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Cancer Chemotherapy and Pharmacology

, Volume 25, Issue 6, pp 418–424 | Cite as

The effect of ara-C-induced inhibition of DNA synthesis on its cellular pharmacology

  • Li-Ming Wang
  • J. Courtland White
  • Robert L. Capizzi
Original Articles Cytosine Arabinoside Cytotoxicity DNA Synthesis, Pharmacology

Summary

The cytotoxicity of ara-C is believed to result from incorporation of ara-CTP into DNA and inhibition of DNA synthesis. Since complete inhibition of DNA synthesis would prevent further incorporation of ara-CTP, ara-C may have a self-limiting effect on its own cytotoxicity, particularly at the high concentrations typical of highdose ara-C clinical protocols. In this study, the incorporation of [3H]-dThd and [3H]-ara-C into DNA were compared. Within 1 h of exposure of L5178Y cells to ara-C, the rate of [3H]-dThd incorporation into the acid-insoluble fraction was reduced by 98%. Despite this nearly complete block in [3H]-dThd incorpration, DNA synthesis was not completely inhibited since [3H]-ara-C continued to be incorporated for up to 6 h, although a plateau in ara-CDNA synthesis was observed between 2 and 3 h exposure when ara-CTP levels were maximal. The effect of ara-C on [3H]-dThd incorporation into DNA was due in part to an indirect effect of ara-C on the metabolism of intracellular [3H]-dThd to [3H]-dTTP. Within 30 min exposure to 10 μM ara-C, the rate of cellular [3H]-dTTP synthesis was slowed to only 15% of the control rate. This was not due to inhibition of [3H]-dThd transport, since the intracellular and extracellular concentrations of the nucleoside were equal. The effect of ara-C on [3H]-dTTP synthesis resulted from significant changes in deoxynucleoside 5′-triphosphate (dNTP) pools. dTTP, dATP, and dGTP levels were increased, whereas the dCTP concentration was decreased. When dThd kinase from L5178Y cells was assayed with increased dTTP levels induced by ara-C vs the dTTP level in control cells, its activity was reduced by 72%. Thus, the [3H]-dThd incorporation experiment overestimated the extent of inhibition of DNA synthesis by ara-C due to increased feedback inhibition of dThd kinase and increased competition for DNA polymerase between the elevated unlabeled dTTP pool and the decreased levels of [3H]-dTTP. In vitro assay of DNA polymerase in the presence of the ara-CTP concentration achieved after 0.5 or 3 h exposure to 10 μM ara-C (60 μM and 200 μM, respectively), plus the mixture of dNTPs found intracellularly at these times, resulted in 57% and 80% inhibition of the polymerase, respectively. This inhibition may account for the plateau in the accumulation of ara-CDNA that was observed at 3 h and suggests that ara-C incorporation may be self-limiting at high cellular concentrations of ara-CTP. The ara-C-induced decline in dCTP noted above was apparently a secondary effect resulting from the inhibition of ribonucleotide reductase by the elevated dTTP and dATP. CDP reductase activity in the presence of dATP and dTTP at the concentrations found in ara-C-treated cells was 58% of the activity observed in the presence of nucleotide levels found in control cells. The decrease in dCTP levels was associated with a reciprocal increase in the rate of [3H]-ara-C phosphorylation following subsequent exposure to unlabeled ara-C. Thus, ara-C self-potentiated its own uptake in these cells. These observations of the self-limiting and self-potentiating effects of high concentrations of ara-C may be relevant to the selection of the optimal dose and the duration of exposure in the clinical use of high-dose ara-C infusions.

Keywords

dTTP L5178Y Cell Cellular Pharmacology High Cellular Concentration dTTP Pool 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

ara-C

I-β-d-arabinofuranosyl, cytosine (cytosine arabinoside)

ara-CTP

ara-C triphosphate

NTP

unspecified nucleoside 5′-triphosphate

dNTP

deoxynucleoside 5′-triphosphate

PBS

phosphate-buffered saline

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Reference

  1. 1.
    Capizzi RL, Powell BL (1987) Sequential high dose ara-C and asparaginase versus high dose ara-C alone in the treatment of patients with relapsed and refractory acute leukemias. Semin Oncol 14 [Suppl 1]:40Google Scholar
  2. 2.
    Capizzi RL, Papirmeister B, Mullins JM, Cheng E (1974) The detection of chemical mutagens using the L5178Y/asn murine leukemia in vitro and in a host-mediated assay. Cancer Res 34:3073Google Scholar
  3. 3.
    Capizzi RL, Yang JL, Cheng E, Bjornsson T, Sahasrabudhe D, Tan RS, Cheng YC (1983) Alteration of the pharmacokinetics of high-dose ara-C by its metabolite, high ara-U, in patients with acute leukemia. J Clin Oncol 1:763Google Scholar
  4. 4.
    Capizzi RL, Oliver L, Friedman H, Davis R, Mayer R, Schiffer C, Lunghofer B, Royer G, Van Echo DA (1988) Variations in ara-C plasma concentration at steady-state during remission induction and intensification therapy of AML. A population pharmacokinetic study by CALGB. Proc Am Soc Clin Oncol 7:57Google Scholar
  5. 5.
    Eriksson S, Thelander L, Akerman M (1979) Allosteric regulation of calf thymus ribonucleoside diphosphate reductase. Biochemistry 18:2948Google Scholar
  6. 6.
    Gale RP (1979) Advances in the treatment of acute myelogenous leukemia. N Engl J Med 300:1189Google Scholar
  7. 7.
    Hubscher U, Kuenzle CC, Spadari S (1977) Variation of DNA polymerases-alpha,-beta,-gamma during perinatal tissue growth and differentiation. Nucleic Acids Res 4:2917Google Scholar
  8. 8.
    Ives DH, Durham JP (1970) Deoxycytidine kinase: III. Kinetics and allosteric regulation of the calf thymus enzyme. J Biol Chem 245:2285Google Scholar
  9. 9.
    Ives DH, Morse PA Jr, Potter VR (1963A) Feedback inhibition of thymidine kinase by thymidine triphosphate. J Biol Chem 238:1467Google Scholar
  10. 10.
    Leclerc JM, Momparler RL (1984) Effect of the interval between exposures to cytarabine on its cytotoxic action on HL-60 myeloid leukemia cells. Cancer Treat Rep 68:1143Google Scholar
  11. 11.
    Lee L-S, Cheng Y-C (1976) Human deoxythymidine kinase: I. Purification and general properties of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia. J Biol Chem 251:2600Google Scholar
  12. 12.
    Khym J (1975) An analytical system for rapid separation of tissue nucleotides at low pressures on conventional anion exchangers. Clin Chem 21:1245Google Scholar
  13. 13.
    Kufe D, Major P, Egan E, Beardsley P (1981) Incorporation of ara-C into L1210 DNA as a correlate of cytotoxicity. J Biol Chem 235:3235Google Scholar
  14. 14.
    Kufe DW, Major P, Egan EM, Beardsley GP (1980) Correlation of cytotoxicity with incorporation of ara-C into DNA. J Biol Chem 255:8997Google Scholar
  15. 15.
    Kufe DW, Munroe D, Herrick D, Egan E, Spriggs D (1984) Effects of 1-β-d-arabinofuranosylcytosine incorporation on eukaryotic DNA template function. Mol Pharmacol 26:128Google Scholar
  16. 16.
    Major P, Egan EM, Beardsley GP, Minden MD, Kufe D (1981) Lethality of human myeloblasts correlates with the incorporation of ara-C in DNA. Proc Natl Acad Sci USA 78:3235Google Scholar
  17. 17.
    Major PP, Egan EM, Herrick DJ, Kufe DW (1982) Effect of ara-C incorporation on deoxyribonucleic acid synthesis in cells. Biochem Pharmacol 31:2937Google Scholar
  18. 18.
    Ochs J, Sinkule JA, Danks MK, Look T, Bowman WP, Rivera G (1984) Continuous infusion high dose cytosine arabinoside in refractory childhood leukemia. J Clin Onco 12:1092Google Scholar
  19. 19.
    Pogolotti AL, Santi DV (1982) High-pressure liquid chromatography ultraviolet analysis of intracellular nucleotides. Anal Biochem 126:335Google Scholar
  20. 20.
    Powell BL, Barberry R, Contento M, Gregory B, Wang L, Rhinehart-Clark A, Capizzi RL (1988) Interdose interval modulates ara-C cytotoxicity in HL-60 cells: correlation with bromodeoxyuridine (BrdUrd) incorporation into DNA. Proc AM Assoc Cancer Res 29:472Google Scholar
  21. 21.
    Raich PC (1978) Prediction of therapeutic response in acute leukemia. Lancet I:74–76Google Scholar
  22. 22.
    Scolnick EM, Aaronson SA, Tadaro GJ, Parks WS (1971) RNA dependent DNA polymerase activity in mammalian cells. Nature 229:318Google Scholar
  23. 23.
    Steeper JR, Stuart CD (1970) A rapid assay for CDP reductase activity in mammalian cell extracts. Anal Biochem 34:123Google Scholar
  24. 24.
    Wang LM, White JC, Capizzi RL (1988) Ara-C self-potentiates its uptake in L5178Y leukemia cells. Proc Am Assoc Cancer Res 29:472Google Scholar
  25. 25.
    White JC, Hines LH (1987) Role of uridine triphosphate in the phosphorylation of 1-β-d-arabinofuranosylcytosine in Ehrlich ascites tumor cells. Cancer Res 47:1820Google Scholar
  26. 26.
    White JC, Rathmell JP, Capizzi RL (1987) Membrane transport influences the rate of accumulation of cytosine arabinoside in human leukemia cells. J Clin Invest 79:380Google Scholar
  27. 27.
    Yang JL, Cheng EH, Capizzi RL, Cheng YC, Kute T (1985) Effect of uracil arabinoside on metabolism and cytotoxicity of cytosine arabinoside in L5178Y murine leukemia. J Clin Invest 75:141Google Scholar

Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • Li-Ming Wang
    • 1
  • J. Courtland White
    • 1
  • Robert L. Capizzi
    • 1
  1. 1.Cancer Center of Wake Forest University, at the Bowman Gray School of MedicineWinston-SalemUSA

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