Abstract
The nucleoside analogs fludarabine and gemcitabine inhibit cellular DNA synthesis by two different mechanisms: (1) direct termination of DNA strand elongation after the triphosphate of each drug is incorporated into DNA; and (2) indirect inhibition of DNA synthesis by decreasing cellular dNTPs through inhibition of ribonucleotide reductase. The present study demonstrated that incorporation of the analogs into DNA is critical for the cytotoxic action of these drugs in human T lymphoblastoid CEM cells. S phase cells, which actively incorporated the analogs into DNA, were most sensitive to the cytotoxic action of these compounds. A relatively short-term (5–24 h) cessation of cellular DNA synthesis without analog incorporation was not sufficient to cause cell death. The drug-treated cells died through apoptosis characterized by generation of internucleosomal DNA fragmentation and apoptotic morphology. Induction of high molecular mass (50–500 kb) DNA fragmentation was also observed in cells undergoing apoptosis; this type of DNA degradation was strongly correlated with the analog-induced cell death process. Inhibition of the analog incorporation into DNA by aphidicolin blocked both types of DNA fragmentation and apoptotic morphology, indicating the essential role of analog incorporation into DNA in drug-induced cell death.
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Arends MJ, Morris RG, Wyllie AH (1990) Apoptosis, the role of the endonuclease. Am J Pathol 136:593
Barry MA, Behnke CA, Eastman A (1990) Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochem Pharmacol 40:2353
Cohen JJ, Duke RC (1984) Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death. J Immunol 132:38
Evans DL, Dive C (1993) Effects of cisplatin on the induction of apoptosis in proliferating hepatoma cells and nonproliferating immature thymocytes. Cancer Res 53:2133
Fairbairn LJ, Cowling GJ, Reipert BM, Dexter TM (1993) Suppression of apoptosis allows differentiation and development of a multipotent hemopoietic cell line in the absence of added growth factors. Cell 74:823
Fesus L, Davis PJA, Piacentini M (1991) Apoptosis: molecular mechanisms in programmed cell death. Eur J Cell Biol 56:170
Gandhi V, Plunkett W (1988) Modulation of arabinosylnucleoside metabolism by arabinosylnucleotides in human leukemia cells. Cancer Res 48:329
Gorczyca W, Gong J, Ardelt B, Traganos F, Darzynkiewicz Z (1993) The cell cycle related differences in susceptibility of HL-60 cells to apoptosis induced by various antitumor agents. Cancer Res 53:3186
Gunji H, Kharbanda S, Kufe D (1991) Induction of internucleosomal DNA fragmentation in human myeloid leukemia cells by 1-β-d-arabinofuranosylcytosine. Cancer Res 51:741
Heinemann V, Xu Y-Z, Chubb S, Sen A, Hertel LW, Grindey GB, Plunkett W (1990) Inhibition of ribonucleotide reduction in CCRF-CEM cells by 2′,2′-difluorodeoxycytidine. Mol Pharmacol 38:567
Huang P, Plunkett W (1992) A quantitative assay for fragmented DNA in apoptotic cells. Anal Biochem 207:163
Huang P, Plunkett W (1993) Replicon-sized DNA fragmentation induced by nucleoside analogs. Proc Am Assoc Cancer Res 34:289
Huang P, Plunkett W (1994) Drug-induced apoptosis: relationship between DNA fragmentation and cell death. Proc Am Assoc Cancer Res 35:313
Huang H, Siciliano MJ, Plunkett W (1989) Gene deletion, a mechanism of induced mutation by arabinosyl nucleosides. Mutat Res 210:291
Huang P, Chubb S, Plunkett W (1990) Termination of DNA synthesis by 9-β-d-arabinofuranosyl-2-fluoroadenine, a mechanism for cytotoxicity. J Biol Chem 265:16617
Huang P, Chubb S, Hertel LW, Grindey GB, Plunkett W (1991) Action of 2′,2′-difluorodeoxycytidine on DNA synthesis. Cancer Res 51:6110
Itoh N, Yonehara S, Ishii A, Yonehara M, Mizushima S-I, Sameshima M, Hase A, Seto Y, Nagata S (1991) The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66:233
Kamiya K, Huang P, Plunkett W (1993) Excision of incorporated fludarabine from DNA by human DNA polymerase ɛ. Proc Am Assoc Cancer Res 34:349
Kaufmann SH (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
Kaufmann SH, Karp JE, Jones RJ, Miler KB, Schneider E, Zwelling LA, Cowan K, Wendel K, Burke PJ (1994) Topoisomerase II levels and drug sensitivity in adult acute myelogenous leukemia. Blood 83:517
Major PP, Egan EM, Beardsley GP, Minden MD, Kufe DW (1981) Lethality of human myeloblasts correlates with the incorporation of arabinofuranosylcytosine into DNA. Proc Natl Acad Sci USA 78:3235
Matherly LH, Schuetz JD, Westin E, Goldman ID (1989) A method for the synchronization of cultured cells with aphidicolin: application to the large-scale synchronization of L1210 cells and the study of the cell cycle regulation of thymidylate synthase and dihydrofolate reductase. Anal Biochem 182:338
McConkey DJ, Hartzell P, Duddy SK, Hakansson H, Orrenius S (1988) 2,3,7,8-Tetrachlorodibenzo-p-dioxin kills immature thymocytes by Ca2+-mediated endonuclease activation. Science 242:256
Michaelson J (1991) The significance of cell death. In: Tomei LD, Cope FO (eds) Apoptosis, the molecular basis of cell death. Cold Spring Harbor Laboratory Press, New York, p 31
Oberhammer F, Wilson JW, Dive C, Morris ID, Hickman JA, Wakeling AE, Walker PR, Sikorska M (1993) Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J 12:3679
Parker WB, Bapat AR, Shen J-X, Townsend AJ, Cheng C-Y (1988) Interaction of 2-halogenated dATP analogs (F, Cl, and Br) with human DNA polymerases, DNA primase, and ribonucleotide reductase. Mol Pharmacol 34:485
Robertson LE, Chubb S, Meyn RE, Story M, Ford R, Hittelman WN, Plunkett W (1993) Induction of apoptotic cell death in chronic lymphocytic leukemia by 2-chloro-2′-deoxyadenosine and 9-β-d-arabinosyl-2-fluoroadenine. Blood 81:143
Sandoval A, Plunkett W (1994) Inhibition of UV-induced DNA repair in quiescent human peripheral blood lymphocytes by fludarabine stimulates apoptosis. Proc Am Assoc Cancer Res 35:395
Sassanfar M, Roberts JW (1990) Nature of the SOS-inducing signal in Escherichia coli, the involvement of DNA replication. J Mol Biol 212:79
Sellins KS, Cohen JJ (1987) Gene induction by γ-irradiation leads to DNA fragmentation in lymphocytes. J Immunol 139:3199
Tartaglia LA, Ayres TM, Wong GH, Goeddel DV (1993) A novel domain within the 55 kd TNF receptor signals cell death. Cell 74:845
Thompson HJ, Strange R, Schedin PJ (1992) Apoptosis in the genesis and prevention of cancer. Cancer Epidemiol Biomarkers Prev 1:597
Trauth BC, Klas C, Peters AMJ, Matzku S, Moller P, Falk W, Debatin K-M, Krammer PH (1989) Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science 245:301
Walker GC (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev 48:60
Walker PR, Smith C, Youdale T, Leblanc J, Whitfield JF, Sikorska M (1991) Topoisomerase II-reactive chemotherapeutic drugs induce apoptosis in thymocytes. Cancer Res 51:1078
Walker PR, Kokileva L, LeBlanc J, Sikorska M (1993) Detection of the initial stages of DNA fragmentation in apoptosis. Biotechniques 15:1032
Wang Z, Wu X, Friedberg EC (1993) Nucleotide-excision repair of DNA in cell-free extracts of the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci USA 90:4907
Warters RL (1990) Radiation-induced apoptosis in a murine T-cell hybridoma. Cancer Res 52:883
Williams GT, Smith CA, Spooncer E, Dexter TM, Taylor TR (1990) Haemopoietic colony stimulating factors promote cell survival by suppressing apoptosis. Nature 343:76
Wyllie AH, Kerr JFR, Currir AR (1980) Cell death: the significance of apoptosis. Int Rev Cytol 68:251
Yang SW, Huang P, Plunkett W, Becker FF, Chan JYH (1992) Dual mode of inhibition of purified DNA ligase I from human cells by 9-β-D-arabinofuranosyl-2-fluoroadenine triphosphate. J Biol Chem 267:2345
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This work was supported in part by grants CA28596 and CA55164 from the National Cancer Institute, Department of Health and Human Services, and by grant DHP-1 from the American Cancer Society
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Huang, P., Plunkett, W. Fludarabine- and gemcitabine-induced apoptosis: incorporation of analogs into DNA is a critical event. Cancer Chemother. Pharmacol. 36, 181–188 (1995). https://doi.org/10.1007/BF00685844
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DOI: https://doi.org/10.1007/BF00685844