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:

Hematoprotection and enrichment of transduced cells in vivo after gene transfer of MGMTP140K into hematopoietic stem cells

Cancer Gene Therapy volume 9, pages 737–746 (2002)Cite this article

Abstract

The overexpression of mutant forms of O6-methylguanine-DNA-methyltransferase (MGMT), resistant to the MGMT inhibitor O6-benzylguanine (BG), protects hematopoietic cells from the toxicity of combined BG plus O6-alkylating agent chemotherapy. To evaluate the feasibility of this approach for clinically relevant O6-alkylating agents, combined therapy with BG and two chloroethylnitrosourea-type drugs, ACNU or BCNU, or the triazene derivative temozolomide (TMZ) was investigated in a murine bone marrow transplant model allowing transgenic expression of the highly BG-resistant MGMTP140K mutant. Whereas 20/20 control animals transplanted with nontransduced cells died of progressive myelosuppression during therapy, nearly all animals transplanted with MGMTP140K-transduced cells survived treatment with BG/ACNU (12/15), BG/TMZ (10/10), or BG/BCNU (5/5). In surviving animals, hematological parameters improved during chemotherapy and pretreatment levels were reestablished during or shortly after therapy. All animals showed enrichment of transgenic granulocytes (range: 15- to 101-fold) and lymphocytes (range: 16- to 55-fold) in peripheral blood, bone marrow, and spleen. No significant differences were observed between individual treatment groups. Serial transplants demonstrated protection in secondary recipients and confirmed the transduction of transplantable stem cells. Thus, these data demonstrate efficient protection from hematotoxicity and substantial enrichment of transgenic cells following MGMTP140K gene transfer and treatment with different O6-alkylating drugs.

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
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Koc ON, Allay JA, Lee K et al. Transfer of drug resistance genes into hematopoietic progenitors to improve chemotherapy tolerance Semin Oncol 1996 23: 46–65

    CAS  PubMed  Google Scholar 

  2. Maze R, Hanenberg H, Williams DA . Establishing chemoresistance in hematopoietic progenitor cells Mol Med Today 1997 3: 350–358

    Article  CAS  PubMed  Google Scholar 

  3. Moritz T, Williams DA . Transfer of drug resistance genes to hematopoietic precursors In: Bertino JR, ed Encyclopedia of Cancer vol 3: 2nd ed New York: Academic Press 1996 1665–1676

    Google Scholar 

  4. Moritz T, Williams DA . Marrow protection — transduction of hematopoietic cells with drug resistance genes Cytotherapy 2001 3: 67–84

    Article  CAS  PubMed  Google Scholar 

  5. Brent TP, Remack JS . Formation of covalent complexes between human O6-alkylguanine-DNA alkyltransferase and BCNU-treated defined length synthetic oligodeoxynucleotides Nucleic Acids Res 1988 16: 6779–6788

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Gerson SL, Miller K, Berger NA . O6-alkylguanine-DNA alkyltransferase activity in human myeloid cells J Clin Invest 1985 76: 2106–2114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Gerson SL, Trey JE, Miller K et al. Comparison of O6-alkylguanine-DNA alkyltransferase activity based on cellular DNA content in human, rat and mouse tissues Carcinogenesis 1986 7: 745–749

    Article  CAS  PubMed  Google Scholar 

  8. Gerson SL, Phillips W, Kastan M et al. Human CD34+ hematopoietic progenitors have low, cytokine-unresponsive O6-alkylguanine-DNA alkyltransferase and are sensitive to O6-benzylguanine plus BCNU Blood 1996 88: 1649–1655

    CAS  PubMed  Google Scholar 

  9. Moritz T, Mackay W, Glassner BJ et al. Retrovirus-mediated expression of a DNA repair protein in bone marrow protects hematopoietic cells from nitrosourea-induced toxicity in vitro and in vivo Cancer Res 1995 55: 2608–2614

    CAS  PubMed  Google Scholar 

  10. Wang G, Weiss C, Sheng P et al. Retrovirus-mediated transfer of the human O6-methylguanine-DNA methyltransferase gene into a murine hematopoietic stem cell line and resistance to the toxic effects of certain alkylating agents Biochem Pharmacol 1996 51: 1221–1228

    Article  CAS  PubMed  Google Scholar 

  11. Jelinek J, Fairbairn LJ, Dexter TM et al. Long-term protection of hematopoiesis against the cytotoxic effects of multiple doses of nitrosourea by retrovirus-mediated expression of human O6-alkylguanine-DNA-alkyltransferase Blood 1996 87: 1957–1961

    CAS  PubMed  Google Scholar 

  12. Allay JA, Dumenco LL, Koc ON et al. Retroviral transduction and expression of the human alkyltransferase cDNA provides nitrosourea resistance to hematopoietic cells Blood 1995 85: 3342–3351

    CAS  PubMed  Google Scholar 

  13. Allay JA, Davis BM, Gerson SL . Human alkyltransferase-transduced murine myeloid progenitors are enriched in vivo by BCNU treatment of transplanted mice Exp Hematol 1997 25: 1069–1076

    CAS  PubMed  Google Scholar 

  14. Maze R, Carney JP, Kelley MR et al. Increasing DNA repair methyltransferase levels via bone marrow stem cell transduction rescues mice from the toxic effects of 1,3-bis 2-chloroethyl)-1-nitrosourea, a chemotherapeutic alkylating agent Proc Natl Acad Sci USA 1996 93: 206–210

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Maze R, Kapur R, Kelley MR et al. Reversal of 1,3-bis (2-chloroethyl)-1-nitrosourea–induced severe immunodeficiency by transduction of murine long-lived hemopoietic progenitor cells using O6-methylguanine DNA methyltransferase complementary DNA J Immunol 1997 158: 1006–1013

    CAS  PubMed  Google Scholar 

  16. Dolan ME, Mitchell RB, Mummert C et al. Effect of O6-benzylguanine analogues on sensitivity of human tumor cells to the cytotoxic effects of alkylating agents Cancer Res 1991 51: 3367–3372

    CAS  PubMed  Google Scholar 

  17. Spiro TP, Gerson SL, Liu L et al. O6-benzylguanine: a clinical trial establishing the biochemical modulatory dose in tumor tissue for alkyltransferase-directed DNA repair Cancer Res 1999 59: 2402–2410

    CAS  PubMed  Google Scholar 

  18. Dolan ME, Roy SK, Fasanmade AA et al. O6-benzylguanine in humans: metabolic, pharmacokinetic, and pharmacodynamic findings J Clin Oncol 1998 16: 1803–1810

    Article  CAS  PubMed  Google Scholar 

  19. Koc ON, Reese JS, Szekely EM et al. Human long-term culture initiating cells are sensitive to benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea and protected after mutant (G156A) methylguanine methyltransferase gene transfer Cancer Gene Ther 1999 6: 340–348

    Article  CAS  PubMed  Google Scholar 

  20. Reese JS, Davis BM, Liu L et al. Simultaneous protection of G156A methylguanine DNA methyltransferase gene-transduced hematopoietic progenitors and sensitization of tumor cells using O6-benzylguanine and temozolomide Clin Cancer Res 1999 5: 163–169

    CAS  PubMed  Google Scholar 

  21. Sawai N, Zhou S, Vanin EF et al. Protection and in vivo selection of hematopoietic stem cells using temozolomide, O6-benzylguanine, and an alkyltransferase-expressing retroviral vector Mol Ther 2001 3: 78–87

    Article  CAS  PubMed  Google Scholar 

  22. Ragg S, Xu-Welliver M, Bailey J et al. Direct reversal of DNA damage by mutant methyltransferase protein protects mice against dose-intensified chemotherapy and leads to in vivo selection of hematopoietic stem cells Cancer Res 2000 60: 5187–5195

    CAS  PubMed  Google Scholar 

  23. Davis BM, Koc ON, Gerson SL . Limiting numbers of G156A O(6)-methylguanine-DNA methyltransferase–transduced marrow progenitors repopulate nonmyeloablated mice after drug selection Blood 2000 95: 3078–3084

    CAS  PubMed  Google Scholar 

  24. Wilczynski SW, Erasmus JJ, Petros WP et al. Delayed pulmonary toxicity syndrome following high-dose chemotherapy and bone marrow transplantation for breast cancer Am J Respir Crit Care Med 1998 157: 565–573

    Article  CAS  PubMed  Google Scholar 

  25. Bailey CC, Marsden HB, Jones PH . Fatal pulmonary fibrosis following 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) therapy Cancer 1978 42: 74–76

    Article  CAS  PubMed  Google Scholar 

  26. Colvin M, Cowens JW, Brundrett RB et al. Decomposition of BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) in aqueous solution Biochem Biophys Res Commun 1974 60: 515–520

    Article  CAS  PubMed  Google Scholar 

  27. Vijayan VK, Sankaran K . Relationship between lung inflammation, changes in lung function and severity of exposure in victims of the Bhopal tragedy Eur Respir J 1977 9: 1977

    Article  Google Scholar 

  28. Plowman J, Waud WR, Koutsoukos AD et al. Preclinical antitumor activity of temozolomide in mice: efficacy against human brain tumor xenografts and synergism with 1,3-bis (2-chloroethyl)-1-nitrosourea Cancer Res 1994 54: 3793–3799

    CAS  PubMed  Google Scholar 

  29. Friedman HS, Dolan ME, Pegg AE et al. Activity of temozolomide in the treatment of central nervous system tumor xenografts Cancer Res 1995 55: 2853–2857

    CAS  PubMed  Google Scholar 

  30. Hawley RG, Lieu FH, Fong AZ et al. Versatile retroviral vectors for potential use in gene therapy Gene Ther 1994 1: 136–138

    CAS  PubMed  Google Scholar 

  31. Dick JE, Magli MC, Huszar D et al. Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice Cell 1985 42: 71–79

    Article  CAS  PubMed  Google Scholar 

  32. Hanenberg H, Xiao XL, Dilloo D et al. Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells Nat Med 1996 2: 876–882

    Article  CAS  PubMed  Google Scholar 

  33. Bierhuizen MF, Westerman Y, Visser TP et al. Enhanced green fluorescent protein as selectable marker of retroviral-mediated gene transfer in immature hematopoietic bone marrow cells Blood 1997 90: 3304–3315

    CAS  PubMed  Google Scholar 

  34. Flasshove M, Bardenheuer W, Schneider A et al. Type and position of promoter elements in retroviral vectors have substantial effects on the expression level of an enhanced green fluorescent protein reporter gene J Cancer Res Clin Oncol 2000 126: 391–399

    Article  CAS  PubMed  Google Scholar 

  35. Preuss I, Haas S, Eichhorn U et al. Activity of the DNA repair protein O6-methylguanine-DNA methyltransferase in human tumor and corresponding normal tissue Cancer Detect Prev 1996 20: 130–136

    CAS  PubMed  Google Scholar 

  36. Silber JR, Bobola MS, Ghatan S et al. O6-methylguanine-DNA methyltransferase activity in adult gliomas: relation to patient and tumor characteristics Cancer Res 1998 58: 1068–1073

    CAS  PubMed  Google Scholar 

  37. Magull-Seltenreich A, Zeller WJ . Sensitization of human colon tumour cell lines to carmustine by depletion of O6-alkylguanine-DNA alkyltransferase J Cancer Res Clin Oncol 1995 121: 225–229

    Article  CAS  PubMed  Google Scholar 

  38. Dolan ME, Stine L, Mitchell RB et al. Modulation of mammalian O6-alkylguanine-DNA alkyltransferase in vivo by O6-benzylguanine and its effect on the sensitivity of a human glioma tumor to 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea Cancer Commun 1990 2: 371–377

    Article  CAS  PubMed  Google Scholar 

  39. Harris LC, Marathi UK, Edwards CC et al. Retroviral transfer of a bacterial alkyltransferase gene into murine bone marrow protects against chloroethylnitrosourea cytotoxicity Clin Cancer Res 1995 1: 1359–1368

    CAS  PubMed  Google Scholar 

  40. Davis BM, Reese JS, Koc ON et al. Selection for G156A O6-methylguanine DNA methyltransferase gene-transduced hematopoietic progenitors and protection from lethality in mice treated with O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea Cancer Res 1997 57: 5093–5099

    CAS  PubMed  Google Scholar 

  41. Xu-Welliver M, Kanugula S, Pegg AE . Isolation of human O6-alkylguanine-DNA alkyltransferase mutants highly resistant to inactivation by O6-benzylguanine Cancer Res 1998 58: 1936–1945

    CAS  PubMed  Google Scholar 

  42. Davis BM, Roth JC, Liu L et al. Characterization of the P140K, PVP (138–140)MLK, and G156A O6-methylguanine-DNA methyltransferase mutants: implications for drug resistance gene therapy Hum Gene Ther 1999 10: 2769–2778

    Article  CAS  PubMed  Google Scholar 

  43. Koc ON, Reese JS, Davis BM et al. DeltaMGMT-transduced bone marrow infusion increases tolerance to O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea and allows intensive therapy of 1,3-bis(2-chloroethyl)-1-nitrosourea–resistant human colon cancer xenografts Hum Gene Ther 1999 10: 1021–1030

    Article  CAS  PubMed  Google Scholar 

  44. Preuss I, Thust R, Kaina B . Protective effect of O6-methylguanine-DNA methyltransferase (MGMT) on the cytotoxic and recombinogenic activity of different antineoplastic drugs Int J Cancer 1996 65: 506–512

    Article  CAS  PubMed  Google Scholar 

  45. Jansen M, Bardenheuer W, Sorg UR et al. Protection of hematopoietic cells from O6-alkylation damage by O6-methylguanine DNA methyltransferase gene transfer: studies with different O6-alkylating agents and retroviral backbones Eur J Haematol 2001 67: 2–13

    Article  CAS  PubMed  Google Scholar 

  46. Stahl W, Eisenbrand G . Comparative study on the influence of two 2-chloroethylnitrosoureas with different carbamoylating potential towards glutathione and glutathione-related enzymes in different organs of the rat Free Radical Res Commun 1991 14: 271–278

    Article  CAS  Google Scholar 

  47. Hanenberg H, Hashino K, Konishi H et al. Optimization of fibronectin-assisted retroviral gene transfer into human CD34+ hematopoietic cells Hum Gene Ther 1997 8: 2193–2206

    Article  CAS  PubMed  Google Scholar 

  48. Kiem HP, Morris J, Heyward S et al. Gene transfer into baboon hematopoietic repopulating cells using recombinant human fibronectin fragment CH-296 Blood 1997 90: 236a

    Google Scholar 

  49. Moritz T, Dutt P, Xiao X et al. Fibronectin improves transduction of reconstituting hematopoietic stem cells by retroviral vectors: evidence of direct viral binding to chymotryptic carboxy-terminal fragments Blood 1996 88: 855–862

    CAS  PubMed  Google Scholar 

  50. Abonour R, Williams DA, Einhorn L et al. Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells Nat Med 2000 6: 652–658

    Article  CAS  PubMed  Google Scholar 

  51. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease Science 2000 288: 669–672

    Article  CAS  PubMed  Google Scholar 

  52. Schiedlmeier B, Kühlcke K, Eckert HG et al. Quantitative assessment of retroviral transfer of the human multidrug resistance 1 gene to mobilized peripheral blood progenitor cells engrafted in nonobese diabetic/severe combined immunodeficient mice Blood 2000 95: 1237–1248

    CAS  PubMed  Google Scholar 

  53. Peters WP, Dansey R . New concepts in the treatment of breast cancer using high-dose chemotherapy Cancer Chemother Pharmacol 1997 40: Suppl S88–S93

    Article  PubMed  Google Scholar 

  54. Weiss RB . Introduction: dose-intensive therapy for adult malignancies Semin Oncol 1999 26: 1–5

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank A Feldmann and M Stuhl for excellent technical assistance and I Demirer and C Wartchow for their help in preparing the manuscript. rhIL-6 was a generous gift from Novartis, and TMZ was kindly supplied by Schering-Plough. The work was supported by Grant W 2/95 Op of “Deutsche Krebshilfe, Dr Mildred Scheel Stiftung” to MF, and a grant of Deutsche Forschungsgesellschaft (DFG), Forschergruppe “Selektive Therapie und Therapieresistenz: Grundlagen und Klinik” to MF and TM.

Author information

Authors and Affiliations

  1. Department of Internal Medicine (Cancer Research), West German Cancer Center, University of Essen Medical School, Essen, Germany

    Michael Jansen, Ursula R Sorg, Michael Flasshove, Siegfried Seeber & Thomas Moritz

  2. Herman B Wells Center of Pediatric Research, Howard Hughes Medical Institute, Indianapolis, Indiana, USA

    Susanne Ragg & David A Williams

Authors
  1. Michael Jansen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ursula R Sorg
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Susanne Ragg
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michael Flasshove
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Siegfried Seeber
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. David A Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Thomas Moritz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Thomas Moritz.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jansen, M., Sorg, U., Ragg, S. et al. Hematoprotection and enrichment of transduced cells in vivo after gene transfer of MGMTP140K into hematopoietic stem cells. Cancer Gene Ther 9, 737–746 (2002). https://doi.org/10.1038/sj.cgt.7700490

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.cgt.7700490

Keywords

This article is cited by

Search

Advanced search

Quick links