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. 2000 Aug 17;83(6):792–799. doi: 10.1054/bjoc.2000.1358

Deoxyuridine triphosphatase (dUTPase) expression and sensitivity to the thymidylate synthase (TS) inhibitorD9331

S D Webley 1, A Hardcastle 1, R D Ladner 2, A L Jackman 1, G W Aherne 1
PMCID: PMC2363540  PMID: 10952785

Abstract

Uracil DNA misincorporation and misrepair of DNA have been recognized as important events accompanying thymidylate synthase (TS) inhibition. dUTPase catalyses the hydrolysis of dUTP to dUMP, thereby maintaining low intracellular dUTP. We have addressed the relationship between dUTPase expression and cellular sensitivity to TS inhibition in four human lung tumour cell lines. Sensitivity (5-day MTT assay) to the growth inhibitory effects of the non-polyglutamatable, specific quinazoline TS inhibitor ZD9331, varied up to 20-fold (IC 50 3–70 nM). TS protein expression correlated with TS activity (r2= 0.88 P= 0.05). Intracellular concentrations of drug following exposure to ZD9331 (1 μM, 24 h) varied by ~2-fold and dTTP pools decreased by > 80% in all cell lines. No clear associations across the cell lines between intracellular drug concentrations, TS activity/expression, or TTP depletion could be made. dUTPase activity varied 17-fold and correlated with dUTPase protein expression (r2= 0.94 P= 0.03). There was a striking variation in the amount of dUTP formed following exposure to ZD9331 (between 1.3 and 57 pmole 10–6cells) and was in general inversely associated with dUTPase activity. A large expansion in the dUTP pool was associated with increased sensitivity to a 24-h exposure to ZD9331 in A549 cells that have low dUTPase activity/expression. dUTPase expression and activity were elevated (approximately 3-fold) in two variants of a human lymphoblastoid cell line with acquired resistance to TS inhibitors, further suggesting an important role for this enzyme in TS inhibited cells. © 2000 Cancer Research Campaign

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Selected References

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  1. Aherne G. W., Hardcastle A., Raynaud F., Jackman A. L. Immunoreactive dUMP and TTP pools as an index of thymidylate synthase inhibition; effect of tomudex (ZD1694) and a nonpolyglutamated quinazoline antifolate (CB30900) in L1210 mouse leukaemia cells. Biochem Pharmacol. 1996 May 17;51(10):1293–1301. doi: 10.1016/0006-2952(96)00035-4. [DOI] [PubMed] [Google Scholar]
  2. Beck W. R., Wright G. E., Nusbaum N. J., Chang J. D., Isselbacher E. M. Enhancement of methotrexate cytotoxicity by uracil analogues that inhibit deoxyuridine triphosphate nucleotidohydrolase (dUTPase) activity. Adv Exp Med Biol. 1986;195(Pt B):97–104. doi: 10.1007/978-1-4684-1248-2_16. [DOI] [PubMed] [Google Scholar]
  3. Calvert A. H., Jones T. R., Dady P. J., Grzelakowska-Sztabert B., Paine R. M., Taylor G. A., Harrap K. R. Quinazoline antifolates with dual biochemical loci of action. Biochemical and biological studies directed towards overcoming methotrexate resistance. Eur J Cancer. 1980 May;16(5):713–722. doi: 10.1016/0014-2964(80)90214-5. [DOI] [PubMed] [Google Scholar]
  4. Canman C. E., Radany E. H., Parsels L. A., Davis M. A., Lawrence T. S., Maybaum J. Induction of resistance to fluorodeoxyuridine cytotoxicity and DNA damage in human tumor cells by expression of Escherichia coli deoxyuridinetriphosphatase. Cancer Res. 1994 May 1;54(9):2296–2298. [PubMed] [Google Scholar]
  5. Caradonna S. J., Adamkiewicz D. M. Purification and properties of the deoxyuridine triphosphate nucleotidohydrolase enzyme derived from HeLa S3 cells. Comparison to a distinct dUTP nucleotidohydrolase induced in herpes simplex virus-infected HeLa S3 cells. J Biol Chem. 1984 May 10;259(9):5459–5464. [PubMed] [Google Scholar]
  6. Cohen S. S. On the nature of thymineless death. Ann N Y Acad Sci. 1971 Nov 30;186:292–301. doi: 10.1111/j.1749-6632.1971.tb31155.x. [DOI] [PubMed] [Google Scholar]
  7. Copur S., Aiba K., Drake J. C., Allegra C. J., Chu E. Thymidylate synthase gene amplification in human colon cancer cell lines resistant to 5-fluorouracil. Biochem Pharmacol. 1995 May 17;49(10):1419–1426. doi: 10.1016/0006-2952(95)00067-a. [DOI] [PubMed] [Google Scholar]
  8. Curtin N. J., Harris A. L., Aherne G. W. Mechanism of cell death following thymidylate synthase inhibition: 2'-deoxyuridine-5'-triphosphate accumulation, DNA damage, and growth inhibition following exposure to CB3717 and dipyridamole. Cancer Res. 1991 May 1;51(9):2346–2352. [PubMed] [Google Scholar]
  9. Estlin E. J., Balmanno K., Calvert A. H., Hall A. G., Lunec J., Newell D. R., Pearson A. D., Taylor G. A. The relationship between intrinsic thymidylate synthase expression and sensitivity to THYMITAQ in human leukaemia and colorectal carcinoma cell lines. Br J Cancer. 1997;76(12):1579–1585. doi: 10.1038/bjc.1997.600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Findlay M. P., Cunningham D., Morgan G., Clinton S., Hardcastle A., Aherne G. W. Lack of correlation between thymidylate synthase levels in primary colorectal tumours and subsequent response to chemotherapy. Br J Cancer. 1997;75(6):903–909. doi: 10.1038/bjc.1997.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fisher T. C., Milner A. E., Gregory C. D., Jackman A. L., Aherne G. W., Hartley J. A., Dive C., Hickman J. A. bcl-2 modulation of apoptosis induced by anticancer drugs: resistance to thymidylate stress is independent of classical resistance pathways. Cancer Res. 1993 Jul 15;53(14):3321–3326. [PubMed] [Google Scholar]
  12. Freemantle S. J., Jackman A. L., Kelland L. R., Calvert A. H., Lunec J. Molecular characterisation of two cell lines selected for resistance to the folate-based thymidylate synthase inhibitor, ZD1694. Br J Cancer. 1995 May;71(5):925–930. doi: 10.1038/bjc.1995.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Horio Y., Takahashi T., Kuroishi T., Hibi K., Suyama M., Niimi T., Shimokata K., Yamakawa K., Nakamura Y., Ueda R. Prognostic significance of p53 mutations and 3p deletions in primary resected non-small cell lung cancer. Cancer Res. 1993 Jan 1;53(1):1–4. [PubMed] [Google Scholar]
  14. Houghton J. A., Ebanks R., Harwood F. G., Tillman D. M. Inhibition of apoptosis after thymineless stress is conferred by oncogenic K-Ras in colon carcinoma cells. Clin Cancer Res. 1998 Nov;4(11):2841–2848. [PubMed] [Google Scholar]
  15. Houghton J. A., Harwood F. G., Houghton P. J. Cell cycle control processes determine cytostasis or cytotoxicity in thymineless death of colon cancer cells. Cancer Res. 1994 Sep 15;54(18):4967–4973. [PubMed] [Google Scholar]
  16. Houghton J. A., Harwood F. G., Tillman D. M. Thymineless death in colon carcinoma cells is mediated via fas signaling. Proc Natl Acad Sci U S A. 1997 Jul 22;94(15):8144–8149. doi: 10.1073/pnas.94.15.8144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jackman A. L., Alison D. L., Calvert A. H., Harrap K. R. Increased thymidylate synthase in L1210 cells possessing acquired resistance to N10-propargyl-5,8-dideazafolic acid (CB3717): development, characterization, and cross-resistance studies. Cancer Res. 1986 Jun;46(6):2810–2815. [PubMed] [Google Scholar]
  18. Jackman A. L., Kelland L. R., Kimbell R., Brown M., Gibson W., Aherne G. W., Hardcastle A., Boyle F. T. Mechanisms of acquired resistance to the quinazoline thymidylate synthase inhibitor ZD1694 (Tomudex) in one mouse and three human cell lines. Br J Cancer. 1995 May;71(5):914–924. doi: 10.1038/bjc.1995.178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jackman A. L., Kimbell R., Aherne G. W., Brunton L., Jansen G., Stephens T. C., Smith M. N., Wardleworth J. M., Boyle F. T. Cellular pharmacology and in vivo activity of a new anticancer agent, ZD9331: a water-soluble, nonpolyglutamatable, quinazoline-based inhibitor of thymidylate synthase. Clin Cancer Res. 1997 Jun;3(6):911–921. [PubMed] [Google Scholar]
  20. Jackson R. C., Jackman A. L., Calvert A. H. Biochemical effects of a quinazoline inhibitor of thymidylate synthetase, N-(4-(N-(( 2-amino-4-hydroxy-6-quinazolinyl)methyl)prop-2-ynylamino) benzoyl)-L-glutamic acid (CB3717), on human lymphoblastoid cells. Biochem Pharmacol. 1983 Dec 15;32(24):3783–3790. doi: 10.1016/0006-2952(83)90150-8. [DOI] [PubMed] [Google Scholar]
  21. Johnston P. G., Fisher E. R., Rockette H. E., Fisher B., Wolmark N., Drake J. C., Chabner B. A., Allegra C. J. The role of thymidylate synthase expression in prognosis and outcome of adjuvant chemotherapy in patients with rectal cancer. J Clin Oncol. 1994 Dec;12(12):2640–2647. doi: 10.1200/JCO.1994.12.12.2640. [DOI] [PubMed] [Google Scholar]
  22. Johnston P. G., Lenz H. J., Leichman C. G., Danenberg K. D., Allegra C. J., Danenberg P. V., Leichman L. Thymidylate synthase gene and protein expression correlate and are associated with response to 5-fluorouracil in human colorectal and gastric tumors. Cancer Res. 1995 Apr 1;55(7):1407–1412. [PubMed] [Google Scholar]
  23. Johnston P. G., Mick R., Recant W., Behan K. A., Dolan M. E., Ratain M. J., Beckmann E., Weichselbaum R. R., Allegra C. J., Vokes E. E. Thymidylate synthase expression and response to neoadjuvant chemotherapy in patients with advanced head and neck cancer. J Natl Cancer Inst. 1997 Feb 19;89(4):308–313. doi: 10.1093/jnci/89.4.308. [DOI] [PubMed] [Google Scholar]
  24. Ju J., Kane S. E., Lenz H. J., Danenberg K. D., Chu E., Danenberg P. V. Desensitization and sensitization of cells to fluoropyrimidines with different antisenses directed against thymidylate synthase messenger RNA. Clin Cancer Res. 1998 Sep;4(9):2229–2236. [PubMed] [Google Scholar]
  25. Kobayashi H., Takemura Y., Miyachi H., Skelton L., Jackman A. L. Effect of hammerhead ribozyme against human thymidylate synthase on the cytotoxicity of thymidylate synthase inhibitors. Jpn J Cancer Res. 1995 Nov;86(11):1014–1018. doi: 10.1111/j.1349-7006.1995.tb03014.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ladner R. D., McNulty D. E., Carr S. A., Roberts G. D., Caradonna S. J. Characterization of distinct nuclear and mitochondrial forms of human deoxyuridine triphosphate nucleotidohydrolase. J Biol Chem. 1996 Mar 29;271(13):7745–7751. doi: 10.1074/jbc.271.13.7745. [DOI] [PubMed] [Google Scholar]
  27. Lenz H. J., Leichman C. G., Danenberg K. D., Danenberg P. V., Groshen S., Cohen H., Laine L., Crookes P., Silberman H., Baranda J. Thymidylate synthase mRNA level in adenocarcinoma of the stomach: a predictor for primary tumor response and overall survival. J Clin Oncol. 1996 Jan;14(1):176–182. doi: 10.1200/JCO.1996.14.1.176. [DOI] [PubMed] [Google Scholar]
  28. Lindahl T. DNA glycosylases, endonucleases for apurinic/apyrimidinic sites, and base excision-repair. Prog Nucleic Acid Res Mol Biol. 1979;22:135–192. doi: 10.1016/s0079-6603(08)60800-4. [DOI] [PubMed] [Google Scholar]
  29. O'Connor B. M., Jackman A. L., Crossley P. H., Freemantle S. E., Lunec J., Calvert A. H. Human lymphoblastoid cells with acquired resistance to C2-desamino-C2-methyl-N10-propargyl-5,8-dideazafolic acid: a novel folate-based thymidylate synthase inhibitor. Cancer Res. 1992 Mar 1;52(5):1137–1143. [PubMed] [Google Scholar]
  30. Parsels L. A., Parsels J. D., Wagner L. M., Loney T. L., Radany E. H., Maybaum J. Mechanism and pharmacological specificity of dUTPase-mediated protection from DNA damage and cytotoxicity in human tumor cells. Cancer Chemother Pharmacol. 1998;42(5):357–362. doi: 10.1007/s002800050829. [DOI] [PubMed] [Google Scholar]
  31. Pestalozzi B. C., Peterson H. F., Gelber R. D., Goldhirsch A., Gusterson B. A., Trihia H., Lindtner J., Cortés-Funes H., Simmoncini E., Byrne M. J. Prognostic importance of thymidylate synthase expression in early breast cancer. J Clin Oncol. 1997 May;15(5):1923–1931. doi: 10.1200/JCO.1997.15.5.1923. [DOI] [PubMed] [Google Scholar]
  32. Rhee M. S., Wang Y., Nair M. G., Galivan J. Acquisition of resistance to antifolates caused by enhanced gamma-glutamyl hydrolase activity. Cancer Res. 1993 May 15;53(10 Suppl):2227–2230. [PubMed] [Google Scholar]
  33. Skelton L. A., Ormerod M. G., Titley J. C., Jackman A. L. Cell cycle effects of CB30865, a lipophilic quinazoline-based analogue of the antifolate thymidylate synthase inhibitor ICI 198583 with an undefined mechanism of action. Cytometry. 1998 Sep 1;33(1):56–66. doi: 10.1002/(sici)1097-0320(19980901)33:1<56::aid-cyto7>3.0.co;2-9. [DOI] [PubMed] [Google Scholar]
  34. Spears C. P., Gustavsson B. G., Mitchell M. S., Spicer D., Berne M., Bernstein L., Danenberg P. V. Thymidylate synthetase inhibition in malignant tumors and normal liver of patients given intravenous 5-fluorouracil. Cancer Res. 1984 Sep;44(9):4144–4150. [PubMed] [Google Scholar]
  35. Strahler J. R., Zhu X. X., Hora N., Wang Y. K., Andrews P. C., Roseman N. A., Neel J. V., Turka L., Hanash S. M. Maturation stage and proliferation-dependent expression of dUTPase in human T cells. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):4991–4995. doi: 10.1073/pnas.90.11.4991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. van Triest B., Pinedo H. M., van Hensbergen Y., Smid K., Telleman F., Schoenmakers P. S., van der Wilt C. L., van Laar J. A., Noordhuis P., Jansen G. Thymidylate synthase level as the main predictive parameter for sensitivity to 5-fluorouracil, but not for folate-based thymidylate synthase inhibitors, in 13 nonselected colon cancer cell lines. Clin Cancer Res. 1999 Mar;5(3):643–654. [PubMed] [Google Scholar]

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