7-Hydroxystaurosporine (UCN-01) is a
protein kinase inhibitor that is under development as an
anticancer agent in the United States and Japan. Long-term exposure of human A549
non-small cell lung cancer cells to
UCN-01 furnished cells (A549/UCN) with acquired resistance against
UCN-01. In this study, the sensitivity of these cells toward the growth-arresting properties of certain conventional
cytotoxic agents was explored. Cells were not cross-resistant against
adriamycin,
Taxol,
staurosporine, and
UCN-02, but they displayed 14- and 4.4-fold resistance against
cisplatin and
mitomycin C, respectively. Previous studies on the mechanism(s) of action of
UCN-01 suggest that induction of apoptosis and G1 phase accumulation are important for its anticancer activity; therefore, we compared induction of apoptosis and cell cycle distribution caused by
UCN-01 in wild-type A549 and A549/UCN cells using flow cytometry.
UCN-01 (0.4 microM) induced apoptosis (62%
terminal deoxynucleotidyl transferase-mediated nick end labeling-positive cells) in A549 cells, but not in A549/UCN cells. The percentages of cells that accumulated in G1 when exposed to
UCN-01 (0.4 microM) were 22% in A549 cells and 67% in A549/UCN cells. These results suggest that acquired resistance of
cancer cells against
UCN-01 is characterized by attenuation of apoptosis induction associated with reinforcement of the G1 checkpoint and that apoptosis regulation is drastically altered in A549/UCN cells as compared with A549 cells.
Cyclin-dependent kinase (CDK) inhibitor
proteins p21 and p27 in A549/UCN cells were up-regulated, which was accompanied by overexpression of G1
cyclins D1 and E, but
UCN-01 hardly affected levels of these
proteins. In contrast,
cyclin A,
cyclin B1,
retinoblastoma, and CDK2
proteins were apparently down-regulated, without changes in CDK4/6.
UCN-01 hardly affected the expression level of
cyclin B1 and induced dephosphorylation of
retinoblastoma in both cell types.
UCN-01 induced down-regulation of
cyclin A level and CDK2 activity accompanied with its dephosphorylation in A549/UCN cells, but not in A549 cells. The antiapoptotic
protein bcl-2 was apparently up-regulated in A549/UCN cells, however, bcl-xL, another antiapoptotic
protein, was down-regulated, without changes in bak and bax. Taken together, these results are consistent with the notion that induction of apoptosis and block of cell cycle in G1 are important determinants of the sensitivity of
cancer cells to
UCN-01 and suggest that inhibition of CDK2 activity accompanied by its dephosphorylation and decrease of expression level of
cyclin A might play an important role in the G1 phase accumulation induced by
UCN-01.