Cell Biology and Toxicology

, Volume 1, Issue 2, pp 75–86 | Cite as

Elevation of dCTP pools in xeroderma pigmentosum variant human fibroblasts alters the effects of DNA repair arrest by arabinofuranosyl cytosine

  • William C. Dunn
  • James D. Regan
  • Ronald D. Snyder


DNA excision repair inhibition by arabinofuranosyl cytosine (ara-C) or by ara-C/hydroxyurea (HU) was measured in log phase and confluent cultures of normal and xeroderma pigmentosium (XP)-variant human fibroblasts following insult by ultraviolet (UV) light (20 J/m2). Repair inhibition was determined by measuring the accumulation of DNA single-strand breaks/108 daltons following cell culture exposure to ara-C or ara-C/HU in a series of 3 hr. pulses up ro 24 hr. after UV insult. Both normal and XP-variant derived cells showed a wide range of sensitivity to ara-C in log phase cells (0.2–9.4 breaks/108 daltons DNA), although strand break accumulation was constant for each specific cell line. The same cells were more sensitive to ara-C/HU with a 2–14 fold increase in DNA strand breaks depending upon the cell line assayed. In confluent cultures of normal cells, maximum sensitivity to ara-C and ara-C/HU was achieved with similar levels of repair inhibition observed (16.1 and 16.5 breaks/108 daltons, respectively). The same level of repair inhibition was observed in confulent XP-variants receiving ara-C/HU, but was reduced by 62–68% in cells treated with ara-C alone. Ara-C repair arrest was more rapidly reversed by competing concentrations of exogenous deoxycytidine (dCyd) in XP-variant compared to normal cells, especially in confluent cell cultures. In ara-C/HU treated cells, the level of dCyd reversal was reduced in the XP-variant when compared to cells exposed to ara-C alone. However, the same addition of HU had relatively little effect on dCyd reversal in normal cells. The measurements of dNTP levels indicate an elevated level of intracellular deoxycytosine triphosphate in XP-variant vs normal cells. The implications of these results are discussed as they relate to possible excision repair anomalies in the XP-variant.

Key words

ara-C dCTP pools DNA repair arrest XP-variant 



arabinofuranosul cytosine


deoxycytosine triphosphate




deoxynucleoside triphosphate






xeroderma pigmentosium


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. AYUSAWA, D., IWATA, K., and SENO, T. (1981). Alteration of ribonucleotide reductase in aphidicolin-resistant mutants of mouse FM3A cells with associated resistance to arabinosyladenine and arabinosylcytosine. Somat. Cell Genet.7: 27–42.Google Scholar
  2. BURK, P.G., LUTZNER, M.A., CLARKE, D.D., and ROBBINS, J.G. (1971). Ultraviolet-stimulated thymidine incorporation in xeroderma pigmentosum lymphocytes. J. Lab. Clin. Med.77: 759–767.Google Scholar
  3. CIARROCCHI, G., JOSE, J.G., and LINN, S. (1979). Further characterization of a cell-free system for measuring replicative and repair DNA synthesis with cultured human fibroblasts and evidence for the environment of DNA polymerase α in DNA repair. Nucleic Acids Res.7: 1205–1219.Google Scholar
  4. CLARKSON, J. (1978). Enhancement of repair replication in mammalian cells by hydroxyurea. Mutat. Res.52: 273–284.Google Scholar
  5. CLEAVER, J.E., THOMAS, G.H., and PARK S.D. (1979). Xeroderma pigmentosum variants have a slow recovery of DNA synthesis after irradiation with ultraviolet light. Biochem. Biophys. Acta.564: 122–131.Google Scholar
  6. CLEAVER, J.E., ARUTHYUNYAN, R.M., SARKISIAN, T.,Kaufmann, W.T., GREENE, A.E., and CORIELL, L. (1980). Similar defects in DNA repair and replication in the pigmented xerodermoid and the xeroderma pigmentosum variants. Carcinogenesis.1:647–655.Google Scholar
  7. CLEAVER, J.E. (1981). Sensitivity of excision repair in normal human, xeroderma pigmentosum variant and Cockayne's syndrome fibroblasts to inhibition by cytosine arabinoside. J. Cell Physiol.108: 163–173.Google Scholar
  8. COLLINS, A.R.S., DOWNES, C.S., and JOHNSON, R.T. (1980). Cell cycle-related variations in UV damage and repair capacity in Chinese hamster (CHO-K1) cells, J. Cell Physiol.103: 179–191.Google Scholar
  9. de SAINT VINCENT, R.B., DECHAMPS, M., and BUTTIN, G. (1980). The modulation of the thymidine triphosphate pool of Chinese hamster cells by dCMP deaminase and UDP reductase. J. Biol Chem.255: 162–167.Google Scholar
  10. DUNN, W.C. and REGAN, J.D. (1979). Inhibition of DNA excision repair in human cells by arabinofuranosyl cytosine: effect on normal and xeroderma pigmentosum cells. Mol. Pharmacol.15: 367–374.Google Scholar
  11. ENGSTROM, Y., ERIKSSON, S., THELANDER, L., and AKERMAN, M. (1979). Ribonucleotide reductase from calf thymus. Purification and properties. Biochemistry.18: 2941–2948.Google Scholar
  12. FORNACE, A.J., KOHN, K.W., and KANN, H.E. (1976). DNA single-strand breaks during repair of UV damage in human fibroblasts and abnormalities of repair in xeroderma pigmentosum. Proc. Nat. Acad. Sci., USA74: 39–43.Google Scholar
  13. FRANCIS, A.A., BLEVINS, R.D., CARRIER, W.L., SMITH, D.P., and REGAN, J.D. (1977). Inhibition of DNA repair in ultraviolet-irradiated cells by hydroxyurea. Biochem. Biophys. Acta.563: 385–392.Google Scholar
  14. FURTH, J.J. and COHEN, S.S. (1968). Inhibition of mammalian DNA polymerase by the 5’-triphosphate of 1-β-D-arabinofuranosyl cytosine and the 5’-triphosphate of 9-β-D-arabinofuranosyladenine. Cancer Res.28: 2061–2067.Google Scholar
  15. GRAHAM, F.L. andWitmore, G.F. (1970). The effect of 1-β-D-arabinofurandylcytosine on growth, viability, and DNA synthesis of mouse L-cells. Cancer Res.30: 2627–2635.Google Scholar
  16. HASHEM, N., BOOTSMA, D., KEIJZER, W., GREENE, A., CORIELL, L., THOMAS, G.H., and CLEAVER, J.E. (1980). Clinical characteristics, DNA repair, and complementation groups in xeroderma pigmentosum patients from Egypt. Cancer Res.40: 13–18.Google Scholar
  17. HISS, E.A. and PRESTON, R.J. (1977). The effect of cytosine arabinoside on the frequency of single strand breaks in DNA of mammalian cells following irradiation or chemical treatment. Biochem. Biophys. Acta.478: 1–8.Google Scholar
  18. HUNTING, D. and HENDERSON, J.F. (1981). Determination of deoxyribonucleotide triphosphates using DNA polymerase: A critical evaluation. Can. J. Biochem.59: 723–727.Google Scholar
  19. JOHNSON, R.T. and COLLINS, A.R.S. (1978). Reversal of the changes in DNA and chromosome structure which follow the inhibition of UV-induced repair in human cells. Biochem. Biophys. Res. Commun.80: 361–369.Google Scholar
  20. KAUFMANN, W.K. and CLEAVER, J.E. (1981). Mechanisms of inhibition of DNA replication by ultraviolet light in normal human and xeroderma pigmentosum fibroblasts. J. Mol. Biol.149: 171–187.Google Scholar
  21. KRAUSS, S.W. and LINN, S. (1982). Changes in DNA polymerases α, β and ψ during the replicative life span of cultured human fibroblasts. Biochemistry.21: 1002–1009.Google Scholar
  22. KUNZ, B.A. (1982). Genetic effects of deoxyribonucleotide pool inbalances. Environmental Mutagenesis.4: 695–725.Google Scholar
  23. LEHMANN, A.R., KIRK-Bell, S., ARLETT, C.F., PATTERSON, M.C., LOHMAN, P. H. M., deWeerd-Kastelein, E. D., andBootsma, D. (1975). Xeroderma pigementosum cells with normal levels of excision repair have a defect in DNA synthesis after UV-irradiation. Proc. Natl. Acad. Sci. USA.72: 219–223.Google Scholar
  24. Mattern, M. R.,Paone, R. F., andDay, R. S. (1982). Eukaryotic DNA repair is blocked at different steps by inhibitors of DNA topoisomerases and of DNA polymerases α and β. Biochem. Biophys. Acta.697: 6–13.Google Scholar
  25. Miller, M. R. andChinault, D. N. (1982). Evidence that DNA polymerases α and β participate differentially in DNA repair synthesis induced by different agents. J. Biol. Chem.257-: 49–69.Google Scholar
  26. Reddy, G. P. V. andPardee, A. B. (1980). Multienzyme complex for metabolic channeling in mammalian DNA replication. Proc. Natl. Acad. Sci. USA.77: 3312–3316.Google Scholar
  27. Robbins, J. H,,Kraemer, K. H.,Lutzner, M. A.,Festoff, B. V., andCoon, H. G. (1974). Xeroderma pigmentosum: an inherited disease with sun sensitivity, multiple cutaneous neoplasms, and abnormal DNA repair. Ann. Int. Med.80: 221–248.Google Scholar
  28. Rossignol, J. M.,Gentil, A.,Lacharpagne, J., and deRecondo, A. M. (1980). Involvement of DNa polymerase β in a N-methyl-N′-nitro-N′-nitrosoduanidine (MNNG)-induced DNA repair process. Biochem. Internatl.1: 253–261.Google Scholar
  29. Siedlecki, J. A.,Szyszko, J.,Pietzykowska, I., andZmudzka, B. (1980). Evidence implying DNA polymerase β function in excision repair. Nucleic Acid Res.8: 361–375.Google Scholar
  30. Sookg, K. L.,Nordenskjold, B. A., andBjursell, K. G. (1973). Deoxyribonucleotide-triphosphate pools and DNA synthesis in synchronized hamster cells. Eur. J. Biochem.33: 428–432.Google Scholar
  31. Snyder, R. D (1983) Deoxynucleoside triphosphate pools in human diploid fibroblasts and their modulation by hydroxyurea. Biochem. Pharmacol. (In press).Google Scholar
  32. Snyder, R. D. (1984). Inhibitors of ribonucleotide reductase alter DNA repair in human fibroblasts through specific depletion of depletion of purine deoxynucleotide triphosphate. Cell Biol. Toxicol.1: 81–94.Google Scholar
  33. Snyder, R. D.,Carrier, W. L., andRegan, J. D. (1981). Application of arabinofuranosyl cytosine in the kinetic analysis and quantitation of DNA repair in human cells after ultraviolet irradiation. Biophys. J.35: 339–350.Google Scholar
  34. Walters, R. A.,Lobey, R. A.,Ratliff, R. L. (1973). Cell cycle dependent variations of deoxynucleoside triphosphate pools in Chinese hamster cells. Biochem. Biophys. Acta.319: 336–347.Google Scholar
  35. Wist, E.,Krokan, H., andPrydz, H. (1976). Effect of 1-β-D-arabinofuranosylcytosine triphosphate on DNA synthesis in isolated HeLa cell nuclei. Biochemistry.15: 3647–3642.Google Scholar

Copyright information

© Princeton Scientific Publishers, Inc 1985

Authors and Affiliations

  • William C. Dunn
    • 1
  • James D. Regan
    • 1
  • Ronald D. Snyder
    • 2
  1. 1.Biology DivisionOak Ridge National LaboratoryTennessee
  2. 2.Stauffer Chemical CompanyFarmington, Connecticut

Personalised recommendations