unresponsiveness. Serum can be replaced by high density lipoproteins but not by low density lipoproteins or lipoprotein deficient serum. The present data show that estradiol and HDL interact in the control of MCF-7 cell proliferation.     

Influence of increased extracellular calcium concentration and donor age on density-dependent growth inhibition of human fibroblasts

Elevated calcium chloride concentration [( CaCl2]) has been shown to increase saturation density for an established mouse fibroblast line and for human fetal lung fibroblasts (WI-38). In order to examine the effect of increased [CaCl2] on human fibroblasts from donors of varying age, fibroblasts were grown in medium (basal level of 1.8 mM CaCl2) supplemented with fetal bovine serum (FBS) until confluent. Compared to controls in basal medium, newborn foreskin fibroblasts exposed to additional CaCl2 had a 110-450% increased cell yield that was independent of [CaCl2] within the range of an additional 1.5-5.0 mM. The effect was maintained over an eightfold range of FBS concentration. Initial growth rate was unaffected, but a prolongation of exponential phase occurred for cultures exposed to increased [CaCl2]. Confluent cultures refed medium with increased [CaCl2] were stimulated 5- to 10-fold more than cultures refed basal medium. An additional 2 mM CaCl2 resulted in a 210% increase for young adult-derived fibroblasts versus a 29% increase for old adult-derived fibroblasts (P less than 0.001). These data indicate that increased [CaCl2] decreases density-dependent growth inhibition of postnatal human dermal fibroblasts in vitro and that this effect is donor age dependent.     

Initiation of Cartilage Cell Culture from Skate (<Emphasis Type="Italic">Raja porasa</Emphasis> Günther)

Abstract Cartilage tissues from the proboscis of skate (Raja porasa Günther) were used to initiate primary cultures of cartilage cells. Aseptically dissected cartilage tissues were immersed in MEM medium free of fetal bovine serum (FBS), pH 7.6, and minced into small pieces (1 mm³ on average). After hydrolysis with collagenase II, hyaluronidase, and trypsin for 2 hours at room temperature, the acquired cartilage cells were rinsed twice with 20% FBS-supplemented MEM medium and then inoculated into 25-cm³ cell culture flasks, and incubated at 24°C. The primary cultures were initiated successfully, and the cartilage cells grew gradually into a confluent monolayer at day 10. Effects of growth factors were also tested in this study, and it was found that 20 ng/ml of basic fibroblast growth factor and 100 ng/ml of insulin-like growth factor II together had the most prominent stimulating effect on the growth and division of cartilage cells in the series of concentration combinations employed. The induced cartilage cells cultured formed a confluent monolayer at day 7.     

Shaking 3T3 cells: further studies on diffusion boundary effects

Confluent quiescent cultures of 3T3 cells can be stimulated to grow—with or without a medium change—simply by shaking (20 mm excursions, 3 per sec). Shaking without medium change, or with low serum concentration in fresh medium (4%), has the greatest effect on thymidine labeling and mitotic index compared with unshaken cultures, but high serum concentration (20%) stimulates both shaken and unshaken cultures equally. Even without medium change, the majority of cells can be stimulated to incorporate thymidine and to divide over 3 days, though initial shaking for 7 hr is sufficient to initiate growth. We conclude that growth in confluent 3T3 cell cultures is limited by a diffusion barrier in the microenvironment, probably affecting uptake of a serum component. The limitation can be overcome by increased fluid velocity as an alternative to raising the serum concentration.Shaking also causes increased cell movement and a change in cell morphology (which may be partly due to alterations in serum caused by shaking). Despite increased movement in the cell sheet, emigration from the border of a wound is diminished by shaking, suggesting that chemotaxis may be involved.To test whether loss of contact between the migrating cells is essential for growth stimulation at the border of a confluent layer, wounds were made in unshaken cultures in the presence of cytochalasin B, which prevents locomotion of cells and maintains the original topography. Nevertheless, the thymidine labeling index was still increased along the wound boundary, and included cells which were near but not on the edge. This supports the view that short range effects on cell growth at discontinuities in cell populations can be due to modification of a diffusion barrier and not simply alteration in cell contacts.     

Long-term cultures of chick embryo fibroblasts transformed by the Schmidt-Ruppin strain (D) of Rous sarcoma virus.

Attempts have been made to keep in vitro, for extended periods of time, cultures of chick embryo fibroblasts transformed by the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup D. Roller cultures of transformed chick cells kept in serum-deficient medium can be maintained without subcultivation for up to 6 months. The confluent cultures continuously release viruses and viable tumor cells into the medium. The released cells can be plated and have characteristics of growth and morphology which are relatively stable with time until the culture degenerates. Cells released at later stages of the culture produced substantially more viruses than those released earlier, suggesting that cell selection or differentiation occurs during long-term cultivation in low serum concentration. Long-term cultures of untransformed chick embryo fibroblasts can also be maintained in the same way. The release of viable cells by these confluent cultures, however, is negligible.     

Cell-cell contact and growth regulation of pinocytosis in 3T3 cells

In sub-confluent cultures of Balb/c-3T3 cells, pinocytosis rates were increased after exposure to specific growth factors (serum; platelet-derived growth factor, PDGF; epidermal growth factor, EGF). Conversely, as cells became growth-inhibited with increasing culture density, there was a corresponding decline in pinocytosis rate per cell. In order to test whether density-inhibition of pinocytosis was influenced either by the growth cycle or by cell contact independently of growth, cells were induced into a quiescent state at a range of subconfluent and confluent densities. Under such conditions, cell density did not significantly inhibit pinocytosis rate. When confluent quiescent cultures in 2.5% serum were exposed to 10% serum, the resulting round of DNA synthesis was accompanied by enhanced pinocytosis per cell, even though the cells were incontact with one another. Furthermore, in a SV40-viral transformed 3T3 cell line, both the growth fraction and the pinocytosis rate per cell remained unchanged over a wide range of culture densities. These studies indicate that density-dependent inhibition of pinocytosis in 3T3 cells appears to be secondary to growth-inhibition rather than to any direct physical effects of cell–cell contact.     

Regulation of the cell cycle of 3T3 cells in culture by a surface membrane-enriched cell fraction

Addition of a suspension of a surface membrane enriched fraction prepared from confluent 3T3 cells to sparse 3T3 cells in culture results in a concentration dependent and saturable decrease in the rate of DNA synthesis. The inhibition of cell growth by membranes resembles the inhibition of cell growth observed at confluent cell densities by a number of criteria: (1) In both cases the cells are arrested in the G₁ protion of the cell cycle; (2) the inhibition by membranes or by high local cell density can to a large extent be compensated for by raising the serum concentration or by addition of fibroblast growth factor plus dexamethasone. Membranes prepared from sparse cultures inhibit less well than membranes from confluent cultures in a manner which suggests that binding of membranes to cells is not by itself sufficient to cause inhibition of cell growth. The inhibitory activity has a subcellular distribution similar to phosphodiesterase (a plasma membrane marker) and appears to reside in one or more intrinsic membrane components. Maximally, membranes can arrest about 40% of the cell population in each cell cycle. Plasma membranes obtained from sparse 3T3 cells are less inhibitory than membranes obtained from confluent cells. This suggests either that the inhibitory component(s) in the plasma membrane responsible for growth inhibition may be in part induced by high cell density, or that this component(s) may be lost from these membranes during purification.     

Changes in adenosine deaminase activity in ageing cultured human cells and the role of zinc

The level of adenosine deaminase (ADA; EC 3.5.4.4) was estimated at different passages in six confluent fibroblast cultures established from forearm skin biopsies of healthy adult normal volunteers. After determination of the zinc concentration in standard growth medium, ADA activity was estimated at different passages of subculture in media with different zinc concentrations. The results indicated that the specific activity of ADA in control confluent skin fibroblast cultures (passage 2) cultivated in standard growth medium containing 15.4 microM zinc (similar to that present in normal human plasma) was equal to 226.6+/-19.64 micromol min(-1) mg(-1) protein. The results showed that there were no significant changes in ADA specific activity in any of the control cultures as the zinc concentration of the medium was increased. To characterize the passage of subculture at which fibroblasts enter the ageing phase, three marker enzymes were assayed namely, phosphofructokinase, lactate dehydrogenase and glycogen phosphorylase. The result showed that the cells enter the ageing phase at passage 20 and beyond. Further investigation showed that ADA activity of serially subcultured confluent cultures cultivated in standard growth medium significantly dropped at passages 20, 25 and 30. ADA activity however was not significantly altered in cells at passage 2, 10 and 15 cultivated in standard growth medium and in the presence of higher zinc levels (23.1, 34.6, 53.8 and 73.1 microM). Furthermore there was significant lowering of ADA activities in cells at passages 20, 25 and 30 when cells were cultured in the presence of 15.4, 23.1 and 34.6 microM zinc. Such lowered activities of ADA were restored to normal when the cells were cultured in the presence of higher zinc concentration equal to 53.8 and 73.1 microM. From the results we concluded that it is possible to restore ADA activity in aged skin fibroblasts to normal levels by raising the zinc concentration in the culture medium to four or five times the control normal plasma zinc level.     

Modulation of the platelet-derived growth factor induced replicative response

Quiescent cultures of density arrested BALB/c-3T3 cells have been sensitized to the growth stimulatory action of the platelet-derived growth factor (PDGF). Sensitization was achieved by depriving the cultures of PDGF prior to growth stimulation and was noted after transfer of cultures from medium supplemented with 10% serum to medium containing either an equivalent concentration of platelet-poor plasma or a low concentration (0.5%) of serum. Sensitized cultures required less pure PDGF for growth stimulation than nonsensitized ones. In addition such cultures required less mitogen to synthesize a PDGF modulated major excreted protein (MEP). The mechanism of sensitization was investigated. Sensitized cultures did not bind more PDGF than non-sensitized ones. Rather, sensitization appeared to result from the loss of cells that occurred when cultures were deprived of PDGF. Such a loss increased the amount of PDGF available per cell, causing a higher percentage of cells to enter the S phase. Similarly, the amount of PDGF per cell regulated MEP synthesis. Furthermore, in non-sensitized cultures (containing the same number of cells), the absolute quantity rather than the concentration of PDGF regulated DNA synthesis. It appears that the amount of PDGF per cell modulates mitogenesis.     

Modulation of Ca2(+)-dependent intercellular adhesion in bovine aortic and human umbilical vein endothelial cells by heparin-binding growth factors

Cultured endothelial cells have been shown to possess two mechanisms of intercellular adhesion: Ca2(+)-dependent and Ca2(+)-independent. We report here that growth of bovine aortic endothelial cells (BAEC) in complete medium containing purified basic fibroblast growth factor (bFGF, 6 ng/ml) results in loss of Ca2(+)-dependent intercellular adhesion. In the presence of heparin (90 micrograms/ml), this effect is reproduced upon treatment with acidic fibroblast growth factor (aFGF, 6 ng/ml) or endothelial cell growth supplement (ECGS, 100 micrograms/ml), in both human umbilical vein endothelial cells (HUVEC) and BAEC. Treatment at these doses with aFGF in the absence of heparin or with heparin alone is without significant effect. Loss of Ca2(+)-dependent adhesion following treatment of cells with heparin-binding growth factors (HBGFs) is prevented by pre-treatment of cell layers with cycloheximide. The Ca2(+)-independent adhesion mechanism is unaffected by HBGF treatment. Exposure of endothelial cells to HBGFs, moreover, prevents the eventual establishment of quiescence in growing cultures and restimulates replication in confluent cultures that have reached a final density-inhibited state. Addition of bFGF alone or aFGF + heparin at these doses results in a 4-fold increase in DNA synthesis over untreated control cultures at saturation density as reflected by thymidine index. A single addition of bFGF (6 ng/ml) to untreated quiescent confluent BAEC monolayers results in an increase in 3H-TdR incorporation reaching a peak at 22 hours with a parallel loss of Ca2(+)-dependent adhesiveness. Fluorescent staining with rhodamine-phalloidin demonstrates an altered distribution of polymerized F-actin in the bFGF-treated monolayers, marked by disruption of the dense peripheral microfilament bands retained by untreated confluent monolayers. Together, these results indicate that the mitogenic effect of HBGFs in cultured endothelial cells is associated with a "morphogenic" set of responses, perhaps dependent on breakdown of calcium-dependent cell-cell contacts.     

Critical variables controlling cell proliferation in primary cultures of rat tracheal epithelial cells

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.