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 chemotherapeutic agents, thus improving the efficacy of cancer treatment. The transfection of the proapoptotic bax gene, which results in the overexpression of bax protein, augments the growth inhibition of A253 cells by BNP1350. Increased drug response was associated with the induction of DNA fragmentation in the size of 30-200 Kb, generating a cleaved fragment of 18 kDa from full-length 21 kDa bax and the cleavage of PARP. A253/vec cells treated with 0.07 microM(IC50) of BNP1350 accumulated in G2 phase at 24 h after drug removal. In contrast, A253/Bax cells treated with an equimolar concentration of BNP1350 primarily displayed a G1 phase accumulation with a concurrent decrease in G2 phase. Certain cell cycle regulatory protein expression and activities were altered following drug exposure in both cell lines under similar conditions. Cdk2- and cdc2-associated H1 kinase activities were markedly increased in the A253/Bax cell line with marginal increased activity in the A253/vec cell line. A chk1 activity assay was performed with GST-cdc25C (200-256) or GST-cdc25C(S216A) (200-256) fusion proteins as the substrate. Increased chk1 activity was observed in the A253/vec cell line, with little change in the A253/Bax cell line, when exposed to equimolar concentrations of BNP1350 (0.07 microM). A Western blot of immunoprecipitated chk1 indicated that increased chk1 phosphorylation following DNA damage induced by BNP1350 was accompanied by the observed G2 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 the BNP1350-treated A253/Bax cells, but not in the A253/vec cell line. Following exposure to BNP1350, increased binding of 14-3-3 proteins to chk1 occurred 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 the abrogation of G2 arrest is involved in sensitizing A253 cells to BNP1350 by bax gene transfer. These findings suggest that bax gene transfer sensitizes A253 cells to BNP1350 through apoptosis promoting and G2/M DNA damage checkpoint regulatory pathways.
Yin
M
M
Department of Pharmacology and Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York, NY 14263, USA.
Hapke
G
G
Guo
B
B
Azrak
R G
RG
Frank
C
C
Rustum
Y M
YM
D017209:Apoptosis; D016203:CDC2 Protein Kinase / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D019926:Cyclin B / Q000378:metabolism; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D016195:G2 Phase; D015971:Gene Expression Regulation, Enzymologic; D018014:Gene Transfer Techniques; D006801:Humans; D020128:Inhibitory Concentration 50; D008175:Lung Neoplasms / Q000201:enzymology* / Q000235:genetics; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2; D059004:Topoisomerase I Inhibitors; D014162:Transfection; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein; D020687:cdc25 Phosphatases / Q000378:metabolism*
C494826:BAX protein, human; D018797:Cell Cycle Proteins; D019926:Cyclin B; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D059004:Topoisomerase I Inhibitors; D051028:bcl-2-Associated X Protein; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D016203:CDC2 Protein Kinase; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases