Centriole deciliation associated with the early response of 3T3 cells to growth factors but not to SV40.

BALB/c-3T3 cells which are growth-arrested by high cell density or low serum have ciliated, unduplicated centrioles. Stimulation of these quiescent cells by serum is associated with a rapid (within 1–2 hr) deciliation of the centriole, followed by reciliation within 6–10 hr. This transient deciliation of the centriole is induced by the platelet-derived growth factor (PDGF) component of serum. The cells treated with PDGF became competent to replicate their DNA; most PDGF treated cells, however, did not progress from Go toward S phase unless they were incubated with the platelet-poor plasma component of serum. Addition of CaCl₂ or Fibroblast Growth Factor to the media mimicked PDGF by producing both centriole deciliation and competence to replicate DNA. In fact, over a range of concentrations of each of these factors, only doses which produced centriole deciliation were capable of producing competence for DNA synthesis. Plasma alone or factors such as Multiplication Stimulating Activity produced neither centriole deciliation nor competence; these agents were, however, required for the optimum progression of competent cells into DNA synthesis. In contrast, infection with SV40 induced host cell DNA synthesis without an initial transient deciliation of the centriole. Thus while growth factors may have to induce centriole deciliation for 3T3 cells to synthesize DNA, abortive transformation by SV40 overrides this requirement.     

Post-transcriptional control of protein synthesis in Balb/c-3T3 cells by platelet-derived growth factor and platelet-poor plasma

Platelet-derived growth factor (PDGF) and platelet-poor plasma, which lacks PDGF, both induce a rapid increase in the rate of total protein synthesis within quiescent, density-arrested Balb/c-3T3 cells. This stimulation of protein synthesis is associated with an increased aggregation of ribosomes into polyribosomes. Nuclear functions are not required for this response, as demonstrated by the observation that this stimulation of protein synthesis occurs in cells pretreated with actinomycin D and in enucleated cells (cytoplasts). The response to PDGF persists even after PDGF has been removed from the culture medium, but in contrast, when plasma is removed from the medium, polysomes disaggregate and protein synthesis declines. PDGF and plasma do not function synergistically to increase protein synthesis, whereas they do to induce optimum DNA synthesis. Thus stimulation of the translational apparatus may be necessary for the mitogenic response of Balb/c-3T3 cells to growth factors, but it is not by itself sufficient.     

Control of the Balb/c-3T3 cell cycle by nutrients and serum factors: analysis using platelet-derived growth factor and platelet-poor plasma.

Much controversy regarding the relationship between nutrients and serum in regulation of cell growth can be reconciled by recognizing that serum contains multiple factors which regulate different events in the cell cycle. Serum was fractionated into a platelet-derived growth factor (PDGF), which induces cells to become competent to synthesize DNA, and plasma which allows competent cells to traverse G0/G1 and enter the S phase. Nutrients are not required for the cellular response to PDGF; however amino acids are required for plasma to promote the entry of PDGF-treated, competent cells into S phase. The nutrient independent, PDGF-modulated, growth regulatory event (competence) is located 12 hours prior to the G1/S phase boundary in quiescent, density-arrested Balb/c-3T3 cells. The nutrient dependent, plasma-modulated event is located six hours prior to the G1/S phase boundary and corresponds in concentration of amino acids required for DNA synthesis. Infection of density-arrested Balb/c3T3 cells with SV40 overrides both the nutrient independent and the nutrient dependent growth regulatory events.     

Identification of a BALB/c-3T3 cell protein modulated by platelet-derived growth factor.

The platelet-derived growth factor (PDGF) stimulates density-arrested BALB/c-3T3 cells to synthesize a protein (pII; Mr, 35,000) that is constitutively synthesized by spontaneously transformed BALB/c-3T3 (ST2-3T3) cells which do not require PDGF for growth. Antisera against a major excreted protein family (MEP) of retrovirus-transformed cells quantitatively precipitated cellular pII. PDGF-stimulated pII has the same molecular weight, a similar charge, and similar antigenic determinants as authentic MEP isolated from ST2-3T3 or retrovirus-transformed cells. MEP represented about 2% of the nonnuclear proteins synthesized by ST2-3T3 cells and 0.3 to 0.6% of the proteins synthesized by PDGF-treated BALB/c-3T3 cells, a three- to sixfold increase over the background. In BALB/c-3T3 cells, less PDGF was required for pII (MEP) synthesis than for DNA synthesis. PDGF induced a selective increase in pII (MEP) within 40 min. Such preferential synthesis was inhibited by brief treatment with actinomycin D, suggesting a requirement for newly formed RNA. The constitutive synthesis of pII (MEP) by ST2-3T3 cells was not inhibited by actinomycin D. Five spontaneously or chemical carcinogen-transformed tumorigenic BALB/c-3T3 cell lines were studied; they neither required PDGF for growth nor responded to it. These cell lines became arrested at confluence with a G1 DNA content. Each of these independently isolated lines synthesized pII (MEP) constitutively. Thus, the synthesis of pII (MEP) may be required, but is not sufficient, for PDGF-modulated DNA synthesis.     

The growth requirements of SV40 virus transformed Balb/c-3T3 cells in serum-free monolayer culture

The growth requirements of SV40 transformed Balb/c-3T3 cells have been studied in the absence of serum. For growth in serum-free medium, the cells require (i) insulin, (ii) transferrin, and (iii) cis-unsaturated fatty acids added in combination with fatty acid free bovine serum albumin. The growth rate, saturation density, and morphology of cells grown in this serum-free medium are the same as those of cells grown in serum supplemented medium. This mixture also supports the growth of SV40 transformed Swiss-3T3 cells and SV40 transformed primary mouse embryo cells, but does not support the growth of untransformed Balb/c-3T3 cells. The addition of fibronectin to this mixture allows routine subculture, repeated passage, and indefinite propagation of SV40 transformed Balb/c-3T3 cells. Cells grown in this medium for a period of two months retain their ability to induce tumors when injected into athymic nude mice.     

Control of cytolysis of BALB/c-3T3 cells by platelet-derived growth factor: a model system for analyzing cell death

In culture medium supplemented with 10% clotted blood serum, the saturation density of BALB/c-3T3 cells is determined jointly by cell replication and cell loss. By prelabelling cellular DNA with ³H-thymidine and also by time lapse photography, we studied cell loss independently of replication. Cell loss was accelerated when BALB/c-3T3 cells were transferred from serum-supplemented medium, which contains the platelet derived growth factor (PDGF), to medium supplemented with platelet-poor plasma which lacks it. Loss occurred via the disintegration of cell attached to the surface of the tissue culture dish. Cytolysis of individual cells occurred rapidly; less than 15 minutes transpired between the first indication of a perturbance (by phase contrast microscopy) and fragmentation of the cell cytoplasm. Kinetic analysis was consistent with random cell death rather than a fixed lifetime. The percentage of cells undergoing cytolysis was governed by the cell density; at high densities, such as are present in confluent cultures, a higher percentage of cell loss was noted than at low density. Cell death was antagonized by partially purified or electrophoretically homogenous preparations of-PDGF. Pure PDGF stimulated cell survivial at ng/ml in a concentration dependent fashion. The process of cell replication was not necessary for survival because PDGF prevented cytolysis in the presence of methotrexate, an inhibitor of DNA synthesis. A brief (4 hour) treatment with PDGF prevented cell death; such PDGF treated cells displayed increased survival after being taken up with trypsin and planted onto a fresh surface in plasma supplemented medium. Pituitary fibroblast growth factor, a functional analogue of PDGF for induc of DNA synthesis in BALB/c-3T3 cells, also functioned as an anticytolytic agent. By contrast, epidermal growth factor and insulin did not. Cytolysis of SV40-transformed cells occurred at a constitutively low rate and was insensitive to PDGF.     

PDGF stimulates transient phosphorylation of 180,000 dalton protein

Cell-free extracts of platelet-derived growth factor (PDGF) treated, density-arrested, quiescent BALB/c-3T3 cells are capable of phosphorylating a 180,000 dalton protein (PP180). The phosphorylation of PP180 was observed in SDS polyacrylamide gel electrophoresis profiles of Nonidet P-40 solubilized cell preparations that had been incubated with [gamma-32P]ATP. When quiescent BALB/c-3T3 cell cultures were incubated at 37 degrees C with PDGF, phosphorylation of PP180 in cell extracts could be detected after a 3-min exposure of the intact cells to PDGF, which was maximal after 10-15 minutes and had diminished by 30-60 min. PDGF stimulation of PP180 phosphorylation also was observed in extracts of cells that had been incubated with PDGF at 4 degrees C; however, in contrast to PDGF exposure at 37 degrees C, the ability of cell extracts to phosphorylate PP180 did not decrease even after 4 hr of cell exposure to PDGF at 4 degrees C. When cells exposed to PDGF at 4 degrees C were transferred to 37 degrees C for 30 min, the ability of cell extracts to phosphorylate PP180 decreased to a nonstimulated level. After cells stimulated by PDGF showed a diminished ability to phosphorylate PP180, immediate restimulation with PDGF did not induce the ability to phosphorylate PP180. Incubation for 11 hr at 37 degrees C was required before readdition of PDGF allowed observable phosphorylation of PP180 in cell extracts, but maximum PDGF stimulation of the phosphorylation of PP180 was found after the cells were incubated for 24 hr in culture conditions. The amount of the stimulation of PP180 phosphorylation was dependent on the concentration of PDGF. The stimulation of DNA synthesis by PDGF was correlated to the phosphorylation of PP180. This phosphorylation activity was not observed in extracts of cells that had been treated with epidermal growth factor (EGF), somatomedin C, insulin, plasma, or fibroblast growth factor (FGF). This novel experimental approach allows the investigation of a PDGF-stimulated phosphorylation activity in relation to the cell cycle and growth regulation.     

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.     

Epidermal growth factor (EGF) and somatomedin C regulate G1 progression in competent BALB/c-3T3 cells

The activity in platelet-poor plasma that allowed density-arrested BALB/c-3T3 cells rendered competent by a transient exposure to platelet-derived growth factor (PDGF) to traverse G1 and enter the S phase has been termed progression activity. Epidermal growth factor (EGF) and somatomedin C-supplemented medium was shown to be capable of replacing the progression activity of 5% platelet-poor plasma (PPP) for competent density-inhibited BALB/c-3T3 cells. Exposure of competent cells to medium supplemented with EGF and somatomedin C reduced the 12 h minimum G1 lag time found in plasma-supplemented medium by 2 h. It is suggested that the reduction in the minimum time required for progression through G1 is due to the availability of free, unbound somatomedin C. Complete G1 traverse required both EGF and somatomedin C; however, the traverse of the last 6 h of G1 and entry into the S phase required only somatomedin C. Though EGF and somatomedin C could replace the G1 phase progression activity of plasma, medium supplemented with EGF and somatomedin C did not support complete cell cycle traverse or growth of sparse cultures of BALB/c-3T3 cells.     

Distribution of tyrosinated and acetylated tubulin in centrioles during mitosis of 3T3 and SV40-3T3 cells

We performed a comparative electron microscopic analysis of centriolar and cytoplasmic microtubules stained with antibodies to acetylated or tyrosinated α-tubulin during the cell cycle of mouse nonmalignant Balb 3T3 (clone A31) and virus-transformed heteroploid SV40-3T3 cell lines. It was shown that the pattern of centriole immunostaining changed during the cell cycle in 3T3 (A31) cells, but not in tumorigenic SV40-3T3 cells. Remarkable changes in the centriole immunostaining pattern were observed during interphase-mitosis or mitosis-interphase transitions when the microtubule system and protein organization of centrosomes underwent drastic rearrangements. A high level of tyrosinated tubulin in centrioles was observed at all stages of the cell cycle except when entering mitosis, whereas a high level of acetylated tubulin was visualized in centrioles at all stages of the cell cycle except at the end of mitosis.     

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.     

Similarities between fibroblast-derived growth factor and platelet-derived growth factor

Platelet-derived growth factor (PDGF), one of the most potent mitogens in serum for non-transformed cells, shares many biological and physical properties with fibroblast-derived growth factor (FDGF), a polypeptide produced by BHK cells transformed by SV40. Thus FDGF and PDGF have biological activity which is recoverable from sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, at positions indicating similar molecular weights. Further, the biological activity of both factors is heat-stable but sensitive to mercaptoethanol. FDGF and PDGF have similar abilities to induce DNA synthesis synergistically in the presence of either insulin, epidermal growth factor (EGF), vasopressin or colchicine. In contrast to other growth factors, (i) either FDGF or PDGF can induce DNA synthesis in the absence of other mitogens in 3T3 cells maintained in serum-free medium and (ii) a transient exposure of cultures to FDGF or PDGF causes a persistent stimulation of DNA synthesis. Either FDGF or PDGF enhances colony formation of non-transformed cells cultured in suspension in the presence of EGF and serum. FDGF is not PDGF adsorbed by SV40-BHK cells from serum, since SV40-BHK cells plated and grown in the absence of serum still produce FDGF. In view of the similarities between PDGF and FDGF, we suggest that they may belong to the same family of growth factors.     

Colchicine acts as a progression factor to initiate DNA synthesis in quiescent Balb/c 3T3 cells

In quiescent Balb/c 3T3 cells, competence factors such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and platelet-derived growth factor (PDGF) synergize with progression factors such as insulin to initiate DNA synthesis. In this study, we found that colchicine, a microtubule-disrupting agent, acted synergistically with TPA, but not with insulin, to induce the maximal stimulation of DNA synthesis. Colchicine also synergized with PDGF in the presence of epidermal growth factor to elicit nearly the optimal induction of DNA synthesis. Moreover, it acted synergistically with fibroblast growth factor, another competence factor. These results suggest that colchicine acts as a progression factor like insulin in quiescent Balb/c 3T3 cells.     

Induction of the nuclear protein ''cyclin'' in quiescent mouse 3T3 cells stimulated by serum and growth factors. Correlation with DNA synthesis

The effect of serum and growth factors [platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)] on the synthesis of the nuclear protein cyclin and its correlation with DNA synthesis has been studied in quiescent mouse 3T3 cells by means of quantitative two-dimensional gel electrophoresis. Serum must be present in the medium for at least 8-12 h to induce maximal synthesis of cyclin (6- to 7-fold increase compared with quiescent cells). The stimulation of cyclin synthesis is dose-dependent and correlates directly with DNA synthesis. In addition, partially purified PDGF and FGF also induce cyclin and DNA synthesis in a coordinate way. Both growth factors, like serum, exhibit a similar lag phase to induce maximal cyclin (6- to 7-fold) and DNA synthesis (90% of the cells). Pure PDGF at a concentration as low as 10 ng/ml has the same effect as 10% serum. The coordinate induction of cyclin and DNA synthesis can only be observed with growth factors that induce DNA synthesis. These results strengthen the notion that cyclin is an essential component of the events leading to DNA replication.     

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate enhances the proliferative response of Balb/c-3T3 cells to hormonal growth factors.

Stimulation of Balb/c-3T3 cell growth by TPA requires factors found in serum. We examined the interaction between TPA and serum growth factors in the stimulation of cell growth. The number of cells synthesizing DNA (incorporating 3H-thymidine) within 24 to 30 hours after the addition of TPA and the growth factors to density-inhibited Balb/c-3T3 cultures in serum-free medium was determined by autoradiography. With no additions or with TPA (30--300 ng/ml) alone, only 3--7% of cells synthesized DNA. However, TPA synergistically promoted DNA synthesis in combination with each of the defined serum growth fractions, platelet derived growth factor and platelet poor plasma. TPA also synergistically promoted DNA synthesis in combination with purified growth factors including fibroblast growth factor, insulin (10(-6)--10(-5)M), and epidermal growth factor. In all conditions, TPA enhancement of DNA synthesis also resulted in an increase in cell number. Because TPA synergistically enhanced the activity of each growth factor tested, it did not act identically to any of the growth factors.     

Multiplication of Swiss 3T3 cells in a serum-free medium

Gently trypsinized Swiss 3T3 cells inoculated into medium MCDB 402 attach readily to polylysine-coated surfaces and remain viable for several days in the absence of exogenously added protein. Short-term multiplication under defined conditions can be obtained by supplementing the MCDB 402 with fibroblast growth factor (FGF), insulin (INS), and dexamethasone (DEX). Addition of bovine plasma fibronectin further improves attachment and viability. This system does not require initial plating in serum or the addition of poorly defined extracts for cellular attachment or for multiplication. In the complete system minus FGF, cells plated at a low density attach to the culture surface and become quiescent. The addition of FGF or PDGF 48–72 h after plating stimulates a high level of DNA synthesis during the following 24 h. EGF also stimulates DNA synthesis in these cells, but to a lesser extent. Insulin and dexamethasone are not needed for the initial DNA synthesis response to FGF, but are needed for continuing multiplication over a period of several days. This system provides a means for studying the effects of specific mitogens on Swiss 3T3 cells in the absence of undefined supplements, and without complications due to density-dependent inhibition.     

Somatomedin-C and platelet-derived growth factor stimulate human fibroblast replication

Previous studies have demonstrated that serum contains mitogens, such as platelet-derived growth factor (PDGF), which may alter fibroblast responsiveness to growth factors contained in plasma. Somatomedin-C (SM-C) has been identified as one of the plasma growth factors required for mouse Balb/c 3T3 fibroblasts to initiate DNA synthesis. The present experiments were undertaken to explore the interaction between PDGF, human growth hormone (hGH), SM-C, and other growth-promoting agents in stimulating the growth of human fibroblasts. Proliferation of human dermal fibroblasts plated at low density (3,000 cells/cm²) was found to be equally stimulated by continuous exposure to either normal or somatomedin-C-deficient serum. In contrast, when confluent monolayers were sequentially exposed to PDGF, followed either by normal platelet poor plasma (PPP) or hypopituitary PPP, the cells exposed to normal PPP entered the “S” phase of the cell cycle 50% faster. This difference could be abolished by a 6-hour incubation with growth hormone (10 ng/ml) or somatomedin-C (5 ng/ml) preceding the addition of plasma. When medium containing either hGH or Sm-C was changed frequently so as to remove factors secreted by fibroblasts, only those cells exposed to exogenous somatomedin-C entered DNA synthesis. This finding is in agreement with previous findings that human fibroblasts are capable of making Sm-C in response to hGH. These findings support the hypothesis that somatomedin is required for fibroblast replication in vitro, and that growth hormone appears to stimulate replication indirectly through somatomedin production.