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.I, a member of the ets family of t ranscription factors, induces apoptotic cell death along with differen tiation of DMSO stimulation in murine erythroleukemia (MEL) cells. To elucidate the molecular mechanisms of apoptosis, cell-cycle distributi on and expression of several genes encoding apoptosis-promoting and -i nhibiting factors were analyzed during the process of PU.I-induced apo ptosis. FAGS analysis revealed that cells were accumulated in the G(0) /G(1) phase of the cell cycle before apoptosis. Morphological analysis of PI-stained nuclei of the apoptotic cells sorted by a FACScan showe d 22.6% in G(0)/G(1), 35.8% in S and 8.5% in G(2)/M phase by fluoresce nt microscopy after cell sorting, suggesting that PU.I-induced apoptos is in MEL cells occurs in G(0)/G(1) through S phases. Semi-quantitativ e RT-PCR revealed that expression of c-myc and bcl-2 genes was reduced during the apoptotic process, while expression of box and bcl-X-L gen es was not changed. Expression of the p53 gene was reduced rather than enhanced, suggesting that PU.I-induced apoptosis in MEL cells is p53- independent. Apoptosis was inhibited by adding 30% serum in culture, w hile no reduction of c-myc and bcl-2 gene expression was observed. For ced expression of the c-myc, bcl-2 and bcl-X-L genes protected MEL cel ls from apoptosis, Our results suggest that a reduction of at least 2 important apoptosis-inhibiting factors, c-Myc and Bcl-2, is involved i n PU.I-induced apoptosis in MEL cells. (C) 1998 Wiley-Liss, Inc.