INDUCTION OF APOPTOSIS IN HELA-CELLS BY MSSP, C-MYC BINDING-PROTEINS

MSSP (c-myc gene single strand binding proteins) were identified as pr otein factors binding to a putative replication origin/transcriptional enhancer sequence present upstream from the human c-myc gene, and two cDNAs encoding highly homologous proteins, MSSP-1 and MSSP-2, have be en cloned, Scr2, independently cloned as a factor which complements th e cdc2 defective mutant of Schizosaccharomyces pombe, has turned out t o be identical to MSSP-1. MSSP-1/Scr2 and MSSP-2 similarly stimulated the initiation of SV40 DNA replication, and thus were suggested to be involved in regulation of cell cycle movement, especially from the G(1 ) to S phase. Here, we examined the functions of MSSP in apoptosis. MS SP expression plasmids mere transfected to human HeLa cells together, with a beta-galactosidase expression vector. After incubation in the p resence of 2% calf serum, cells were stained with X-gal and morphologi cally apoptotic cells among the beta-galactosidase-positive cells mere counted, Both MSSP-1 and 2 induced apoptosis in a dose-dependent mann er as in the control experiments with c-myc or adenovirus E1A. DNA fra gmentation, a hallmark of apoptosis, was also observed in cells transf ected with MSSP expression plasmids. The results of experiments using various deletion mutants of MSSP indicated that the region containing one of the two RNP consensus motifs, RNP1-B, is required for induction of apoptosis as, well as specific DNA binding activity.