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. 1997;76(12):1579–1585. doi: 10.1038/bjc.1997.600

The relationship between intrinsic thymidylate synthase expression and sensitivity to THYMITAQ in human leukaemia and colorectal carcinoma cell lines.

E J Estlin 1, K Balmanno 1, A H Calvert 1, A G Hall 1, J Lunec 1, D R Newell 1, A D Pearson 1, G A Taylor 1
PMCID: PMC2228199  PMID: 9413945

Abstract

Thymidylate synthase (TS) expression has been characterized for a panel of eight human colorectal carcinoma and five human leukaemia cell lines, to relate differences in intrinsic TS activity, protein and mRNA levels to growth inhibition caused by continuous exposure to THYMITAQ, a specific non-classical antifolate TS inhibitor. Although a 20-fold variation in sensitivity to THYMITAQ was found within the colorectal cell line panel (IC50 0.12-2.7 microM), sensitivity was not related to TS activity, TS protein or TS mRNA levels. For the leukaemic cell lines, only a twofold range in sensitivity to THYMITAQ was observed (IC50 0.87-2.3 microM), and this did not correlate with TS activity, TS protein or TS mRNA levels. Across all of the cell lines, TS activity was linearly related to TS protein levels (r2 = 0.87, P < 0.0001). However, for both the colorectal and leukaemia cell line panels, no relationship was found between TS mRNA/18S rRNA ratios and either TS activity or TS protein, consistent with the importance of post-transcriptional mechanisms in regulating TS activity. Two of the colorectal cell lines (BE and HCT116) and one of the human leukaemic cell lines (HL60), were intrinsically resistant to THYMITAQ (IC50 > 2 microM) in the absence of TS overexpression, suggesting that, subsequent to TS inhibition, events such as DNA repair and tolerance to apoptotic stimuli are also important determinants of sensitivity to THYMITAQ.

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Selected References

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  1. Barbour K. W., Hoganson D. K., Berger S. H., Berger F. G. A naturally occurring tyrosine to histidine replacement at residue 33 of human thymidylate synthase confers resistance to 5-fluoro-2'-deoxyuridine in mammalian and bacterial cells. Mol Pharmacol. 1992 Aug;42(2):242–248. [PubMed] [Google Scholar]
  2. Berger S. H., Barbour K. W., Berger F. G. A naturally occurring variation in thymidylate synthase structure is associated with a reduced response to 5-fluoro-2'-deoxyuridine in a human colon tumor cell line. Mol Pharmacol. 1988 Oct;34(4):480–484. [PubMed] [Google Scholar]
  3. Berger S. H., Berger F. G. Thymidylate synthase as a determinant of 5-fluoro-2'-deoxyuridine response in human colonic tumor cell lines. Mol Pharmacol. 1988 Oct;34(4):474–479. [PubMed] [Google Scholar]
  4. 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]
  5. Canman C. E., Tang H. Y., Normolle D. P., Lawrence T. S., Maybaum J. Variations in patterns of DNA damage induced in human colorectal tumor cells by 5-fluorodeoxyuridine: implications for mechanisms of resistance and cytotoxicity. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10474–10478. doi: 10.1073/pnas.89.21.10474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Chu E., Koeller D. M., Casey J. L., Drake J. C., Chabner B. A., Elwood P. C., Zinn S., Allegra C. J. Autoregulation of human thymidylate synthase messenger RNA translation by thymidylate synthase. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8977–8981. doi: 10.1073/pnas.88.20.8977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Geyer P. K., Johnson L. F. Molecular cloning of DNA sequences complementary to mouse thymidylate synthase messenger RNA. J Biol Chem. 1984 Jun 10;259(11):7206–7211. [PubMed] [Google Scholar]
  13. Jackman A. L., Calvert A. H. Folate-based thymidylate synthase inhibitors as anticancer drugs. Ann Oncol. 1995 Nov;6(9):871–881. doi: 10.1093/oxfordjournals.annonc.a059353. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Jackman A. L., Taylor G. A., Gibson W., Kimbell R., Brown M., Calvert A. H., Judson I. R., Hughes L. R. ICI D1694, a quinazoline antifolate thymidylate synthase inhibitor that is a potent inhibitor of L1210 tumor cell growth in vitro and in vivo: a new agent for clinical study. Cancer Res. 1991 Oct 15;51(20):5579–5586. [PubMed] [Google Scholar]
  16. Jansen G., Schornagel J. H., Westerhof G. R., Rijksen G., Newell D. R., Jackman A. L. Multiple membrane transport systems for the uptake of folate-based thymidylate synthase inhibitors. Cancer Res. 1990 Dec 1;50(23):7544–7548. [PubMed] [Google Scholar]
  17. Johnston P. G., Drake J. C., Steinberg S. M., Allegra C. J. Quantitation of thymidylate synthase in human tumors using an ultrasensitive enzyme-linked immunoassay. Biochem Pharmacol. 1993 Jun 22;45(12):2483–2486. doi: 10.1016/0006-2952(93)90230-t. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. Peters G. J., Laurensse E., Leyva A., Lankelma J., Pinedo H. M. Sensitivity of human, murine, and rat cells to 5-fluorouracil and 5'-deoxy-5-fluorouridine in relation to drug-metabolizing enzymes. Cancer Res. 1986 Jan;46(1):20–28. [PubMed] [Google Scholar]
  22. Peters G. J., van Groeningen C. J., Laurensse E. J., Pinedo H. M. Thymidylate synthase from untreated human colorectal cancer and colonic mucosa: enzyme activity and inhibition by 5-fluoro-2'-deoxy-uridine-5'-monophosphate. Eur J Cancer. 1991;27(3):263–267. doi: 10.1016/0277-5379(91)90512-c. [DOI] [PubMed] [Google Scholar]
  23. Pinedo H. M., Peters G. F. Fluorouracil: biochemistry and pharmacology. J Clin Oncol. 1988 Oct;6(10):1653–1664. doi: 10.1200/JCO.1988.6.10.1653. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Touroutoglou N., Pazdur R. Thymidylate synthase inhibitors. Clin Cancer Res. 1996 Feb;2(2):227–243. [PubMed] [Google Scholar]
  26. Van der Wilt C. L., Smid K., Aherne G. W., Pinedo H. M., Peters G. J. Evaluation of immunohistochemical staining and activity of thymidylate synthase in cell lines. Adv Exp Med Biol. 1993;338:605–608. doi: 10.1007/978-1-4615-2960-6_124. [DOI] [PubMed] [Google Scholar]
  27. Webber S. E., Bleckman T. M., Attard J., Deal J. G., Kathardekar V., Welsh K. M., Webber S., Janson C. A., Matthews D. A., Smith W. W. Design of thymidylate synthase inhibitors using protein crystal structures: the synthesis and biological evaluation of a novel class of 5-substituted quinazolinones. J Med Chem. 1993 Mar 19;36(6):733–746. doi: 10.1021/jm00058a010. [DOI] [PubMed] [Google Scholar]
  28. Webber S., Bartlett C. A., Boritzki T. J., Hillard J. A., Howland E. F., Johnston A. L., Kosa M., Margosiak S. A., Morse C. A., Shetty B. V. AG337, a novel lipophilic thymidylate synthase inhibitor: in vitro and in vivo preclinical studies. Cancer Chemother Pharmacol. 1996;37(6):509–517. doi: 10.1007/s002800050422. [DOI] [PubMed] [Google Scholar]

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