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.     

Inhibition of early platelet-derived growth factor responses in BALB/c-3T3 cells by interferon

Stimulation of total inositol phosphate production, alteration of cytosolic free calcium [( Ca++]i), vinculin disruption from adhesion plaques, and DNA synthesis caused by PDGF were examined in normal and INF pretreated density arrested BALB/c-3T3 fibroblasts. In normal cells, PDGF caused an increase in total inositol phosphates, a rapid, transient increase in [Ca++]i, disappearance of vinculin from adhesion plaques, and stimulation of DNA synthesis. Pretreatment of cells with INF inhibited PDGF-stimulated increases in [Ca++]i, vinculin disruption from adhesion plaques, and DNA synthesis, but had no effect on PDGF-induced increase in total inositol phosphate levels. These findings suggest that INF prevents entry of quiescent BALB/c-3T3 cells into G1 by inhibiting PDGF-induced release of Ca++ from intracellular stores.     

Biochemical and functional discrimination of platelet-derived growth factor alpha and beta receptors in BALB/c-3T3 cells.

Three biologically active isoforms of platelet-derived growth factor (PDGF) exist: PDGF-AB, the predominant form in human platelets; PDGF-BB, the product of the c-sis protooncogene; and PDGF-AA. PDGF-BB and PDGF-AB interact with two distinct PDGF receptors (termed alpha and beta) of similar size, whereas PDGF-AA binds alpha receptors only. To dissect alpha and beta receptor-mediated signals, we compared the biological activities of PDGF-AA and PDGF-BB in density-arrested BALB/c-3T3 cells, which possess a 4:1 ratio of beta to alpha receptors, and assessed the contribution of alpha receptors to PDGF-BB- and PDGF-AB-induced responses. In addition, we describe a convenient method for resolving alpha and beta receptors on one-dimensional protein gels. This protocol involves treatment of cells with neuraminidase, a desialylating agent, and subsequent in vitro autophosphorylation of solubilized cells, and was used to monitor the presence or absence of alpha and beta receptors under various experimental conditions. Our data show that although higher concentrations were required, PDGF-AA stimulated DNA synthesis to the same extent as did PDGF-BB. Both isoforms induced inositol phosphate formation, epidermal growth factor transmodulation, and PDGF receptor autophosphorylation; PDGF-AA, however, was less effective than was PDGF-BB even at doses causing maximal mitogenesis. Pretreatment of cells with PDGF-AA for 30-60 min at 37 degrees C effectively down-regulated alpha receptors as verified by the absence of desialylated alpha receptor phosphorylation. Depletion of alpha receptors did not affect the capacity of PDGF-BB or PDGF-AB to activate the beta receptor tyrosine kinase, as assessed by tyrosine phosphorylation of an endogenous substrate, or stimulate the formation of inositol phosphates. We suggest that alpha and beta receptors independently mediate similar biological responses in BALB/c-3T3 cells, and that alpha receptors are not required for responses induced by PDGF-BB or PDGF-AB.     

Relationship of cytosolic ion fluxes and protein kinase C activation to platelet-derived growth factor induced competence and growth in BALB/c-3T3 cells

Platelet-derived growth factor (PDGF) and other agents that activate protein kinase C (PKC) rapidly alter cytosolic pH (pHi) and intracellular free calcium ([Ca++]i) in BALB/c-3T3 fibroblasts. To define whether changes in pHi or [Ca++]i are linked to PDGF-stimulated mitogenesis, these parameters were assessed in control and PKC depleted fibroblasts. PDGF addition to BALB/c-3T3 fibroblasts resulted in transient acidification of the cytoplasm followed by prolonged cytosolic alkalinization. Exposure of cells to 12-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester that activates PKC, resulted in cytosolic alkalinization without prior acidification. Overnight incubation with 600 nM TPA decreased the total cell PKC histone phosphorylating activity in BALB/c-3T3 fibroblasts by greater than 90%. In PKC-deficient fibroblasts, TPA, and PDGF-induced alkalinization was abolished. In addition, the transient drop in pHi seen initially in control cells treated with PDGF is sustained to the point where pHi is fully 0.6-0.7 pH units below control cell values for up to 30 minutes. PDGF increased [Ca++]i threefold; this transient rise in [Ca++]i was only minimally affected (less than 15%) by lowering of the extracellular calcium level with ethylene glycol bis(b-aminoethyl ether)0 N,N,N' tetraacetic acid (EGTA) or blocking calcium influx with CoCl2. In contrast, 8-(diethylamine)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an agent thought to inhibit calcium release from intracellular stores, substantially inhibited the rise in [Ca++]i caused by PDGF. TPA and 1-oleoyl-2-acetylglycerol (OAG) increased [Ca++]i but in contrast to PDGF this effect was blocked by pretreatment of cells with EGTA or CoCl2. In PKC-deficient fibroblasts, PDGF still increased [Ca++]i and stimulated DNA synthesis as effectively as in controls. TPA and OAG however, no longer increased [Ca++]i. The continued ability of PDGF to stimulate DNA synthesis in the face of sustained acidification and the absence of PKC activity suggests that cytosolic alkalinization and PKC activation are not essential for PDGF-induced competence in BALB/c-3T3 fibroblasts.     

beta-Interferon alters the pattern of proteins secreted from quiescent and platelet-derived growth factor-treated BALB/c-3T3 cells

Mouse beta-interferon (at a concentration of 100 units/ml or higher) inhibited the platelet-derived growth factor (PDGF)-induced replication of quiescent BALB/c-3T3 cells. The interferon treatment did not inhibit, but slightly enhanced, the accumulation of the following three PDGF-induced RNAs: myc RNA, JE RNA, and KC RNA. The treatment with interferon changed the pattern of secreted proteins from quiescent cells and from cells treated with partially purified PDGF; it inhibited the accumulation of the PDGF-induced proteins (including proteins of 63 and 32 kDa) and it induced the accumulation of several other proteins (including proteins of 89, 31.5, 30, and 10.5 kDa) in both quiescent and also in PDGF-treated cells.     

BALB/c-3T3 fibroblasts resistant to growth inhibition by beta interferon exhibit aberrant platelet-derived growth factor, epidermal growth factor, and fibroblast growth factor signal transduction.

Several lines of evidence now exist to suggest an interaction between the platelet-derived growth factor (PDGF) growth-stimulatory signal transduction pathway and the beta interferon (IFN-beta) growth-inhibitory signal transduction pathway. The most direct examples are inhibition of PDGF-mediated gene induction and mitogenesis by IFN-beta and the effects of activators and inhibitors of the IFN-inducible double-stranded RNA-dependent eIF2 kinase on expression of PDGF-inducible genes. To further investigate the nature of this PDGF/IFN-beta interaction, we selected BALB/c-3T3 cells for resistance to growth inhibition by IFN-beta and analyzed the phenotypes of resulting clonal lines (called IRB cells) with respect to PDGF signal transduction. Although selected only for IFN resistance, the IRB cells were found to be defective for induction of growth-related genes c-fos, c-myc and JE in response to PDGF. This block to signal transduction was not due to loss or inactivation of PDGF receptors, as immunoprecipitation of PDGF receptors with antiphosphotyrosine antibodies showed them to be present at equal levels in the BALB/c-3T3 and IRB cells and to be autophosphorylated normally in response to PDGF. Furthermore, treatment with other peptide growth factors (PDGF-AA, fibroblast growth factor, and epidermal growth factor) also failed to induce c-fos, c-myc, or JE expression in IRB cells. All of these growth factors, however, were able to induce another early growth-related gene, Egr-1. The block to signaling was not due to a defect in inositol phosphate metabolism, as PDGF treatment induced normal calcium mobilization and phosphotidylinositol-3-kinase activation in these cells. Activation of protein kinase C by phorbol esters did induce c-fos, c-myc, and JE in IRB cells, indicating that signalling pathways distal to this enzyme remained intact. We have previously shown that IFN-inducible enzyme activities, including double-stranded RNA-dependent eIF2 kinase and 2',5'-oligoadenylate synthetase, are normal in IRB cells. The finding that the induction of multiple growth-related genes by several independent growth factors is inhibited in these IFN-resistant cells suggests that there is a second messenger common to both growth factor and IFN signaling pathways and that this messenger is defective in these cells.     

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.     

Distribution of intracellular free calcium in quiescent BALB/c 3T3 cells stimulated by platelet-derived growth factor

A digital imaging microscope and fluorescent Ca(2+)-sensitive probe (Fura 2) were used to study the spatial location and time course of increases in free intracellular calcium (Cai) induced by platelet-derived growth factor (PDGF). Microinjection of Fura 2 acid avoided problems of incomplete deesterification of Fura 2-acetoxymethyl ester (Fura 2/AM) and dye localization in cellular organelles. PDGF stimulated a rapid increase in Cai (up to 8-fold increase) in both the nucleus and the cytoplasm in approximately half of the quiescent BALB/c 3T3 cells. Cai changes were both spatially and temporally heterogeneous, the latter including both transient (1-2 min) and prolonged increases (greater than 5 min) in the same cell. PDGF stimulated mitogenesis and Cai increases in approximately the same percentage of cells. Moreover, large intracellular concentrations of a Ca2+ buffer (Quin 2) inhibited both Cai increases and mitogenesis stimulated by PDGF. Thus, Ca2+ increases in the nuclear and/or cytosolic compartments appear to be required for the stimulation of mitogenesis by polypeptide growth factors such as PDGF.     

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.     

Repression of p27kip1 synthesis by platelet-derived growth factor in BALB/c 3T3 cells.

We have investigated the regulation of p27kip1, a cyclin-dependent kinase inhibitor, in BALB/c 3T3 cells during growth factor-stimulated transition from quiescence (G0) to a proliferative (G1) state. The level of p27kip1 protein falls dramatically after mitogenic stimulation and is accompanied by a decrease in cyclin E associated p27kip1, as well as a transient increase in cyclin D1-associated p27kip1 that later declines concomitantly with the loss of total p27kip1. Analysis of metabolically labelled cells revealed that cyclin D2, cyclin D3, and cdk4 were also partnered with p27kip1 in quiescent BALB/c 3T3 cells and that this association decreased after platelet-derived growth factor (PDGF) treatment. Furthermore, the decline in p27kip1 and reduced association with cyclin D3, initiated by the addition of PDGF but not plasma-derived factors, suggested that these changes are involved in competence, the first step in the exit from G0. Synthesis of p27kip1 as determined by incorporation of [35S]methionine was repressed upon mitogenic stimulation, and PDGF was sufficient to elicit this repression within 2 to 3 h. Pulse-chase experiments demonstrated the reduced rate of synthesis was not the result of an increased rate of degradation. Full repression of p27kip1 synthesis required the continued presence of PDGF and failed to occur in the presence of the RNA polymerase inhibitor 5,6-dichlorobenzimidazole riboside. These characteristics demonstrate that repression was a late effect of PDGF and was consistent with our finding that conditional expression of activated H-ras did not affect synthesis of p27kip1. Northern (RNA) analysis of p27kip1 mRNA revealed that the repression was not accompanied by a corresponding decrease in p27kip1 mRNA, suggesting that the PDGF-regulated decrease in p27kip1 expression occurred through a translational mechanism.     

Platelet-derived growth factor stimulation of [3H]-glucosamine incorporation in density-arrested BALB/c-3T3 cells

G0/G1 traverse in density-arrested BALB/c-3T3 cells is controlled by multiple serum-derived growth factors. Platelet-derived growth factor (PDGF) initiates a proliferative response, whereas factors present in plasma facilitate progression through G0/G1. In the absence of competence formation, progression factors are unable to stimulate cell cycle traverse. We have identified the stimulation of a biochemical process specific to competence formation in BALB/c-3T3 cells. PDGF treated BALB/c-3T3 cells incorporated 5-10-fold more [3H]-glucosamine (GlcN) into acid-insoluble material as compared to platelet-poor plasma (PPP) treated cultures. Increased GlcN incorporation occurred in density-arrested BALB/c-3T3 cells in response to treatment with other competence factors, fibroblast growth factor, and Ca3 (PO4)2 and was not due to cell-cycle traverse. Stimulation of [3H]-GlcN incorporation by PDGF was time dependent, and increased incorporation of [3H]-GlcN into protein required de novo protein synthesis. Several mechanisms through which PDGF could increase GlcN incorporation into cellular material were examined. Results of these studies suggest an increase in the cellular capacity to glycosylate proteins is a response to or a part of competence formation.     

Platelet-derived growth factor-induced alterations in vinculin and actin distribution in BALB/c-3T3 cells

Exposure of BALB/c-3T3 cells (clone A31) to platelet-derived growth factor (PDGF) results in a rapid time- and dose-dependent alteration in the distribution of vinculin and actin. PDGF treatment (6-50 ng/ml) causes vinculin to disappear from adhesion plaques (within 2.5 min after PDGF exposure) and is followed by an accumulation of vinculin in punctate spots in the perinuclear region of the cell. This alteration in vinculin distribution is followed by a disruption of actin-containing stress fibers (within 5 to 10 min after PDGF exposure). Vinculin reappears in adhesion plaques by 60 min after PDGF addition while stress fiber staining is nondetectable at this time. PDGF treatment had no effect on talin, vimentin, or microtubule distribution in BALB/c-3T3 cells; in addition, exposure of cells to 5% platelet-poor plasma (PPP), 0.1% PPP, 30 ng/ml epidermal growth factor (EGF), 30 ng/ml somatomedin C, or 10 microM insulin also had no effect on vinculin or actin distribution. Other competence-inducing factors (fibroblast growth factor, calcium phosphate, and choleragen) and tumor growth factor produced similar alterations in vinculin and actin distribution as did PDGF, though not to the same extent. PDGF treatment of cells for 60 min followed by exposure to EGF (0.1-30 ng/ml for as long as 8 h after PDGF removal), or 5% PPP resulted in the nontransient disappearance of vinculin staining within 10 min after EGF or PPP additions; PDGF followed by 0.1% PPP or 10 microM insulin had no effect. Treatment of cells with low doses of PDGF (3.25 ng/ml), which did not affect vinculin or actin organization in cells, followed by EGF (10 ng/ml), resulted in the disappearance of vinculin staining in adhesion plaques, thus demonstrating the synergistic nature of PDGF and EGF. These data suggest that PDGF-induced competence and stimulation of cell growth in quiescent fibroblasts are associated with specific rapid alterations in the cellular organization of vinculin and actin.     

Macromolecular synthesis and stability in growth-arrested BALB/c-3T3 cells

Concentrations of methylglyoxal bis-(guanylhydrazone) (mGBG) that inhibited serum-stimulated BALB/c-3T3 cells in late G1 caused a marked inhibition of 3H-leucine incorporation during a 20-min incubation. No decrease was observed in the incorporation of 3H-uridine during a 20-min incubation; however, the amount of acid-insoluble 3H-uridine in mGBG-treated cultures was decreased when the incubation period was longer than 20 min. The amount of the decrease in the accumulation of incorporated 3H-uridine was directly proportional to the length of the incorporation time. Between 10 and 12 h after quiescent BALB/c-3T3 cells were serum-stimulated in mGBG no additional 3H-uridine was accumulated. The stability of the incorporated 3H-uridine, as determined by acid-insoluble radioactivity remaining after the addition of actinomycin D, was less in cells cultured in mGBG. Exogenous spermine or spermidine reversed the inhibition of 3H-uridine accumulation in acid-insoluble material produced by mGBG as well as the decrease in stability of the incorporated 3H-uridine in acid-insoluble material. The effects of mGBG on both the incorporation of 3H-uridine and the stability of the incorporated 3H-uridine can apparently be accounted for by an effect on ribosomal RNA.     

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.     

Intracellular acidification inhibits the proliferative response in BALB/c-3T3 cells

One of the earliest events to occur upon the addition of serum to quiescent cells is an increase in the intracellular pH (pHin). The relationship between this pH change and proliferation is not known. In the present study, we investigate the consequences of acidifying the cytosol using the weak acid, 5', 5"-dimethyl oxazolidine 2,4-dione (DMO). At a concentration of 50 mM, DMO inhibits the serum-induced increases in pHin, DNA synthesis, and cell number. This concentration of DMO is shown not to inhibit the steady-state rate of mitochondrial respiration and not to inhibit DNA synthesis in a pH-independent fashion. The effects of DMO treatments are also shown to be reversible, indicating that this compound is not cytotoxic. These observations indicate that DMO inhibits cell proliferation by lowering intracellular pH. One important event that must occur prior to the initiation of DNA synthesis is an elevated rate of protein synthesis. The rate of protein synthesis in situ is extremely pH sensitive. Addition of 50 mM DMO to serum-stimulated cultures reduces the rate of leucine incorporation to unstimulated levels. These observations suggest that cytoplasmic acidification may inhibit proliferation through its effects on protein synthesis.     

Cell cycle-dependent gene expression in V point-arrested BALB/c-3T3 cells

Density-arrested BALB/c-3T3 cells stimulated to proliferate in an amino acid-deficient medium arrest in mid-G₁ at a point termed the V point. Cells released from V point arrest require 6 hr to traverse late G₁ and enter S phase. As data presented here show that mRNA synthesis is needed for 2–3 hr after release of cells from the V point, after which inhibition of mRNA synthesis does not prevent entry into S phase, we used this mid-G₁ arrest protocol to analyze gene expression in late G₁. We found that although stimulation of cells in amino acid-deficient medium did not inhibit the induction of genes expressed in early G₁, genes normally expressed in late G₁ were expressed only after release from the V point. The expression of late G₁ genes in cells released from the V point was temporally similar, in respect to G₁ location, as was seen in stimulation of quiescent G_o cells. As this protocol effectively divides gene expression into early (pre-V point) and late (post-V point) categories, it should be useful in studies of growth factor-modulated events that regulate traverse of late G₁ and commitment to DNA synthesis. In addition, we used c-myb antisense oligonucleotides to show that c-myb expression, which occurs in late G₁, is required for BALB/c-3T3 fibroblasts to traverse late G₁ and initiate DNA synthesis. © 1993 Wiley-Liss, Inc.     

Tumorigenicity of morphologically distinct transformed foci induced by 3-methylcholanthrene in BALB/c-3T3 cells

4 mm in diameter), invasiveness (smooth vs. invading margins) and other properties (piling vs. spread). In our previous report, we showed that cells from all five types grew in soft agar, transformed normal NIH 3T3 cells and formed foci on normal layer of BALB/c-3T3 cells. In this study, the neoplastic/tumorigenic potential of cells from the five different types of transformed foci was investigated in nude mice. About two million cells from each transformed focus were injected into 4-week-old nude mice. Non-transformed BALB/c-3T3 cells were used as control. The results of this study indicate that all the 45 athymic mice injected with different transformants developed tumors between 2 and 4 weeks after injection. Tumors were not observed in eight mice injected with non-transformed BALB/c-3T3 cells. All tumors were histopathologically confirmed fibrosarcomas. These findings indicate that all five morphologically different foci show tumorigenicity and that any foci of size > or =2 mm regardless of invasiveness and piling could be scored as positive during the cell transformation assay.     

Transforming and carcinogenic potential of cadmium chloride in BALB/c-3T3 cells

A large number of workers are potentially exposed to cadmium during mining and processing. Therefore, there is a concern regarding the potential carcinogenic hazards of cadmium to exposed workers. Studies have been performed to determine if cadmium chloride (CdCl(2)) can induce morphological cell transformation, DNA from CdCl(2)-induced transformed cells can transform other mammalian cells, and the transformed cells induced by CdCl(2) can form tumors in nude mice. BALB/c-3T3 cells were treated with different concentrations of CdCl(2) for 72 h. The frequency of transformed foci from each treatment was determined after cells were cultured for 4 to 5 weeks. DNAs from five CdCl(2)-induced transformed cell lines were isolated and gene transfection assay was performed using NIH-3T3 cells. Non-transformed BALB/c-3T3 cells and cells from 10 transformed cell lines induced by CdCl(2) were injected into both axillary regions of nude mice. Mice were screened once a week for the appearance and size of tumors. CdCl(2) caused a statistically significant, concentration-related increase in the transformation frequency. DNA from all five CdCl(2)-induced transformed cell lines tested was found to induce varying degrees of transfection-mediated transformation in NIH-3T3 cells. All 10 CdCl(2)-induced transformed cell lines formed fibrosarcomas in nude mice within 39 days of inoculation. Within this time period, no tumors were found in nude mice injected with non-transformed BALB/c-3T3 cells. These results indicate that CdCl(2) is capable of inducing morphological cell transformation and that the transformed cells induced by CdCl(2) are potentially tumorigenic.     

Effects of platelet-derived growth factor on Ca in 3T3 cells

The effect of platelet-derived growth factor (PDGF) on cellular Ca²⁺ was examined in BALB/c-3T3 cells. PDGF induced:

1. 1. A decrease in cell ⁴⁵Ca²⁺ content.

2. 2. An apparent increased rate of efflux of preloaded ⁴⁵Ca²⁺.

3. 3. A decrease in residual intracellular ⁴⁵Ca²⁺ remaining after rapid efflux.

4. 4. When added after the rapid phase of efflux of ⁴⁵Ca²⁺ had occurred, an immediate decrease in post-efflux residual intracellular ⁴⁵Ca²⁺.

All of the observed changes in ⁴⁵Ca²⁺ induced by PDGF are consistent with a rapid release of Ca²⁺ from an intracellular Ca²⁺ pool that has the slowest efflux and is relatively inaccessible to extracellular EDTA. When incubated with chlortetracycline (CTC), a fluorescent Ca²⁺ probe, 3T3 cell mitochondria became intensely fluorescent. Addition of PDGF resulted in a rapid decrease in CTC fluorescence intensity in both adherent and suspended 3T3 cells. The effects of PDGF on 3T3 cell Ca²⁺ stores and CTC fluorescence intensity were identical with the effects of the Ca²⁺ ionophore A23187 and of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone. Serum, which contains PDGF, also altered intracellular Ca²⁺ stores, but platelet-poor plasma, which does not contain PDGF, had no effect. EGF, insulin, and tetradecanoyl phorbol acetate (TPA), other factors which stimulate 3T3 cell growth, did not alter 3T3 cell Ca²⁺ stores. Release of Ca²⁺ from intracellular sequestration sites may be a mechanism by which PDGF Stimulates Cell growth.