INCREASED GADD153 MESSENGER-RNA LEVEL IS ASSOCIATED WITH APOPTOSIS INHUMAN LEUKEMIC-CELLS TREATED WITH ETOPOSIDE

Treatment of leukemic cells with topoisomerase inhibitors can lead to growth arrest and subsequent apoptotic cell death, The relationships b etween cell cycle regulation and apoptosis triggering remain poorly un derstood, The gadd153 gene encodes the nuclear protein CHOP 10 that ac ts as a negative modulator of CCAAT/enhancer binding protein transcrip tional factors and inhibits cell cycle progression, We have investigat ed the relationships between gadd153 gene expression and apoptosis ind uction in four human leukemic cell lines with different sensitivities to apoptosis induced by etoposide (VP-16), a topoisomerase II inhibito r, The gadd153 gene was constitutively expressed in the four studied c ell lines, In U937 and HL-60 cells that were very sensitive to apoptos is induction by the drug, VP-16 induced a time- and dose-dependent inc rease of gadd153 gene mRNA expression, Using agarose gel electrophores is and a quantitative filter elution assay, apoptotic DNA fragmentatio n was observed to begin when gadd153 gene expression increased, Equito xic doses of VP-16 (as defined using a 96-h 3-4,5-dimethylthiazol-2,5- diphenyltetraz bromide assay) did not increase the gadd153 mRNA level in K562 and KCL22 cell lines that were more resistant to apoptosis ind uction by the drug, Nuclear run-on and mRNA stability experiments demo nstrated that VP-16 treatment increased gadd153 gene transcription in the sensitive U937 cells, Cycloheximide did not prevent gadd153 expres sion increase, Both gadd153 mRNA level increase and internucleosomal D NA fragmentation were inhibited by N-tosyl-L-phenylalanine chloromethy lketone, a serine threonine protease inhibitor, N-acetyl-leucyl-leucyl -norleucinal, an inhibitor of calpain, N-acetylcysteine, an inhibitor of oxidative metabolism, and overexpression of Bcl-2, Z-VAD and Z-DEVD peptides that inhibit interleukin 1 beta-converting enzyme-like prote ases suppressed DNA fragmentation without preventing gadd153 mRNA incr ease in VP-16-treated U937 cells, These results indicate that gadd153 gene expression increase occurs downstream of events sensitive to N-to syl-L-phenylalanine chloromethylketone, calpain inhibitor I, and Bcl-2 and upstream of interleukin 1 beta-converting enzyme-related protease s activation in leukemic cells in which treatment with VP-16 induces r apid apoptosis.