Regulation of platelet-derived growth factor gene expression by transforming growth factor beta and phorbol ester in human leukemia cell lines.

We studied the expression of the genes encoding the A and B chains of platelet-derived growth factor (PDGF) in a number of human leukemia cell lines. Steady-state expression of the A-chain RNA was seen only in the promonocytic leukemia cell line U937 and in the T-cell leukemia cell line MOLT-4. It has previously been reported that both PDGF A and PDGF B genes are induced during megakaryoblastic differentiation of the K562 erythroleukemia cells and transiently during monocytic differentiation of the promyelocytic leukemia cell line HL-60 and U937 cells. In this study we show that PDGF A RNA expression was induced in HL-60 and Jurkat T-cell leukemia cells and increased in U937 and MOLT-4 cells after a 1- to 2-h stimulation with an 8 pM concentration of transforming growth factor beta (TGF-beta). PDGF A RNA remained at a constant, elevated level for at least 24 h in U937 cells, but returned to undetectable levels within 12 h in HL-60 cells. No PDGF A expression was induced by TGF-beta in K562 cells or in lung carcinoma cells (A549). Interestingly, essentially no PDGF B-chain (c-sis proto-oncogene) RNA was expressed simultaneously with PDGF A. In the presence of TGF-beta and protein synthesis inhibitors, PDGF A RNA was superinduced at least 20-fold in the U937 and HL-60 cells. PDGF A expression was accompanied by secretion of immunoprecipitable PDGF to the culture medium of HL-60 and U937 cells. The phorbol ester tumor promoter tetradecanoyl phorbol acetate also increased PDGF A expression with similar kinetics, but with a mechanism distinct from that of TGF-beta. These results suggest a role for TGF-beta in the differential regulation of expression of the PDGF genes.

Escape from senescence in hybrid cell clones involves deletions of two regions located on human chromosome 1q.

Human normal cells have been shown to undergo a limited number of cell doublings, a phenomenon termed cellular senescence. Human chromosome 1 has been implicated in this process, and several lines of evidence indicate that there is a senescence-inducing gene or genes on human chromosome 1q. Our approach to analyze the senescence-inducing effect of chromosome 1 includes the use of somatic cell hybrid revertants. We show here that fusion of a hypoxanthine phosphoribosyl transferase-negative mouse cell line (A9) containing a human neo-tagged chromosome 1 with an immortal hamster cell line (10W-2) results in cell hybrids that senesce after a few population doublings. Rare revertants that had escaped senescence were obtained after one large fusion experiment. Thirty-five nonsenescent hybrids were obtained from a total of approximately 1 million hybrids, and 25 of these were subjected to further analysis. The presence of a single copy of human chromosome 1 in the revertant hybrids was confirmed by fluorescence in situ hybridization analysis using a chromosome 1-specific painting probe. No visible translocations or deletions of chromosome 1 were observed in any of the hybrids. Deletion mapping revealed that 11 (56%) of the hybrids analyzed had lost one or more markers on chromosome 1q. Two regions with deletions were detected, one of which has been shown to be implicated in the senescence-inducing effect exerted by chromosome 1 following monochromosome transfer (P. J. Vojta et al., manuscript submitted for publication). The present study suggests that two separate loci on human chromosome 1q may be of importance for the induction of senescence. Moreover, this set of nonsenescent revertants could be useful for future detailed analyses of the senescence-inducing loci.

Age related induction of platelet-derived growth factor A-chain mRNA in normal human fibroblasts.

We have previously found that stimulation of normal neonatal fibroblasts with PDGF or EGF leads to a transient induction of PDGF A-chain mRNA and the synthesis of PDGF-AA proteins. This finding may imply the existence of an autocrine feedback mechanism to amplify the mitogenic signal under certain conditions. We have now studied the PDGF-BB mediated PDGF A-chain induction in a set of fibroblasts from young and old donors to clarify if the levels of induction are correlated to the donor age and the replicative capacity of the cells. The PDGF A-chain induction was found to be reduced in cells from old donors compared with cells from embryonic and neonatal donors. The different cell strains were also characterized further with respect to PDGF receptor expression and PDGF binding properties. PDGF beta-receptors were found to be enhanced in old donor cell strains, whereas the PDGF alpha-receptors showed more variability in expression between the strains. The PDGF A-chain mRNA induction was also decreased or absent in late passage human fibroblasts (senescent cells) when compared with early passage cells. These data suggest that the PDGF A-chain mRNA induction is regulated by an age related mechanism in human fibroblasts.

Effect on platelet-derived growth factor-induced mitogenesis of double-stranded RNA: evidence for an autocrine growth inhibition mediated by interferon-beta.

Stimulation of normal human foreskin fibroblasts with platelet-derived growth factor (PDGF) was inhibited by the addition of the synthetic double-stranded RNA polyinosinic-polycytidylic acid (poly-I:C) as measured by incorporation of 3H-thymidine (3H-TdR). Single-stranded polycytidylic or polyinosinic acid had no effect. Double-stranded RNA is an inducer of interferon-beta (IFN-beta) in fibroblasts. On the mRNA level, an expression of IFN-beta 2 but not of IFN-beta 1 was seen after addition of PDGF and/or poly-I:C. The inhibition of PDGF-induced mitogenesis was completely blocked by an antiserum to IFN-beta. Poly-I:C did not interfere with PDGF binding to its receptor, nor did it block protein synthesis, indicating that the inhibition is not due to a nonspecific toxic effect of the double-stranded RNA but rather is mediated by IFN-beta. The present study implies that the IFN-beta system in fibroblasts is a very potent autocrine inhibitory pathway.

Density-dependent inhibitory effect of transforming growth factor-beta 1 on human fibroblasts involves the down-regulation of platelet-derived growth factor alpha-receptors.

We have previously found that transforming growth factor-beta 1 (TGF-beta 1) inhibits the mitogenic activity of platelet-derived growth factor (PDGF) in cultures of human neonatal fibroblasts in a density-dependent fashion. In the present investigation we determined the effect of TGF-beta 1 on the PDGF alpha-receptor, which binds all PDGF isoforms, as well as on the beta-receptor, which binds only PDGF-BB with high affinity. We found that the inhibitory effect of TGF-beta 1 on PDGF-AA-induced mitogenesis was density-dependent; when dense cell cultures were preincubated with TGF-beta 1, there was an complete inhibition of 3H-thymidine incorporation, whereas the effect was less in sparse cultures. A similar density-dependent effect of TGF-beta 1 was seen in PDGF-BB treated cells, although less pronounced. The binding of 125I-labeled PDGF-AA and PDGF-BB to the alpha-receptor was significantly reduced after treatment with TGF-beta 1 in dense cultures, whereas the sparse cultures were less affected. A decrease of alpha-receptor mRNA was also seen. The levels of beta-receptor protein and mRNA were unaffected. We conclude that the growth inhibitory effect of TGF-beta 1 is cell density-dependent and involves down-regulation of PDGF alpha-receptors.

Possible positive autocrine feedback in the prereplicative phase of human fibroblasts.

The growth of normal diploid fibroblasts is generally thought to be tightly controlled by exogenous growth factors such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF). Subversion of a growth factor pathway at a regulatory point is considered to be a key event in neoplastic transformation and tumorigenesis. Thus, simian sarcoma virus has acquired the gene encoding the B-chain of PDGF and there is direct experimental proof that SSV-transformation is mediated by a PDGF-like growth factor. There is accumulating evidence that PDGF-like molecules are also synthesized and released by certain normal cells, suggesting an important role of cellularly produced PDGF in development and tissue regeneration. We now present evidence that a transient expression of the gene encoding the PDGF A-chain, and the synthesis and release of functional A-chain homodimers, is an early event in the prereplicative phase of normal human foreskin fibroblasts exposed to PDGF or EGF. Since these cells are PDGF-responsive, the results imply the existence of a positive autocrine signal that may serve as an amplifier of the mitogenic response under certain conditions.

Effects of epidermal growth factor and platelet-derived growth factor on c-fos and c-myc mRNA levels in normal human fibroblasts.

The mRNA levels of two proto-oncogenes, c-fos and c-myc, were determined in human foreskin fibroblasts exposed to epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) in a serum-free, defined medium (MCDB 104). Untreated, quiescent cells were found to have low or undetectable levels of c-fos and c-myc mRNA. Within 10 min after the addition of EGF or PDGF the c-fos mRNA level increased, reached a peak at 30 min, and then declined to the control level after 60 min. The level of c-myc mRNA increased somewhat later and peaked after 8 h in cultures treated with either of the growth factors. The c-myc mRNA level remained elevated throughout the 24 h of investigation. The concentrations of EGF and PDGF required for a maximal effect on c-fos or c-myc expression were found to be similar to those that give maximal effect on cell proliferation. Both c-fos and c-myc mRNA expression were super-induced by the addition of cycloheximide. The addition of neutralizing PDGF antibodies to cultures that had received PDGF 4 h earlier inhibited the subsequent increase in the c-myc mRNA level, indicating that the effect of PDGF on c-myc expression is not caused by a "hit and run," mechanism. Density-inhibited cells responded to EGF and PDGF by an increase in c-fos and c-myc mRNA levels in the absence of any mitogenic response. The present results conform to the view that the c-fos and c-myc proto-oncogenes may be important (or necessary) but not sufficient for the initiation of DNA synthesis. Moreover, the finding that both EGF and PDGF increase c-fos and c-myc expression supports our previous suggestion that these two growth factors may in part act via a common intracellular pathway in the prereplicative phase of human fibroblasts.

Elevated level of gas3 gene expression is correlated with G0 growth arrest in human fibroblasts.

The gas3/PMP22 gene product is a dual function protein, involved in both peripheral nerve myelination and cell proliferation. gas 3/PMP22 is highly expressed in myelinating Schwann cells and is required for normal PNS development. In addition, a more general function for gas3 is suggested by its expression in non-neural tissues and upregulation by growth arrest in cultured rodent fibroblasts. In the present work, the expression of the gas3 gene has been studied in human fibroblasts. We have confirmed that gas3 mRNA is upregulated when cells are serum starved or grown to high cell density (G0 arrest). When quiescent cells were stimulated by serum or platelet-derived growth factor-BB (PDGF-BB), gas3 mRNA was down regulated. In contrast, we found that the expression of gas3 mRNA was neither upregulated in senescent cells nor in cells arrested in G1 using Lovastatin. Thus, high expression of gas3 is not related to growth inhibition in general, but more probably to the G0 growth arrest state. Furthermore, we found that in two malignant fibrous histiocytoma cell lines, gas3 expression was lower than in normal fibroblasts, suggesting an altered regulation of the gas3 gene in transformed cells.

Induction of cyclic AMP synthesis by forskolin is followed by a reduction in the expression of c-myc messenger RNA and inhibition of 3H-thymidine incorporation in human fibroblasts.

We have studied the effect of increased intracellular levels of cyclic AMP on the growth response to platelet-derived growth factor (PDGF) of human foreskin fibroblasts in culture. It was found that forskolin, a potent stimulator of adenylate cyclase activity, inhibits the stimulatory effect of PDGF on 3H-thymidine incorporation with a dose dependence similar to that observed with regard to cyclic AMP formation. A time-course study indicated that forskolin has no effect on ongoing DNA synthesis but affects events in the prereplicative phase. The cell-cycle block induced by forskolin was found to be reversible; after removal of the drug, DNA synthesis was initiated after a lag period, similar to that of the prereplicative phase of control cells. Forskolin had no effect on PDGF binding, receptor autophosphorylation, or c-fos mRNA expression. However, a reduction in PDGF-induced c-myc mRNA expression was observed in cultures given forskolin. Forskolin was also found to have a marked stimulatory effect on the expression of interferon-beta 2 mRNA expression. However, we were unable to demonstrate that the growth-inhibitory effect of forskolin is mediated by interferon-beta. In conclusion, an increase in cAMP levels leads to a reversible inhibition of PDGF-induced DNA synthesis in human fibroblasts, which may be related to an inhibition of c-myc mRNA expression.

Density-dependent inhibition of cell growth by transforming growth factor-beta 1 in normal human fibroblasts.

We report here that transforming growth factor-beta 1 (TGF-beta 1) inhibits platelet-derived growth factor (PDGF)-induced DNA synthesis in normal human fibroblasts in a cell density-dependent manner; no inhibition was seen in sparse cultures, approximately 50% inhibition in confluent cell cultures, and an almost total inhibition in dense cultures. The PDGF-inducible genes c-myc and c-fos were induced also by TGF-beta 1. Simultaneous addition of TGF-beta 1 and PDGF resulted in sustained, rather than transient, expression of c-fos mRNA; c-fos mRNA was detected as long as 24 hr after addition of PDGF and TFG-beta 1. TGF-beta 1 also induced mRNA for the A chain, but not the B chain, of PDGF. Conversely, PDGF induced TGF-beta 1 mRNA in sparse but not in dense cultures. These data indicate the existence of a complex interdependent regulation of PDGF and TGF-beta mRNA expression which is influenced by the cell density.

Tumor necrosis factor-induced expression of platelet-derived growth factor A-chain messenger RNA in fibroblasts.

Recombinant tumor necrosis factor (rTNF) induced a transient increase in the expression of the PDGF A-chain mRNA in FS-4 fibroblasts, as determined by Northern blot hybridization. The mRNA transcripts were of the expected sizes 1.9, 2.3, and 2.8 kb. Increased levels of the PDGF A-chain mRNA were detected 1, 2, and 4 h after addition of rTNF, the highest level being detected after 2 h. After 8 h the mRNA level was similar to that in control cultures. No PDGF B-chain mRNA (c-sis) could be detected following addition of rTNF. There was also an accumulation of PDGF receptor-competing activity in the medium of rTNF-exposed fibroblasts, suggesting that rTNF also induced the synthesis and release of functional PDGF A-chain homodimers (PDGF-AA). The fibroblast growth stimulatory activity in supernatants from rTNF-exposed fibroblasts could be neutralized by antiserum against rTNF, and thus rTNF seemed to be able to act as a direct mitogen. Hence rTNF-induced PDGF-AA is not the main mediator of rTNF's mitogenicity under the conditions investigated.

Production of cell-associated PDGF-AA by a human sarcoma cell line: evidence for a latent autocrine effect.

The alternative splicing of platelet-derived growth factor A-chain is known to result in 2 different protein products. One variant is encoded by transcripts containing the 69 nts representing exon 6 (PDGF-AA(L)), and one variant is encoded by transcripts in which exon 6 is excluded (PDGF-AA(S). Transfection assays have suggested that the long splice variant of the A-chain is mainly associated with membrane- and matrix-associated heparan sulphate proteoglycans, whereas the shorter variant is soluble. We describe a human sarcoma cell line (U-2197) that expresses a high level of PDGF-A transcripts. Immunoprecipitations revealed cell-associated protein products of mainly 24, 28 and 33 kDa and less abundant forms of 40-45 kDa, while no PDGF was found in the medium. Analysis of extracellular medium in a radioreceptor assay confirmed that PDGF was not secreted by the U-2197 cells. The addition to U-2197 cultures of a carboxy terminal peptide that specifically competes with the binding of the long splice variant of PDGF-AA to extracellular matrix and cell membranes resulted in the release of 3 PDGF-AA-specific dimeric proteins with molecular masses of 33, 37 and 45 kDa. Furthermore, polymerase chain reaction studies discriminating between the long and the short splice variants of the PDGF-A transcripts revealed that U-2197 expressed relatively higher amounts of the long splice variant compared with U-343 MGa Cl 2:6, which is known to secrete PDGF-AA. These cell-associated forms of PDGF, released to the medium by adding carboxy terminal peptide, increased the tyrosine kinase activity of the endogenous PDGF alpha-receptor.

Human melanoma cell lines of primary and metastatic origin express the genes encoding the chains of platelet-derived growth factor (PDGF) and produce a PDGF-like growth factor.

Normal human melanocytes and five human melanoma cell lines were analyzed for production of platelet-derived growth factor (PDGF)-like activity. Three of the melanoma cell lines released an activity that inhibited binding of 125I-labeled PDGF to human foreskin fibroblasts and stimulated [3H]thymidine incorporation in such cells. These activities were inhibited by the addition of anti-PDGF antibodies. All three factor-producing cell lines were derived from the same patient--one originated from the primary tumor (WM 115), and two were from individual lymph-node metastases (WM 239A and WM 266-4). The factor produced by WM 266-4 cells was characterized biochemically in detail. Immunoprecipitated, the metabolically labeled factor migrated in NaDod-SO4/gel electrophoresis as a homogeneous Mr 31,000 species, which under reducing conditions was resolved into two species of Mr 16,500 and Mr 17,000, implying a dimeric structure of the molecule. The factor was purified to homogeneity. Analysis by reverse-phase high-pressure liquid chromatography of reduced and alkylated factor revealed an elution pattern identical to that of PDGF A chains. Thus, the native molecule appears to be a homodimer of PDGF A chains. Blot-hybridization analysis of poly(A)+ RNA from the cell lines with 32P-labeled PDGF A chain and B chain (SIS product) cDNA probes revealed a relative abundance of B chain transcripts in the cell line originating from the primary tumor tissue only but expression of A chain in all three cell lines. We conclude that the two structural genes encoding each of the subunit chains of PDGF can be expressed in human melanoma cells and that the two genes can be independently expressed in such cells.

Growth factors induce early pre-replicative changes in senescent human fibroblasts.

As human fibroblasts in culture senesce their response to platelet-derived growth factor (PDGF) becomes attenuated. To clarify at which level such cells are blocked in the pre-replicative part of the cell cycle, we have analysed PDGF-induced pre-replicative events in senescent (phase III) cultures. We found that phase III cells retain a normal number of PDGF receptors and that these are functional with regard to PDGF-induced receptor autophosphorylation. Phase III cells also respond to PDGF by rapid actin reorganization and increased levels of c-fos and c-myc mRNA, similar to growth-arrested phase II fibroblasts. However, the expression of the nuclear antigen K-67, which in phase II cell is induced in S-phase and continues to be expressed throughout the cell cycle, is not induced in phase III cells in response to PDGF. We conclude that phase III human fibroblasts, although blocked with regard to proliferation, still retain a functional growth factor receptor system, and display early responses when exposed to growth factors, such as changes in the cytoskeleton and the expression of proto-oncogenes.