The receptor for epidermal growth factor (EGF) is a single-chain transmembrane polypeptide of relative molecular mass (Mr) 170,000 (170K) which has been implicated in the regulation of both normal ...and abnormal cell proliferation. It has an externally facing EGF-binding domain and a cytoplasmically facing tyrosine-specific protein kinase site. Although the receptor has been well characterized, the mechanism by which it transmits the growth stimulatory signal from the plasma membrane to the nucleus is unclear. EGF binding to cells has been shown to enhance topoisomerase activity within the cells. Topoisomerases catalyse the interconversion of topological isomers of DNA and thus may influence replication and transcription. Mroczkowski et al. reported that purified EGF receptors of both human and murine origin can nick supercoiled double-stranded (ds) DNA in an ATP-dependent fashion, an activity related to those of topoisomerases. Another related tyrosine kinase, pp60src, has also been reported to have a similar DNA-nicking activity. We have now characterized the EGF receptor-associated DNA-nicking activity by sucrose gradient centrifugation. Our results, presented here, indicate that the DNA-nicking activity is not intrinsic to the EGF receptor, but is found in a distinct molecular species.
The platelet-derived growth factor (PDGF) modulated growth response of the MG-63 human osteosarcoma cell line, which neither expresses c-sis mRNA nor secretes a PDGF analogue, was characterized. ...Scatchard analysis demonstrated that the MG-63 cells have 23,000 receptors per cell with a Kd of 5 X 10(-11) M. The receptor became phosphorylated, in a PDGF concentration-dependent manner, when 32P-orthophosphate-labeled cells were treated with PDGF for 3 h at 4 degrees C. The phosphorylated receptor was identified by autoradiography and gel electrophoresis after isolation of the 32P-labeled receptor using a solid-phase monoclonal antibody directed against phosphotyrosine. Binding of the receptor to the antibody was inhibited by 5 mM phenyl phosphate, further suggesting that PDGF stimulated tyrosine-specific receptor autophosphorylation. In addition, treatment of MG-63 cells with PDGF for 3 h at 37 degrees C induced a 7.5-fold increase in c-myc mRNA accumulation as analyzed on Northern gels. However, MG-63 cells grew equally well in either serum-(which contains PDGF) or plasma-(which does not) supplemented medium. Furthermore, PDGF did not stimulate DNA synthesis in growth arrested MG-63 cells, nor did it potentiate DNA synthesis modulated by somatomedin C. Thus MG-63 cells are a naturally occurring cell variant in which PDGF stimulates c-myc expression but does not modulate mitogenesis.
Treatment of quiescent MG-63 cells with 12-0-tetradecanoylphorbol-13-acetate (TPA) or platelet-derived growth factor (PDGF) stimulates the rapid accumulation of c-myc RNA. We have now determined that ...a similar effect can be induced by cAMP. Treatment with forskolin (an activator of adenylate cyclase), IBMX (a phosphodiesterase inhibitor), PGE
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, and isoproterenol stimulated accumulation of both cAMP and c-myc RNA, but no increase in either cAMP or c-myc RNA was seen with the inactive forskolin analog 1,9-dideoxyforskolin. Forskolin and IBMX acted synergistically in stimulating accumulation of both cAMP and c-myc RNA. However, three lines of evidence indicated that PDGF action is not mediated by cAMP. First, PDGF treatment caused no elevation of cAMP within 1 h, even in the presence of IBMX. Second, the kinetics of c-myc RNA elevation after treatment with PDGF or forskolin were similar, ruling out delayed onset of cAMP stimulation. Finally, simultaneous treatment with forskolin and the calcium ionophore A23187 enhanced the elevation of c-myc RNA levels; no such effect was seen with PDGF. We had previously shown that PDGF action is not affected by prior treatment of MG-63 cells with TPA, a treatment which desensitizes the c-myc response to TPA. Similarly, TPA pretreatment had minimal effect on forskolin or IBMX-induced c-myc expression. These data suggest that cAMP, phorbol esters, and PDGF act independently to stimulate c-myc RNA expression in MG-63 cells. However, nuclear runoff experiments and RNA half-life measurements demonstrated that PDGF, phorbol ester, and cAMP all act to increase the transcription of the MYC gene.
Platelet-derived growth factor (PDGF) stimulates density-arrested BALB/c-3T3 cells to synthesize MEP, a lysosomal protein. This enhanced synthesis appears to be largely regulated by the ...PDGF-modulated accumulation of MEP mRNA, a 1.8-kilobase species. The increase in the MEP transcript, which is dependent on the PDGF concentration, begins 3 to 4 h after PDGF addition and is maximal at 12 h. The accumulation of the MEP transcript is growth-factor specific: PDGF and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, an agent which acts like PDGF, induce MEP RNA accumulation, whereas epidermal growth factor, somatomedin C, insulin, and whole plasma do not. A spontaneously transformed BALB/c-3T3 cell line (ST2-3T3), which does not require PDGF for growth, optimally expresses MEP RNA in the absence of PDGF. The PDGF-modulated increase in MEP RNA is unlike PDGF-modulated c-myc and c-fos RNA accumulation because it is blocked by cycloheximide, suggesting a requirement for de novo protein synthesis. It appears that PDGF modulates a program of gene expression with the accumulation of some transcripts, typified by MEP, being dependent upon the translation of others.
