Abstract
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 μg/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 induced 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. Flow-cytometric 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 G2/M arrest and DNA fragmentation. These findings suggest that okadaic-acid-induced apoptosis occurs as a result of metaphase arrest.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- OA :
-
okadaic acid
- TPA :
-
phorbol 12-myristate 13-acetate
- FBS :
-
fetal bovine serum
References
Bhalla K, Ibrado AM, Tourkina Tang C, Mahoney ME, Huang Y (1993) Taxol induces internucleosomal DNA fragmentation associated with programmed cell death in human myeloid leukemia cells. Leukemia 7:563–568
Bøe R, Gjertsen BT, Vintermyr OK, Houge G, Lanotte M, Døskeland SO (1991) The protein phosphatase inhibitor okadaic acid induces morphological changes typical of apoptosis in mammalian cells. Exp Cell Res 195:237–246
Dive C, Hickman JA (1991) Drug-target interactions: only the first step in the commitment to a programmed cell death? Br J Cancer 64:192–196
Fujiki H, Suganuma M (1993) Tumor promotion by inhibitors of protein phosphatases 1 and 2A: the okadaic acid class of compounds. In: Woude GFV, Klein G (eds) Adv Cancer Res 61: 143–193
Haas-Kogan DA, Kogan SC, Levi D, Dazin P, T'Ang A, Fung YKT, Israel MA (1995) Inhibition of apoptosis by the retinoblastoma gene product. EMBO J 14:461–472
Hickman JA (1992) Apoptosis induced by anticancer drugs. Cancer Metastasis Rev 11:121–139
Huschtscha LI, Jeitner TM, Andersson CE, Bartier WA, Tattersall MHN (1994) Identification of apoptotic and necrotic human leukemic cells by flow cytometry. Exp Cell Res 212:161–165
Inomata M, Kaneko A (1987) Chemosensitivity profiles of primary and cultured human retinoblastoma cells in a human tumor clonogenic assay. Jpn J Cancer Res 78:838–868
Inomata M, Kaneko A, Hoshi A (1986) Improved colony formation of cultured retinoblastoma cells. Invest Ophthalmol Vis Sci 27:1423–1428
Inomata M, Kaneko A, Saijo N, Tokura S (1994) Culture of retinoblastoma cells from clinical specimens: growth-promoting effect of 2-mercaptoethanol. Cancer Res Clin Oncol 120:149–155
Ishida Y, Furukawa Y, DeCaprio JA, Saito M, Griffin DG (1992) Treatment of myeloid leukemic cells with the phosphatase inhibitor okadaic acid induces cell cycle arrest at either G1/S or G2/M depending on dose. J Cell Physiol 150:484–492
Kaufmann SC (1989) Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: a cautionary note. Cancer Res 49:5870–5878
Kerr JFR (1994) Apoptosis Its significance in cancer and cancer therapy. Cancer 73:2013–2026
Kerr JFR, Harmon BV (1991) Definition and incidence of apoptosis: an historical perspective. In: Tomei LD, Cope FO (eds) Apoptosis the molecular basis of cell death. (Current communications in cell and molecular biology, vol 3) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 5–29
Kerr JFR, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239–257
Kigasawa K, Soushi S, Tanaka Y, Obazawa H (1994) Morphologic and chromosomal study of a human retinal pigment epithelial cell line. Jpn J Ophthalmol 38:10–15
Kizaki H, Suzuki K, Tadakuma T, Ishimura Y (1990) Adenosine receptor-mediated accumulation of cyclic AMP-induced T-lymphocyte death through internucleosomal DNA cleavage. J Biol Chem 265:5280–5284
Krishan HA (1975) Rapid flow cytometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol 66:188–193
Lanotte M, Riviere JB, Hermouet S, Houge G, Vintermyr OK, Gjertsen BT, Døskeland SO (1991) Programmed cell death (apoptosis) is induced rapidly and with positive cooperativity by activation of cyclic adenosine monophosphate-kinase I in a myeloid leukemia cell line. J Cell Physiol 146: 73–80
Madigan MC, Penfold PL, Billson FA (1993) Features of apoptosis in retinoblastoma. Annual Meeting Abstract Issue. Invest Ophthal Vis Sci 34:968
McFall RC, Sery TW, Makadon M (1977) Characterization of a new continuous cell line derived from a human retinoblastoma. Cancer Res 37:1003–1010
Murakami Y, Hayashi K, Sekiya T (1991) Detection of aberrations of the p53 alleles and the gene transcript in human tumor cell lines by single-strand conformation polymorphism analysis. Cancer Res 51:3356–3361
Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–279
Piacentini M, Fesus L, Melino G (1993) Multiple cell cycle access to the apoptotic death programme in human neuroblastoma cells. FEBS Lett 320:150–154
Reid TW, Albert DM, Rabson AS, Russell P, Craft J, Chu EW, Tralka TS, Wilcox JL (1974) Characteristics of an established cell line of retinoblastoma. J Natl Cancer Inst 53:347–360
Sen S, D'Incalci M (1992) Apoptosis biochemical events and relevance to cancer chemotherapy. FEBS Lett 307:122–127
Smith CA, Williams GT, Kingston R, Jenkinson EJ, Owen JTT (1989) Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature 357:181–184
Stewart BW (1994) Mechanism of apoptosis: Integration of genetic, biochemical, and cellular indicators. J Natl Cancer Inst 86:1286–1296
Vandré DD, Wills VL (1992) Inhibition of mitosis by okadaic acid: possible involvement of a protein phosphatase 2A in the transition from metaphase to anaphase. J Cell Sci 101:79–91
Wyllie AH (1985) The biology of cell death in tumours. Anticancer Res 5:131–136
Yen A, Varvayanis S (1995) DMSO, sodium butyrate, and TPA induce hypophosphorylation of RB with HL-60 cell differentiation. In Vitro Cell Dev Biol 31:164–167
Zheng B, Woo CF, Kuo JF (1991) Mitotic arrest and enhanced nuclear protein phosphorylation in human leukemia K562 cells by okadaic acid, a potent protein phosphatase inhibitor and tumor promoter. J Biol Chem 266:10031–10034
Author information
Authors and Affiliations
Additional information
This study was supported, in part, by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare, Japan.
Rights and permissions
About this article
Cite this article
Inomata, M., Saijo, N., Kawashima, K. et al. Induction of apoptosis in cultured retinoblastoma cells by the protein phosphatase inhibitor, okadaic acid. J Cancer Res Clin Oncol 121, 729–738 (1995). https://doi.org/10.1007/BF01213319
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01213319