Transcriptional Activation of the c-myc Proto-Oncogene in Murine Keratinocytes Enhances the Response to Epidermal Growth Factor

Abstract

To investigate the relationship between activation of the c-myc proto-oncogene and the controls of cellular growth and differentiation of epidermal cells, a transcriptionally activated c-myc gene (DM-myc) was introduced into the established murine keratinocytes, BALB/MK. Exponential growth rates of myc-transfectants were not significantly different from that of parental BALB/MK cells. C-myc RNA transcripts were not detectable in confluent, mitogen-deprived cultures of parental BALB/MK cells, whereas four out of five clones expressed elevated levels of myc mRNA under these conditions. All of the cell lines, however, displayed density-dependent growth arrest in the G0/1 phase of the cell cycle. Maximal stimulation of quiescent BALB/MK cells with epidermal growth factor (EGF) caused a 70- to 100-fold increase of [methyl-3H]-thymidine incorporation into DNA. In the four subclones that expressed the myc gene, the peak thymidine incorporation into DNA was significantly higher than in BALB/MK cells, ranging from 340- to 650-fold control levels. This increased sensitivity to EGF was not due to autocrine mitogenic activity or to a change of EGF binding. Type beta transforming growth factor strongly inhibited the EGF-induced DNA synthesis in BALB/MK cultures as well as in each of the five transfectants (IC50 4-40 pM). Furthermore, both BALB/MK cells and the transfected subclones could be induced to form cornified cell envelopes by increasing the extracellular concentration of calcium. Thus, the constitutive expression of c-myc in BALB/MK appears to affect predominantly the reinitiation of DNA synthesis by EGF.