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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Inhibition of BALB/c-3T3 cells in late G1: Commitment to DNA synthesis controlled by somatomedin C

Methylglyoxal bis-(guanylhydrazone) (mGBG) blocked the stimulation of DNA synthesis in quiescent, density-inhibited BALB/c-3T3 cells treated with platelet-derived growth factor (PDGF) and platelet-poor plasma (PPP). Competence formation produced by a transient exposure to PDGF was not effected by mGBG. In contrast, mGBG effectively inhibited the PPP-stimulated progression of competent cells through the G₁ phase of the cell cycle, although maximal inhibition was observed when mGBG was present during both the exposure to PDGF- and PPP-supplemented media. When quiescent cells were treated with PDGF and PPP-supplemented media in the presence of mGBG for 12–18 hours and the mGBG was then removed, cells entered the S phase after a 4 hour lag. The rate of entry into the S phase, but not the time necessary for the cells to progress from the mGBG block into the S phase, was dependent on the concentration of PPP present after removal of the mGBG. Either somatomedin C or insulin, but not epidermal growth factor, fibroblast growth factor, or PDGF were able to substitute for PPP in allowing cells to enter the S phase after the cells were released from the mGBG block. A marked inhibition of (³H)-leucine incorporation in serum-stimulated cultures was produced at mGBG concentrations which caused no decrease in the amount of (³H)-uridine incorporated during a short (15 minute) pulse. The ability of hormones to allow cells to progress to the late G₁ phase and become committed to DNA synthesis after a mGBG inhibition was not related to their ability to restore the normal rate of protein synthesis as determined by (³H)-leucine incorporation.     

Effect of heparin-binding growth factors on monovalent cation transport in Balb/C 3T3 cells

The changes in monovalent cation fluxes induced by class 1 and class 2 heparin-binding growth factors purified from bovine brain were studied in confluent quiescent Balb/C 3T3 cells. Both classes of HBGF's rapidly stimulate the activity of the Na+-K+ pump in a concentration-dependent manner that parallels their mitogenic activity. This effect requires the presence of external Na+ and is abolished by 1 mM amiloride. As with other growth factors, HBGF's increase the activity of the Na+-K+ pump by increasing Na+ influx into the cells via the amiloride-sensitive Na+-H+ exchange. These results indicate that rapid activation of cation transport is an early cellular response that follows binding of HBGF's to specific receptors on the surface of target cells.     

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.     

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.     

Increases in cyclic AMP potentiate competence formation in BALB/c-3T3 cells

The ability of platelet-derived growth factor and fibroblast growth factor to stimulate the initiation of DNA synthesis in quiescent BALB/c-3T3 cells was enhanced by cholera toxin. However, the addition of cholera toxin to unsupplemented medium was not mitogenic, nor did cholera toxin increase the mitogenic potential of mediuum supplemented with platelet-poor plasma. The enhancement of serum-induced DNA synthesis by cholera toxin was due to a specific effect on competence formation and not plasma-controlled progression. Cholera toxin increased the rate of competence formation during a transient exposure of quiescent cells to platelet-derived growth factor; this rate was further increased by the addition of isobutylmethylxanthine, a cyclic nucleotide phosphodiesterase inhibitor. Intracellular cyclic AMP concentrations in quiescent BALB/c-3T3 cells were increased 2- to 3-fold after the addition of cholera toxin. The addition of cholera toxin plus 30 m?M isobutylmethylxanthine caused an even greater (7- to 8-fold) increase in the cellular levels of cyclic AMP. That these increases in cyclic AMP concentrations mediated at least part of the increased sensitivity of quiescent cells to competence factors was substantiated by the observation that 0.01 to 1 mM monobutrylcyclic AMP or 8-bromocyclic AMP also caused a concentration-dependent potentiation of competence formation in quiescent cells during a transient exposure to platelet-derived growth factor.     

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.     

Murine mRNA for the beta-subunit of integrin is increased in BALB/c-3T3 cells entering the G1 phase from the G0 state

The amount of murine mRNA for the beta-subunit of integrin is enriched 6-fold when BALB/c-3T3 cells traverse from the G0 state to the G1 phase, whereas it remains at the basal level when the cells are growing continuously. The peak of its appearance is at 10 h after serum stimulation. The increase in integrin mRNA at a specific point in cell proliferation may be correlated with growth-signal transduction.     

A 63 kDa protein is secreted from BALB/c-3T3 cells entering the G1 phase from the G0 state

A 63 kDa protein is detectable in the culture fluid of mouse BALB/c-3T3 cells traversing from the G0 state to the G1 phase, whereas it is undetectable in the culture fluid of quiescent or growing BALB/c-3T3 cells. Secretion of the protein is maximal at 10 h after serum addition. G0-specific ts mutant cells (rat tsJT60) also secrete the 63 kDa protein only when the quiescent cells are stimulated by serum addition at permissive temperature. These facts indicate that the 63 kDa protein is secreted only from cells traversing from the G0 state to the G1 phase.     

Autocrine induction of major histocompatibility complex class I antigen expression results from induction of beta interferon in oncogene-transformed BALB/c-3T3 cells.

By varying growth conditions, we identified a novel mechanism of autocrine regulation of major histocompatibility complex (MHC) class I gene expression by induction of beta interferon gene expression in transformed BALB/c-3T3 cells. Low-serum conditions enhanced MHC class I antigen expression in v-rasKi- and v-mos-transformed BALB/c-3T3 cells but not in untransformed BALB/c-3T3 cells. Transformed and untransformed cells grown under standard serum conditions (10% bovine calf serum) expressed similar cell surface levels of MHC class I antigens. However, low-serum conditions (0.5% bovine calf serum) induced four- to ninefold increases in cell surface levels of MHC class I antigens in both v-rasKi- and v-mos-transformed cells but not in untransformed cells. These increases in MHC class I gene expression were seen at both the mRNA and cell surface protein levels and involved not only the heavy-chain component of the class I antigens but also beta 2 microglobulin. Beta 1 interferon mRNA and beta interferon-inducible 2',5'-oligoadenylate synthetase mRNA were induced by growth under low-serum conditions in transformed BALB/c-3T3 cells, and antibodies to beta interferon blocked the induction of MHC class I antigen expression by serum deprivation in these cells. These results demonstrate that growth under low-serum conditions leads to induction of beta interferon expression in oncogene-transformed cells which then directly mediates autocrine enhancement of MHC class I gene expression.     

Transformed phenotype conferred to NIH/3T3 cells by ectopic expression of heparin-binding growth factor 1/acidic fibroblast growth factor

Summary Heparin-binding growth factor 1 (HBGF-1), also known as acidic fibroblast growth factor, is a potent mitogen and angiogenic factor found in tissues such as brain, kidney and heart. The genomic and cDNA sequences indicate that HBGF-1 does not have a typical signal peptide sequence. HBGF-1 was shown to be localized to the extracellular matrix of cardiac myocytes, but the mechanism of secretion is not presently known. We have cloned the HBGF-1 cDNA which allowed us to directly test the biological activity, mechanism of secretion and transforming potential of the recombinant protein. A previous report showed that the truncated HBGF-1 confers partial transformed phenotype to the recipient fibroblasts. However, expression of full-length HBGF-1 has not been reported. The HBGF-1 coding sequence was cloned into the retroviral expression vector, SVX, and transfected into NIH/3T3 cells. Transfectants expressing full-length HBGF-1 protein at high levels form foci and grow to a higher cell density than the parental NIH/3T3 cells. Western blotting analysis showed that the recombinant HBGF-1 is a unique band of approximately 20 kDa and can be detected in the cell homogenate but not in the conditioned medium. NIH/3T3 cells were conferred anchorage independence when HBGF-1 was provided exogenously. We showed the transformed cells are capable of growing on soft agar even in the absence of exogenously-provided HBGF-1. Transfected cells expressing HBGF-1 also induced tumor formation when injected into nude mice. Thus, NIH/3T3 cells acquired a full spectrum of transformed phenotype when full length HBGF-1 was expressed at high levels. This work was supported by grants from the National Cancer Institute (RO1 CA45611), The March of Dimes Birth Defects Foundation (No. 6-549) and The Ohio Cancer Research Associates, Inc. I.-M.C. is a recipient of The Research Career Development Award (KO4 CA01369) from the National Institutes of Health.