The Chk1-Cdc25C regulation is involved in sensitizing A253 cells to a novel topoisomerase I inhibitor BNP1350 by bax gene transfer

Promotion of apoptosis may potentiate the sensitivity of tumor cells to che motherapeutic agents, thus improving the efficacy of cancer treatment. The transfection of the proapoptotic bax gene, which results in the overexpress ion of bax protein, augments the growth inhibition of A253 cells by BNP1350 . Increased drug response was associated with the induction of DNA fragment ation in the size of 30-200 Kb, generating a cleaved fragment of 18 kDa fro m full-length 21 kDa bax and the cleavage of PARP. A253/vec cells treated w ith 0.07 muM(IC50) of BNP1350 accumulated in G(2) phase at 24 h after drug removal. In contrast, A253/Bax cells treated with an equimolar concentratio n of BNP1350 primarily displayed a G(1) phase accumulation with a concurren t decrease in G(2) phase. Certain cell cycle regulatory protein expression and activities were altered following drug exposure in both cell lines unde r similar conditions. Cdk2- and cdc2-associated HI kinase activities were m arkedly increased in the A253/Bax cell line with marginal increased activit y in the A253/vec cell line. A chk1 activity assay was performed with GST-c dc25C (200 - 256) or GST-cdc25C(S216A) (200-256) fusion proteins as the sub strate. Increased chk1 activity was observed in the A253/vec cell line, wit h little change in the A253/Bax cell line, when exposed to equimolar concen trations of BNP1350 (0.07 MM). A Western blot of immunoprecipitated chk1 in dicated that increased chk1 phosphorylation following DNA damage induced by BNP1350 was accompanied by the observed G(2) accumulation in the A253/vec cell line, while only a slight increase in chk1 phosphorylation was seen in the A253/Bax cell line. A decreased expression of cdc25C was observed in t he BNP1350-treated A253/Bax cells, but not in the A253/vec cell line. Follo wing exposure to BNP1350, increased binding of 14-3-3 proteins to chk1 occu rred in both cell lines, with more being observed in the A253/vec cell line . The data have shown that inhibition of the chk1 pathway accompanied by th e abrogation of G(2) arrest is involved in sensitizing A253 cells to BNP135 0 by bax gene transfer. These findings suggest that bax gene transfer sensi tizes A253 cells to BNP1350 through apoptosis promoting and G(2)/M DNA dama ge checkpoint regulatory pathways.