Differential effects of ACTH or 8-Br-cAMP on growth and replication in a functional adrenal tumor cell line

The effects of ACTH and 8-Br-cAMP on growth and replication of a functional mouse adrenal tumor cell line (Y-1) were investigated. ACTH and 8-Br-cAMP both inhibited DNA synthesis and replication when added to randomly growing cell cultures. ACTH addition and serum deprivation each arrested cells in G₁; an additional point of arrest in G₂ occurred with 8-Br-cAMP. Cells whose growth was arrested in G₁ by ACTH had a significantly larger volume and protein and RNA content compared to cells arrested in G₁ by serum deprivation. When ACTH or 8-Br-cAMP was added with serum to cells arrested by serum deprivation, the wave of DNA synthesis and cell division seen with serum was abolished. ACTH and 8-Br-cAMP had no effect on the serum-induced increases in protein and RNA content, rates of leucine incorporation into protein and uridine incorporation into RNA, and RNA polymerase I activity observed in cells during the pre-replicative period. Partial inhibition of the serum-induced increase in uridine transport occurred. ACTH and cAMP do not appear to inhibit replication by generalized negative pleiotypic effects but rather to inhibit the initiation of DNA synthesis more specifically. The ACTH-arrested Y-1 cell resembles an in vivo hypertrophied adrenal cortical cell.     

Inhibition of HeLa-S3 cell proliferation and biosynthesis by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB)

Treatment of HeLa-S3 cells in suspension cultures with 60 microM 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) for 18-30 hr stops the growth of the cell population when treatment is carried out at 37 degrees C in Eagle's spinner culture medium supplemented with 5% fetal bovine serum. The length of the period of no growth after termination of treatment is directly related to the duration of DRB treatment. Upon resumption of growth, the rate becomes exponential and is not distinguishably different from the control rate (doubling time: 19 hr). The growth of the progeny population of the previously DRB-treated cells is as sensitive to inhibition by DRB as the growth of control populations not treated with DRB. After treatment of cells with DRB for 30 hr at 39.5-40 degrees C, the population which grows out has a prolonged doubling time. DRB treatment at 37 degrees C for 5 hr markedly inhibits uridine uptake and cellular RNA synthesis in the presence either of 5 or 15% serum. After treatment for 48 hr in 15% serum, inhibition of RNA synthesis by DRB is significantly decreased. DRB treatment does not inhibit leucine uptake in HeLa cells growing in suspension cultures. Protein synthesis is moderately inhibited in 5% serum and only slightly inhibited in 15% serum after either 5- or 48-hr period of treatment.     

Reversible alterations in the mitotic cycle of chick embryo cells in various states of growth regulation

Chick embryo cells which have been kept overnight at pH 6.8 in the absence of serum multiply very slowly. Only a small fraction of cells is in the S period at any given time, and the rate of uptake of 2-deoxy-D-glucose is very low. Upon raising the pH to 7.4 and adding serum (“turn-on”) the uptake of 2-deoxy-D-glucose increases immediately; the rate of DNA synthesis increases after a lag of about 4 hours, and represents an increase in the fraction of cells synthesizing DNA. The uptake of 2-deoxy-D-glucose is rapidly returned to its original low rate at any time by again lowering the pH and removing serum (“turn-off”). The synthesis of DNA in the culture remains constant or continues to rise at a markedly reduced rate following the same treatment. Lowering pH or removing serum independently of each other is less efficient at inhibiting the increase in DNA synthesis than the combined treatment but each accomplishes a similar result. Cultures which have been “turnedoff” during the early stages of the rapid increase in DNA synthesis, resume their prior rate of increase immediately if “turned-on” again within 2.5 hours. If the cultures have been “turned-off” for 5.5 hours before restoring the “turn-on,” there is a 2 hour delay before they resume an increased rate of DNA synthesis. The results indicate that chick embryo cells do not become committed to the initiation of DNA synthesis until shortly before, or at the time of the onset of the S period. Up to 96% of the cells in post-confluent cultures growing in conventional medium become labeled upon continuous, prolonged exposure to ³H-thymidine. Seventy-eight percent of the cells in serum-deprived cultures growing at a very low rate become labeled. These and other considerations suggest that the inhibition of cell multiplication by high population density or serum deprivation is caused by a lengthening of the time cells remain in the prereplicative G₁ period rather than by shifting cells into a qualitatively distinct G₀ period. There may, however, be a period common to all cells regardless of growth rate, in which cells are not progressing toward the S period. The length of this variable period would then determine the growth rate of a population of cells.     

Lack of a correlation between polyamine synthesis and DNA synthesis by cultured rat liver cells and fibroblasts

Growth stimulation of either fetal rat liver cells or rat embryo fibroblasts in culture results in considerable increases in intracellular polyamine levels as cells proceed through the cell cycle. Treatment of such cell cultures with appropriate levels of two inhibitors of polyamine synthesis, namely α-hydrazino ornithine and methylglyoxal bis(guanylhydrazone), can essentially completely block these increases in cellular polyamine content. Under such conditions, where the elevation in intracellular polyamine content is prevented, cell cultures are nevertheless able to initiate DNA synthesis and subsequently synthesize DNA at rates comparable to untreated control cultures that have been growth-stimulated. These two cell types therefore contain sufficient polyamines when in a resting state (G₁) to enable them to enter from G₁ into S phase and traverse S phase at normal rates in the absence of further polyamine synthesis. The recruitment of cells into the first cell cycle, through serum stimulation of growth, therefore appears not to be mediated or regulated by the increases in intracellular levels of polyamines that occurs under these conditions. Conversely, the arrest of growth of these cell types resulting from serum deprivation is not mediated by a limitation of intracellular polyamine content.     

Magnesium and phosphate enrichment of culture medium stimulates the proliferation of epidermal cells from newborn and adult mice

The proliferation and differentiation of mouse epidermal cells can be sequentially analyzed by modification of extracellular calcium. Newborn cells cultured in low calcium medium (less than 0.1 mM) proliferate as a monolayer and maintain a typical basal cell phenotype in culture but have a limited proliferative capacity and short lifespan. Elevation of the magnesium content of the culture medium from 1 to 5 mM stimulated the proliferation of newborn mouse (1-3 days old) keratinocytes. Maximal DNA synthesis rates, as determined on day 5 of culture, were up to 2-3-fold higher in the magnesium-enriched cultures. Exposure to high magnesium caused 3-4-fold increases in the DNA content of newborn keratinocyte cultures, and extended the confluent phase of epidermal cell growth to over 10 days. Other divalent cations (strontium, copper, zinc, nickel, beryllium, and barium) did not improve keratinocyte growth in culture. Keratinocytes from the tail skin of adult (3 months old) mice displayed an absolute requirement for high phosphate in the culture medium. The medium containing an optimal (10 mM) phosphate concentration prevented the cell detachment caused by the standard low (1 mM) phosphate medium, and in combination with an elevated magnesium content (10-15 mM) it markedly increased both DNA synthesis rates and DNA content of the adult cell cultures. Optimally growing, newborn or adult cultures contained less cells in the G1 phase of the cell cycle and more cells in S and G2 +M. The addition of phosphate and magnesium per se did not induce keratinocyte differentiation and did not interfere with the high calcium (1 mM)-induced differentiation.     

pH, serum and Zn++ in the regulation of DNA synthesis in cultures of chick embryo cells

Variations in pH, serum concentration and the availability of Zn⁺⁺ in the medium markedly influence the initiation of DNA synthesis in cultured chick embryo cells. This report considers the interplay of these factors with one another and with other factors such as type of medium, cell population density and the malignaut transformation in an attempt to better define the variables of the growth control system. Conditioned medium seems to protect the cells against the inhibitory effects of lowered pH. Increased serum concentration has a similar, but more striking effect. Increased serum concentration and pH, as well as decreased population density, which stimulate DNA synthesis, also lower the sensitivity of DNA synthesis to inhibition by Zn⁺⁺ deprivation. Likewise, cell transformation by infection with Rous sarcoma virus lowers the sensitivity of DNA synthesis to inhibition by Zn⁺⁺ deprivation and by pH reduction. The response of DNA synthesis to pH varies with the type and concentration of buffer used. It is concluded that there are a number of mutually interacting variables involved in the regulation of animal cell multiplication.     

Phosphate and the regulation of DNA replication in normal and virus-transformed 3T3 cells.

3T3 cells were cultured in media with different phosphate concentrations and the effects on DNA synthesis were examined. Even a modest phosphate depletion markedly inhibited DNA synthesis and cell multiplication in proliferating cultures. Furthermore, the decrease in the proportion of DNA-synthesizing cells observed after phosphate starvation followed the same time-course as the decrease seen after serum starvation. Cells starved to quiescence in a medium with a 100-fold decrease in phosphate concentration remained viable but non-proliferating for up to 3 weeks, i.e. they had entered a state of quiescence comparable with that seen after serum starvation. Addition of phosphate to phosphate-depleted cultures restored DNA synthesis within 24h. Furthermore, the kinetics of [3H]thymidine labelling after phosphate addition were nearly identical with the labelling kinetics following addition of serum to serum-depleted cultures. In contrast, phosphate deprivation had no inhibitory effects on DNA synthesis in simian-virus-40-transformed 3T3 cells. Furthermore, the inhibitory effects on DNA synthesis in such cells caused by a complete removal of serum could not be further enhanced by decreasing the phosphate concentration in the culture medium.     

Polyamine metabolism, RNA synthesis, and proliferation in density-Inhibited 3T3 cells.

Density-inhibited cultures of 3T3 cells were stimulated with calf serum or with one of 11 other agents reported to cause cells in culture to divide. In agreement with previous studies, activation of polyamine synthesis and cell number increase showed a similar dose-response to calf serum. In contrast, when the results from all agents were considered together, increases in ornithine decarboxylase activity and putrescine and spermidine concentrations correlated poorly with the stimulation of DNA synthesis and proliferation. However, the increases in polyamine parameters correlated highly with the stimulation of rRNA synthesis by both serum and the other agents. These latter results are consistent with previous evidence of a temporal relationship between polyamine and RNA concentrations and synthesis. Increases in polyamine synthesis were not sufficient to cause cell division in resting 3T3 cells, a result similar to previous observations with rat tissues. Also, results with glucocorticoids demonstrate that induction of cell division in resting 3T3 cells does not require activation of either polyamine or RNA synthesis.     

Myocytes and fibroblasts exhibit functional synergism in mixed cultures of neonatal rat heart cells

Cardiac cells obtained from neonatal rat heart contain a mixed population of cell types that can be enriched in culture in either myocytes or fibroblast-like cells. A metabolic comparison of mixed heart cell cultures with enriched cultures of the same age-in-culture and initial cell density showed that mixed cultures used glucose more rapidly than either enriched myocytes or fibroblasts. Mixed cultures were shown to respond to deprivation of insulin and of serum with decreases in the rate of glucose usage and decreases in the protein content of cells, whereas enriched cultures did not respond in the expected manner to insulin deprivation. Mixed, 11-day-old cells also exhibited greater increases in cellular protein and greater resistance to the stress of starvation than enriched cultures. Palmitate usage, however, was similar in all cultures examined. We conclude that mixed cultures may serve as a better model system to study cardiac metabolism and to monitor the effects of drugs and hormones on the neonatal myocardium. In addition, it is clear from our results that myocytes and fibroblastic-like cells coexist in a metabolically functional synergism.     

CHARACTERIZATION OF LYMPHOCYTE TRANSFORMATION INDUCED BY ZINC IONS

Lymphocyte cultures from all normal human adults are stimulated by zinc ions to increase DNA and RNA synthesis and undergo blast transformation. Optimal stimulation occurs at 0.1 mM Zn⁺⁺. Examination of the effects of other divalent cations reveals that 0.01 mM Hg⁺⁺ also stimulates lymphocyte DNA synthesis. Ca⁺⁺ and Mg⁺⁺ do not affect DNA synthesis in this culture system, while Mn⁺⁺, Co⁺⁺, Cd⁺⁺, Cu⁺⁺, and Ni⁺⁺ at concentrations of 10^-7–10^-3 M are inhibitory. DNA and RNA synthesis and blast transformation begin to increase after cultures are incubated for 2–3 days with Zn⁺⁺ and these processes reach a maximum rate after 6 days. The increase in Zn⁺⁺-stimulated lymphocyte DNA synthesis is prevented by rendering cells incapable of DNA-dependent RNA synthesis with actinomycin D or by blocking protein synthesis with cycloheximide or puromycin. Zn⁺⁺-stimulated DNA synthesis is also partially inhibited by 5'-AMP and chloramphenicol. Zn⁺⁺ must be present for the entire 6-day culture period to produce maximum stimulation of DNA synthesis. In contrast to its ability to independently stimulate DNA synthesis, 0.1 mM Zn⁺⁺ inhibits DNA synthesis in phytohemagglutinin-stimulated lymphocytes and L1210 lymphoblasts.     

Relationships between cytoplasmic microtubular complex,DNA synthesis and cell morphology in mouse embryonic fibroblasts (effects of age,serum deprivation,aphidicolin, cytochalasin B and colchicine)

Aging, aphidicolin, serum deprivation and cytochalasin B induce a decrease in the rate of DNA synthesis, an increase in cell flattening (cell surface increase) and an extension of the cytoplasmic microtubular complex (CMTC). Age and experimental conditions affect the protein content of the cell, but there is no relationship between cell morphology and cell protein content. Serum deprivation, aphidicolin and cytochalasin B are more effective on DNA synthesis and cytoplasmic actin complex (CAC) of late than of early fibroblasts. Despite these facts, the cell morphology of late cells is fairly stable and is not affected by experimental conditions, which exert an “aging effect” upon the cell morphology in earlier cultures. Colchicine acts upon the CMTC, cell morphology and DNA synthesis at all ages of the cultures. It also induces disruption of the CAC, the intensity of the disruption depending on both the length of the treatment and the age of the culture: the sensitivity of the actin-microfilaments to colchicine increases with the mitotic age of the cells. We suggest that the microtubular integrity is needed, but not sufficient, to preserve the organization of the CAC into microfilaments. We propose a logical model comprising feedback loops between the number of the mitotic cycles, the rate of DNA synthesis, the extention rate of the plasma membranes and CMTC in normal fibroblasts. CMTC is associated, in this model, with the expression of negative or positive controls, depending on the grade of its extension (Fig. 9).     

Relationships between cytoplasmic microtubular complex, DNA synthesis and cell morphology in mouse embryonic fibroblasts (effects of age, serum deprivation, aphidicolin, cytochalasin B and colchicine)

Aging, aphidicolin, serum deprivation and cytochalasin B induce a decrease in the rate of DNA synthesis, an increase in cell flattening (cell surface increase) and an extension of the cytoplasmic microtubular complex (CMTC). Age and experimental conditions affect the protein content of the cell, but there is no relationship between cell morphology and cell protein content. Serum deprivation, aphidicolin and cytochalasin B are more effective on DNA synthesis and cytoplasmic actin complex (CAC) of late than of early fibroblasts. Despite these facts, the cell morphology of late cells is fairly stable and is not affected by experimental conditions, which exert an "aging effect" upon the cell morphology in earlier cultures. Colchicine acts upon the CMTC, cell morphology and DNA synthesis at all ages of the cultures. It also induces disruption of the CAC, the intensity of the disruption depending on both the length of the treatment and the age of the culture: the sensitivity of the actin-microfilaments to colchicine increases with the mitotic age of the cells. We suggest that the microtubular integrity is needed, but not sufficient, to preserve the organization of the CAC into microfilaments. We propose a logical model comprising feedback loops between the number of the mitotic cycles, the rate of DNA synthesis, the extention rate of the plasma membranes and CMTC in normal fibroblasts. CMTC is associated, in this model, with the expression of negative or positive controls, depending on the grade of its extension (Fig. 9).     

Subdivision of the G1 phase of human glia cells in culture

Human glia cells blocked post-mitotically by serum deprivation require about 8–12 h of continuous stimulation by growth factors to become committed to DNA synthesis. DNA synthesis begins about 5 h after growth factor withdrawal. The length of time until the S phase began and the length of the apparent commitment period, i.e. the time when cells progressed towards the G1/S transition point even in the absence of growth factors were independent of the nature of the growth factors studied (calf serum, platelet-rich human serum, epidermal growth factor). Epidermal growth factor and calf serum were mutually interchangeable during the pre-commitment period. Increasing cell density reduced the number of cells which entered DNA synthesis, but had no effect on the length of the apparent commitment period or the latent time until DNA synthesis commenced. The requirement for a long exposure to a growth factor may be an important safeguard in normal cells against “accidental” entry into the cell cycle, since malignant glia cells do not show the same requirement.     

Suppression of fibroblast proliferation and lysyl hydroxylase activity by minoxidil

The effect of minoxidil on lysyl hydroxylase activity and proliferation of human skin fibroblasts in culture was examined. Exposure of cells to minoxidil resulted in a specific loss of lysyl hydroxylase activity, the extent of which was dependent on the concentration of minoxidil from 25 to 500 microM and the duration of the treatment from 6 to 48 h. This phenomenon was unaffected by culture conditions, i.e. ascorbic acid status, serum concentration, and cell density. Minoxidil added directly to cell extracts had no effect on lysyl hydroxylase activity, showing a requirement for intact cells. Mixing experiments with extracts of minoxidil-treated cells and controls gave additive results which rule out the possibility that a metabolite derived from minoxidil could be inhibiting the enzyme activity. The effect of minoxidil on fibroblast lysyl hydroxylase activity disappeared in the presence of cycloheximide, an inhibitor of protein synthesis. Moreover, the recovery of the enzyme activity that occurred after removal of minoxidil from the culture medium could be prevented by actinomycin D, an inhibitor of RNA synthesis. These results indicate that minoxidil may inhibit the synthesis of lysyl hydroxylase in the cell. In addition to suppressing fibroblast lysyl hydroxylase activity, minoxidil caused inhibition of cell growth within 48 h in a manner dependent on the concentration from 10 to 1000 microM, the latter resulting in almost complete cessation of cell proliferation. This effect was not accompanied by cytotoxicity as judged by the criteria of dye exclusion, plating efficiency, growth recovery, and protein synthesis. The inhibition of fibroblast proliferation by minoxidil appeared to be related to its ability to inhibit DNA synthesis measured by incorporation of tritiated thymidine into acid-precipitable material.     

The effect of dihydrotestosterone and culture conditions on proliferation of the human prostatic cancer cell line LNCaP.

Cell density, nutritional state, and serum factors modify the growth response of LNCaP human prostatic cancer cells to dihydrotestosterone. Evaluation of growth response to dihydrotestosterone requires logarithmic transformation of cell count or thymidine incorporation data. Under conditions of dose response, growth increases with cell density but no significant interaction of dihydrotestosterone with cell density was found under optimal culture conditions. The frequency of media change was a significant factor in modulating dose response. When cells from cultures maintained at different feeding periods were plated at different cell densities of (trypan blue) viable cells, significant effects of plating density on dihydrotestosterone response were found. Dihydrotestosterone protects cells under the adverse effects of media deprivation. Under the extreme adverse effects of serum deprivation, cells respond to dihydrotestosterone even under conditions of increasing cell loss. The effects of dihydrotestosterone on final cell density were significant. In the absence of serum, the elongated cells of LNCaP assume a round shape, but many remain adherent to the culture dish and can be restored to normal morphology by serum. A number of growth factors fail to restore normal morphology that was completely restored by a combination of fibronectin and dihydrotestosterone. We have not developed a practicable serum-free system for LNCaP.     

Effect of specific FSH or LH deprivation on testicular function of the adult rat.

While the need for FSH in initiating spermatogenesis in the immature rat is well accepted, its requirement for maintenance of spermatogenesis in adulthood is questioned. In the current study, using gonadotropin antisera to neutralize specifically either endogenous FSH or LH, we have investigated the effect of either FSH or LH deprivation for a 10-day period on (i) testicular macromolecular synthesis in vitro, (ii) the activities of testicular germ cell specific LDH-X and hyaluronidase enzymes, and finally (iii) on the concentration of sulphated glycoprotein (SGP-2), one of the Sertoli cell marker proteins. Both immature (35-day-old) and adult (100-day-old) rats have been used in this study. Since LH deprivation leads to a near total blockade of testosterone production, the ability of exogenous testosterone supplementation to override the effects of LH deficiency has also been evaluated. Deprivation of either of the gonadotropins significantly affected in vitro RNA and protein synthesis by both testicular minces as well as single cell preparations. Fractionation of dispersed testicular cells preincubated with labelled precursors of RNA and protein on Percoll density gradient revealed that FSH deprivation affected specifically the rate of RNA and protein synthesis of germ cell and not Leydig cell fraction. LH but not FSH deprivation inhibited [3H]thymidine incorporation into DNA. The inhibitory effect of LH could mostly be overriden by testosterone supplementation. LDH-X and hyaluronidase activities of testicular homogenates of adult rats showed significant reduction (50%; P less than .05) following either FSH or LH deprivation. Again testosterone supplementation was able to reverse the LH inhibitory effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

RELATIONSHIPS BETWEEN NUCLEIC ACID SYNTHETIC PATTERNS AND ENCYSTMENT IN AGING UNAGITATED CULTURES OF ACANTHAMOEBA CASTELLANII

Changes in the levels of DNA and RNA syntheses have been studied in unagitated cultures of Acanthamoeba castellanii during the phases of logarithmic multiplication (LM) and population growth deceleration (PGD). Pulse-labeling experiments show that the rate of DNA synthesis decreases at the same time that DNA per cell is known to drop by 50%. The drop in DNA content has been explained by demonstrating with hydroxyurea that the majority of LM amebas can replicate once when DNA synthesis is inhibited and, therefore, must be in G₂, whereas the PGD amebas cannot multiply in the presence of inhibitor and, therefore, must be in G₁. The inhibition of DNA synthesis in LM or PGD cells has been shown to induce encystment. The rate of RNA synthesis, as illustrated by pulse-labeling experiments, increases 25% in late LM-early PGD while RNA per cell increases 75%. The rate of synthesis then decreases 65%. The majority of accumulated RNA has been demonstrated to be ribosomal by disc electrophoresis. By using actinomycin D at different stages during the RNA build-up, the ability of the amebas to encyst has been shown to depend on the presence of this RNA. The observations on DNA and RNA are discussed with respect to the occurrence of cysts in the cultures during PGD.     

Conditional responsiveness of a chemically transformed cell line to growth factor stimulated DNA synthesis

BP3T3, a clonal benzo(a)pyrene-transformed BALB/c-3T3 cell line, is conditionally responsive to growth factor stimulation. Density arrested cell populations deprived of growth factors by pretreatment with 0.5% platelet-poor plasma synthesized DNA both in response to ng/ml concentrations of PDGF, EGF, and somatomedin C, and in response to insulin, plasma, and serum. The above agents acted singly to induce DNA synthesis, but synergism is suggested because a higher percentage of cells were stimulated to enter the S phase when the growth factors were added in combination. Desensitization to growth factors occurred when cultures were pretreated with the high concentration of growth factors present in 10% serum (or plasma). In desensitized cultures none of the above agents, added singly or in combination, stimulated DNA synthesis. This effect appears to be global because pretreatment with one growth factor (e.g., insulin) inhibited the action of another (e.g., PDGF). Cell density appears to play a critical role in regulating DNA synthesis. Unlike nontransformed BALB/c-3T3 cells whose density is regulated by the serum concentration, the density of BP3T3 cells reached a plateau when cultures were grown in a serum (or plasma) concentration of 3% or greater. Such density arrested cultures were growth factor unresponsive; however, the cells rapidly responded to growth factors by synthesizing DNA and replicating when reseeded at a lower cell density. Thus the growth of BP3T3 cells is regulated by both growth factors and cell density.     

The role of increased proteolysis in the atrophy and arrest of proliferation in serum-deprived fibroblasts

When cultured fibroblasts are deprived of serum, the degradation of long-lived proteins and RNA increases, the cells stop proliferating, and they decrease in size. To determine the role of the increased protein catabolism in these responses, we studied the effects of inhibitors of intralysosomal proteolysis in Balb/c 3T3 cells. When these cells were placed in serum-deficient medium (0.5% serum), the rate of degradation of long-lived proteins increased about twofold within 30 min. This increase was reduced by 50-70% with inhibitors of lysosomal thiol proteases (Ep475 and leupeptin) or agents that raise intralysosomal pH (chloroquine and NH4Cl). By contrast, these compounds had little or no effect on protein degradation in cells growing in 10% serum. Thus, in accord with prior studies, lysosomes appear to be the site of the increased proteolysis after serum deprivation. When 3T3 cells were deprived of serum for 24-48 hours, the rate of protein synthesis and the content of protein and RNA and cell volume decreased two- to fourfold. The protease inhibitor, Ep475, reduced this decrease in the rate of protein synthesis and the loss of cell protein and RNA. Cells deprived of serum and treated with Ep475 for 24-48 hours had about twice the rate of protein synthesis and two- to fourfold higher levels of protein and RNA than control cells deprived of serum. The Ep475-treated cells were also about 30% larger than the untreated cells. Thus, the protease-inhibitor prevented much of the atrophy induced by serum deprivation. The serum-deprived fibroblasts also stopped proliferating and accumulated in the G1 phase of the cell cycle. The cells treated with Ep475 accumulated in G1 in a manner identical to untreated serum-deprived cells. Other agents which inhibited protein breakdown in serum-deprived cells also did not prevent the arrest of cell proliferation. Thus the enhancement of proteolysis during serum deprivation appears necessary for the decrease in size and protein synthesis, but probably not for the cessation of cell proliferation. When cells deprived of serum in the presence or absence of Ep475 were stimulated to proliferate by the readdition of serum, the larger Ep475-treated cells began DNA synthesis 1-2 hours later than the smaller untreated cells. Thus, after treatment with Ep475, the rate of cell cycle transit following serum stimulation was not proportional to the cell's size, protein, or RNA content, or rate of protein synthesis.     

Coordinate control of Balb/c3T3 cell survival and multiplication by serum or calcium pyrophosphate complexes.

Balb/c3T3 cells in crowded cultures detach from the dish when deprived of serum, and the survivors incorporate 3H-thymidine at a reduced rate. The detachment becomes pronounced two hours after removal of serum, and reaches its maximum rate between two and four hours. Cells in sparse culture are not detached by serum removal, and their rate of 3H-thymidine incorporation is only slightly reduced. As the sparse cultures grow into more crowded cultures, and the serum is depleted, increasing numbers detach. The detached cells are incapable of reattaching when placed in a new dish with ample fresh serum. The cells are leaky to cellular constituents and appear to be dead. Detachment is a consequence rather than the cause of cell death, and can be produced by agents which inhibit cellular energy metabolism. The cells on the dish which survive serum deprivation are fully viable and grow rapidly when serum is added. When they become crowded they are as sensitive to serum deprivation as was the original population. They are therefore not selected for a low serum requirement but apparently survive because they spread into the space vacated by the detaching cells and then behave as sparse cultures in response to serum variations. Insoluble complexes of Ca2+ and pyrophosphate (Ca2+-PPi) show the same concentration dependence in promoting cell survival as in stimulating 3H-thymidine incorporation, showing that a single substance can be responsible for both activities. It is concluded that survival and growth are part of the coordinate response of 3T3 cells to single external effectors. The results are discussed in terms of a simple model in which the coordinate response is regulated by the availability of Mg2+ for transphosphorylation reactions within the cell, and the availability depends on the binding affinity of cellular membranes for Mg2+. The difference between survival and multiplication is postulated to be in the intensity and duration rather than the kind of stimulus.