Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Protection of hematopoietic stem cells from pemetrexed toxicity by retroviral gene transfer with a mutant dihydrofolate reductase-mutant thymidylate synthase fusion gene

Cancer Gene Therapy volume 11, pages 767–773 (2004)Cite this article

Abstract

Myelosuppression is one of the major side effects of most anticancer drugs. To confer myeloprotection, our laboratory generated drug-resistant mutants of select target human enzymes for gene transfer to the bone marrow. Mutants of two of these enzymes, dihydrofolate reductase (DHFR F/S) and thymidylate synthase (TS G52S), were previously shown to confer resistance to methotrexate and 5-FU, respectively, and recently a fusion cDNA of both mutant enzymes (DHFR F/S-TS G52S) was shown to confer dual resistance to both antimetabolites. In this study, we examined the sensitivity of the DHFR F/S-TS G52S fusion protein to the multitargeted antifolate, pemetrexed (LY231514, Alimta), which targets both DHFR and TS and is currently in phase III trials for the treatment of solid tumors and in combination with cisplatin has been shown to be an advance in the treatment of mesothelioma. The Ki for the DHFR F/S portion of the purified fusion protein to pemetrexed was increased by greater than 9000-fold when compared to wtDHFR (8000 versus 0.86 nM), while the Ki for the TS G52S portion of the fusion protein to pemetrexed was similar to that of wtTS (2.8 versus 3.1 nM). When the fusion gene was retrovirally transduced into NIH 3T3 fibroblasts, the IC50 to pemetrexed was three- to four-fold higher than cells transduced with DHFR F/S or TS G52S alone (163 versus 53 and 45 nM, respectively). Similarly, expression of the DHFR F/S–TS G52S fusion gene in retrovirally transduced mouse marrow cells resulted in an increased survival of CFU-GM colonies when compared to cells transduced with either of the mutants alone. Co-expression of mutant DHFR and TS enzymes has additive effects in conferring resistance to pemetrexed-induced toxicity. This construct may be useful for conferring myeloprotection to patients receiving this drug.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4

Similar content being viewed by others

References

  1. Capiaux GM, Budak-Alpdogan T, Takebe N, et al. Retroviral transduction of a mutant dihydrofolate reductase thymidylate synthase fusion gene into murine marrow cells confers resistance to both methotrexate and 5-fluorouracil. Human Gene Ther. 2003;14:435–446.

    Article  CAS  Google Scholar 

  2. Shih C, Chen VJ, Gossett LS, et al. LY231514, a pyrrolo[2,3-d]pyrimidine-based antifolate that inhibits multiple folate-requiring enzymes. Cancer Res. 1997;57:1116–1123.

    CAS  PubMed  Google Scholar 

  3. Curtin NJ, Hughes AN . Pemetrexed disodium, a novel antifolate with multiple targets. Lancet Oncol. 2001;2:298–306.

    Article  CAS  Google Scholar 

  4. Teicher BA, Chen V, Shih C, et al. Treatment regimens including the multitargeted antifolate LY231514 in human tumor xenografts. Clin Cancer Res. 2000;6:1016–1023.

    CAS  PubMed  Google Scholar 

  5. Banerjee D, Bertino JR . Myeloprotection with drug resistance genes. Lancet Oncol. 2002;3:154–158.

    Article  CAS  Google Scholar 

  6. Bonadonna G, Zambetti M, Valagussa P . Sequential or alternating Doxorubicin and CMF regimens in breast cancer with more than 3 positive nodes. 10 year results. JAMA. 1995;273:542–547.

    Article  CAS  Google Scholar 

  7. Bertino JR . Biomodulation of 5-fluorouracil with antifolates. Semin Oncol. 1997;24:S18-52–S18-56.

  8. Nordic Gastrointestinal Tumor Adjuvant Therapy Group. Superiority of sequential methotrexate, fluorouracil, and leucovorin to fluorouracil alone in advanced symptomatic colorectal carcinoma: a randomized trial. J Clin Oncol. 1989;7:1437–1446.

  9. de Mulder PH . The chemotherapy of head and neck cancer. Anticancer Drugs. 1999;10:S33–S37.

    Article  CAS  Google Scholar 

  10. Markowitz D, Goff S, Band A . Construction and use of a safe and efficient amphotropic packaging cell line. Virology. 1988;167:400–406.

    Article  CAS  Google Scholar 

  11. Hillcoat BL, Nixon PF, Blakley RL . Effect of substrate decomposition on the spectrophotometric assay of dihydrofolate reductase. Anal Biochem. 1967;21:179–189.

    Article  Google Scholar 

  12. Ellis KJ, Morrison JF . Buffers of constant ionic strength for studying pH dependent processes. Methods Enzymol. 1982;87:405–426.

    Article  CAS  Google Scholar 

  13. Wahba AJ, Friedkin M . Direct spectophotometric evidence for the oxidation of tetrahydrofolate during the enzymatic synthesis of thymidylate. J Biol Chem. 1961;236:11–12.

    Google Scholar 

  14. Ercikan-Abali EA, Waltham MC, Dicker AP, et al. Variants of human dihydrofolate reductase with substitutions at leucine-22: effect on catalytic and inhibitor binding properties. Mol Pharmacol. 1996;49:430–437.

    CAS  PubMed  Google Scholar 

  15. Tong Y, Liu-Chen X, Ercikan-Abali EA, Capiaux GM, Banerjee D, Bertino JR . Isolation and characterization of thymitaq (AG337) and 5-fluoro-2-deoxyuridylate-resistant mutants of human thymidylate synthase from ethyl methanesulfonate-exposed human sarcoma HT1080 cells. J Biol Chem. 1998a;273:11611–11618.

    Article  CAS  Google Scholar 

  16. Williams JW, Morrison JF . The kinetics of reversible tight binding inhibition. Methods Enzymol. 1979;63:437–467.

    Article  CAS  Google Scholar 

  17. Williams JW, Morrison JF, Duggleby RG . Methotrexate, a high affinity pseudosubstrate of dihydrofolate reductase. Biochemistry. 1979;18:2567–2573.

    Article  CAS  Google Scholar 

  18. Duggleby RG . Regression analysis of nonlinear Arrhenius plots: an empirical model and a computer program. Comput Biol Med. 1984;14:447–455.

    Article  CAS  Google Scholar 

  19. Allay JA, Persons DA, Galipeau J, et al. In vivo selection of retrovirally transduced hematopoietic stem cells. Nat Med. 1998;4:1136–1143.

    Article  CAS  Google Scholar 

  20. Ercikan-Abali EA, Mineishi S, et al. Active site-directed double mutants of dihydrofolate reductase. Cancer Res. 1996a;56:4142–4145.

    CAS  Google Scholar 

  21. Mineishi S, Nakahara S, Takebe N, Banerjee D, Zhao SC, Bertino JR . Co-expression of the herpes simplex virus thymidine kinase gene potentiates methotrexate resistance conferred by transfer of a mutated dihydrofolate reductase gene. Gene Therapy. 1997;4:570–576.

    Article  CAS  Google Scholar 

  22. Tong Y, Liu-Chen X, Ericikan-Abali EA, et al. Probing the folate-binding site of human thymidylate synthase by site-directed mutagenesis. J Biol Chem. 1998b;273:31209–31214.

    Article  CAS  Google Scholar 

  23. Sauerbrey A, McPherson JP, Ahao S, Banerjee D, Bertino JR . Expression of a novel double-mutant dihydrofolate reductase-cytidine deaminase fusion gene confers resistance to both methotrexate and cytosine arabinoside. Hum Gene Ther. 1999;10:2495–2504.

    Article  CAS  Google Scholar 

  24. Takebe N, Zhao SC, Ural AU, et al. Retroviral transduction of human dihydropyrimidine dehydrogenase cDNA confers resistance to 5-fluorouracil in murine hematopoietic progenitor cells and human CD 34+-enriched peripheral blood progenitor cells. Cancer Gene Ther. 2001;8:966–973.

    Article  CAS  Google Scholar 

  25. Coutre LA, Mullen CA, Morgan RA . Retroviral vectors containing chimeric promoter/enhancer elements exhibit cell-type-specific gene expression. Hum Gene Ther. 1994;5:667–677.

    Article  Google Scholar 

  26. Paz-Ares L, Bezares S, Tabernero JM, Castellanos D, Cortes-Funes H . Review of a promising new agent — pemetrexed disodium. Cancer. 2003;97(Suppl):2056–2063.

    Article  CAS  Google Scholar 

  27. Vogelzang NJ, Rusthoven JJ, Symanowski J, et al. Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma. J Clin Oncol. 2003;21:2636–2644.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants P01 CA59350, RO1 CA-08010, and R01-CA 44355(FM).

Author information

Author notes

  1. Naoko Takebe

    Present address: Department of Medicine and Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD, 21201, USA

  2. Debabrata Banerjee & Joseph R Bertino

    Present address: Cancer Institute of New Jersey, Robert Wood Johnson Medical School, 195 Little Albany Street, New Brunswick, NJ, 08903, USA

Authors and Affiliations

  1. Graduate School of Medical Sciences, Cornell University, New York, 10021, NY, USA

    Gina M Capiaux & Joseph R Bertino

  2. Program of Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, 10021, NY, USA

    Gina M Capiaux, Tulin Budak-Alpdogan, Onder Alpdogan, William Bornmann, Naoko Takebe, Debabrata Banerjee & Joseph R Bertino

  3. New York State Department of Health, Wadsworth Center, Albany, 12201, NY, USA

    Frank Maley

Authors
  1. Gina M Capiaux
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tulin Budak-Alpdogan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Onder Alpdogan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. William Bornmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Naoko Takebe
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Debabrata Banerjee
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Frank Maley
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Joseph R Bertino
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Joseph R Bertino.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Capiaux, G., Budak-Alpdogan, T., Alpdogan, O. et al. Protection of hematopoietic stem cells from pemetrexed toxicity by retroviral gene transfer with a mutant dihydrofolate reductase-mutant thymidylate synthase fusion gene. Cancer Gene Ther 11, 767–773 (2004). https://doi.org/10.1038/sj.cgt.7700683

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.cgt.7700683

Keywords

This article is cited by

Search

Advanced search

Quick links