Down-regulation of c-myc and bcl-2 gene expression in PU.1-induced apoptosis in murine erythroleukemia cells.

We found that over-expression of PU.1, a member of the ets family of transcription factors, induces apoptotic cell death along with differentiation of DMSO stimulation in murine erythroleukemia (MEL) cells. To elucidate the molecular mechanisms of apoptosis, cell-cycle distribution and expression of several genes encoding apoptosis-promoting and -inhibiting factors were analyzed during the process of PU.1-induced apoptosis. FACS analysis revealed that cells were accumulated in the G0/G1 phase of the cell cycle before apoptosis. Morphological analysis of PI-stained nuclei of the apoptotic cells sorted by a FACScan showed 22.6% in G0/G1, 35.8% in S and 8.5% in G2/M phase by fluorescent microscopy after cell sorting, suggesting that PU.1-induced apoptosis in MEL cells occurs in G0/G1 through S phases. Semi-quantitative RT-PCR revealed that expression of c-myc and bcl-2 genes was reduced during the apoptotic process, while expression of bax and bcl-X(L) genes was not changed. Expression of the p53 gene was reduced rather than enhanced, suggesting that PU.1-induced apoptosis in MEL cells is p53-independent. Apoptosis was inhibited by adding 30% serum in culture, while no reduction of c-myc and bcl-2 gene expression was observed. Forced expression of the c-myc, bcl-2 and bcl-X(L) genes protected MEL cells from apoptosis. Our results suggest that a reduction of at least 2 important apoptosis-inhibiting factors, c-Myc and Bcl-2, is involved in PU.1-induced apoptosis in MEL cells.