Skip to main content

Advertisement

Log in

Cytotoxic and biochemical implications of combining AZT and AG-331

  • Original Article
  • Published:
Cancer Chemotherapy and Pharmacology Aims and scope Submit manuscript

Abstract

We have reported that noncytotoxic concentrations of 3′-azido-3′-deoxythymidine (AZT) increase the cytotoxicity of ICI Dl694, a folate-based thymidylate synthase (TS) inhibitor, with increasing AZT incorporation into DNA. We postulated that the inhibition of TS by ICI D1694 would decrease 5′-deoxythymidine triphosphate (dTTP) pools, which compete with AZT triphosphate (AZT-TP) as a substrate for DNA polymerase. Furthermore, the inhibition of TS would increase the activity of both thymidine kinase (TK) and thymidylate kinase (TdK). Each of these consequences of TS inhibition would favor more incorporation of AZT into DNA. Thus, we reasoned that other TS inhibitors should also result in increased AZT incorporation into DNA and, perhaps, in increased cytotoxicity. N 6-[4-(Morpholinosulfonyl) benzyl]-N 6-methyl-2,6-diaminobenz[cd]indole glucuronate (AG-331) differs from ICI D1694 in that it is a de novo designed lipophilic TS inhibitor, it does not require a specific carrier for cellular uptake, and it does not undergo intracellular polyglutamation. This potent TS inhibitor causes minimal cytotoxicity in MGH-U1 human bladder cancer cells. A 24-h exposure to 5 μM AG-331 causes almost complete TS inhibition but only 35% cell kill. The combination of AZT and AG-331 in MGH-U1 cells resulted in an enhanced antitumor effect relative to that of each agent alone; 50 μM AZT, noncytotoxic alone, increased the cell kill of induced by AG-331 from 35% to 50%. Biochemical studies of this combination revealed that simultaneous treatment with 5 μM AG-331 plus 1.8 μM [3H]-AZT produced as much as a 68% ± 7% increase in AZT incorporation into DNA. This observation was associated with an increase in DNA single-strand breaks, measured as comet tail moment, of up to 6.6-fold. These studies support our original premise that TS inhibition favors increased incorporation of AZT into DNA and that the combination causes more cell kill than either drug alone in MGH-U1 cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

AZT:

3′-Azido-3′-deoxythymidine

TS:

thymidylate synthase

dTTP:

5′-deoxythymidine triphosphate

AZT-TP:

AZT triphosphate

TK:

thymidine kinase

TdK:

thymidylate kinas

AG-331:

N 6-[4-(morpholinosulfonyl)benzyl]-N 6-methyl-2,6-diaminobenz[cd]indole glucuronate

dTMP:

thymidylate

TdR:

thymidine

References

  1. Danenberg PV (1977) Thymidylate synthetase: a target enzyme in cancer chemotherapy. Biochim Biophys Acta 473: 73

    PubMed  CAS  Google Scholar 

  2. Houghton JA, Houghton PJ, Woonten RS (1979) Mechanism of induction of gastrointestinal toxicity in the mouse by 5-fluorouracil, 5-fluorouridine and 5-fluoro-2′-deoxyuridine. Cancer Res 39: 2406

    PubMed  CAS  Google Scholar 

  3. Weber G (1983) Biochemical strategy of cancer cells and the design of chemotherapy: G.H.A. Clowes Memorial Lecture. Cancer Res 43: 3466

    CAS  Google Scholar 

  4. Pressacco J, Erlichman C (1993) Combination studies with 3′-azido-3′-deoxythymidine (AZT) plus ICI D1694: cytotoxic and biochemical effects. Biochem Pharmacol 46: 1989

    Article  PubMed  CAS  Google Scholar 

  5. Darnowski JW, Goulette FA (1993) Increased azido-deoxythymidine metabolism in the presence of fluorouracil reflects increased thymidine kinase activity. Proc Am Assoc Cancer Res 34: 302

    Google Scholar 

  6. Jones TR, Varney MD, Webber SE, Welsh KM, Webber S, Matthews DA, Appelt K, Smith WS, Janson C, Bacquet R, Lewis KK, Marzoni GP, Kathardekar V, Howland E, Booth C, Herrmann S, Ward R, Sharp J, Moomaw E, Bartlett C, Morse C (1990) New lipophilic thymidylate synthase inhibitors designed from the X-ray structure of the E. coll enzyme. Proc Am Assoc Cancer Res 31: 340

    Google Scholar 

  7. Nicander B, Reichard P (1983) Dynamics of pyrimidine deoxynucleoside triphosphate pools in relationship to DNA synthesis in 3T6 mouse fibroblasts. Proc Natl Acad Sci, USA 80:1347

    Article  PubMed  CAS  Google Scholar 

  8. Erlichman C, Vidgen D (1984) Cytotoxicity of adriamycin in MGH-U1 cells grown as monolayer cultures, spheroids and xenografts in immune-deprived mice. Cancer Res 44: 5369

    PubMed  CAS  Google Scholar 

  9. Mackillop WJ, Stewart SS, Buick RN (1982) Density/volume analysis in the study of cellular heterogeneity in human ovarian carcinoma. Br J Cancer 45: 812

    PubMed  CAS  Google Scholar 

  10. Chu M-Y, Fischer GA (1968) The incorporation of 3H-cytosine arabinoside and its effect on murine leukemic cells (L5178Y). Biochem Pharmacol 17: 553

    Google Scholar 

  11. Olive PL, Banath JP, Evans HH (1993) Cell killing and DNA damage by etoposide in Chinese hamster V79 monolayers and spheroids: influence of growth kinetics, growth environment and DNA packaging. Br J Cancer 67: 522

    PubMed  CAS  Google Scholar 

  12. Olive PL, Banath JP, Durand RE (1990) Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the “comet” assay. Radiat Res 122: 86

    Article  PubMed  CAS  Google Scholar 

  13. Tosi P, Calabresi P, Goulette FA, Renaud CA, Darnowski JW (1992) Azidothymidine-induced cytotoxicity and incorporation into DNA in the human colon tumor cell line HCT-8 is enhanced by methotrexate in vitro and in vivo. Cancer Res 52: 4069

    PubMed  CAS  Google Scholar 

  14. Frick LW, Nelson DJ, St Clair MH, Furman PA, Krenitsky TA (1988) Effects of 3′-azido-3′-deoxythymidine on the deoxynuc-leotide triphosphate pools of cultured human cells. Biochem Biophys Res Commun 154: 124

    Article  PubMed  CAS  Google Scholar 

  15. Sommadossi J-P, Carlisle R, Zhou Z (1989) Cellular pharmacology of 3′-azido-3′-deoxythymidine with evidence of incorporation into DNA of human bone marrow cells. Mol Pharmacol 36:9

    PubMed  CAS  Google Scholar 

  16. Vazquez-Padua MA, Starnes MC, Cheng Y-C (1990) Incorporation of 3′-azido-3′-deoxythymidine into cellular DNA and its removal in a human leukemic cell line. Cancer Commun 2:55

    PubMed  CAS  Google Scholar 

  17. Furman PA, Fyfe JA, St Clair MH, Weinhold K, Rideout JL, Freeman GA, Lehrman SN, Bolognesi DP, Broder S, Mitsuya H, Barry DW (1986) Phosphorylation of 3′-azido-3′-deoxy-thymidine and selective interaction of 5′-triphosphate with human immunodeficiency virus reverse transcriptase. Proc Natl Acad Sci USA 83: 8333

    Article  PubMed  CAS  Google Scholar 

  18. Pressacco J, Hedley DW, Hu Q, Chow S, Newcombe D, Erlichman C (1994) D1694 biochemical modulation of idoxuridine (IUDR) causes synergistic cytotoxicity. Proc Am Assoc Cancer Res 35: 326

    Google Scholar 

  19. Mitrovski B, Johnston PG, Erlichman C (1994) Cytotoxic and biochemical effects of a lipophilic (AG-331) and a non-lipophilic (D1694) thymidylate synthase inhibitor in MGH-U1 cells. Proc Am Assoc Cancer Res 35: 300

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Additional information

Supported by a grant from the NCI (Canada)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pressacco, J., Mitrovski, B. & Erlichman, C. Cytotoxic and biochemical implications of combining AZT and AG-331. Cancer Chemother. Pharmacol. 35, 387–390 (1995). https://doi.org/10.1007/s002800050251

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s002800050251

Key words

Navigation