Molecular mechanisms ofc-myc deregulation during induction of transformation in temperature-sensitive cell lines

We have previously reported accelerated transcription and rapid accumulation of c-myc mRNAs upon induction of transformation in a temperature-sensitive mouse cell line (Gallant et al., 1989). Here we have used both mouse and human cell lines transformed with a temperature-sensitive mutant of the Simian virus 40 (SV40) virus, to investigate whether a shift in promoter utilization within the c-myc gene locus is part of a general mechanism that deregulates c-myc expression during transformation induction. We devised a simple and sensitive method using reverse transcription followed by radioactive polymerase chain reaction (RT-PCR) to measure the relative change in c-myc mRNAs arising from each of the four known promoters. We show that a 3- to 5-fold increase in c-myc transcripts from the P1 and P3 promoters occurs in both human and mouse cell lines within 30 minutes of the shift to the permissive temperature. The major P2-initiated transcripts are not significantly effected. However, exon 3-containing RNAs increase more gradually up to 24 hour post-induction, and P1 and P3 transcripts, while remaining elevated, still contribute relatively little to the total c-myc RNA population. Nuclear run-on transcription assays showed that a transient acceleration of transcription occurs for P1 and P3 promoters with peak transcription rates (4-fold increase for P1 and 6 fold increase for P3) at 5 hour post-induction in both cases, reverting to normal by 24 hours. This indicates that this accelerated transcription is accompanied by transcript stabilization which maintains higher steady state levels of c-myc transcripts at later times. The transient acceleration of both P1 and P3 transcription was preceded by 7 fold increase in the transcription rate of 7SK, a class III RNA gene, occuring within 30 minutes of induction. The present study suggests that the transcripts initiated from the minor promoters (P1 and P3) play an indirect role in the deregulated expression of the c-myc gene in transformed cells and that 7SK RNA may participate in P1 and/or P3 activation.