INDUCTION OF APOPTOSIS IN CULTURED RETINOBLASTOMA CELLS BY THE PROTEIN PHOSPHATASE INHIBITOR, OKADAIC ACID

The induction of apoptosis in cultured retinoblastoma cells by diverse drugs was examined by analyzing DNA fragmentation, a hallmark of apoptosis. First, the ability of six retinoblastoma cell lines to undergo apoptosis was surveyed using etoposide (30 mu g/ml, 20 h exposure). The NCC-RbC-60 cell line, established in this laboratory, showed DNA fragmentation clearly, whereas the other cell lines tested, including the representative retinoblastoma cell line, Y-79, did not show distinct DNA fragmentation. Biochemical modulators, such as A23187, forskolin, retinoic acid, phorbol 12-myristate 13-acetate and okadaic acid, were examined to ascertain whether they could induce apoptosis in NCC-RbC-60 and Y-79 cells after exposure for 20 h. Only okadaic acid induced DNA fragmentation in all the retinoblastoma cell lines tested and it induced DNA fragmentation in Y-79 cells in a time- and concentration-dependent manner. Flowcytometric analysis and microscopic examination revealed that Y-79 cells treated with okadaic acid for 24-48 h accumulated at the G2/M, especially M, phases, before undergoing DNA fragmentation. Other mitotic poisons, nocodazole, colcemid and taxol, also induced apoptosis in Y-79 cells. In the K1034 cell line, established from non-malignant retinal pigmental epithelium, okadaic acid failed to induce both G(2)/M arrest and DNA fragmentation. These findings suggest that okadaic-acid-induced apoptosis occurs as a result of metaphase arrest.