Abstract
Purpose: Retroviral transfer of cDNA sequences that confer drug resistance can be used to protect against chemotherapy-induced hematopoietic toxicity and for the selective expansion of gene-modified cells. To successfully expand genetically engineered cells in vivo, an appropriate balance must be achieved between systemic toxicity induced by the selecting agent and the expansion of modified cells. Method: In this study, we investigate retroviral transfer of cytosolic 5′-nucleotidase I (cN-I) for protection and selection of gene-modified cells when treated with 2-chloro-2′-deoxyadenosine (2-CdA) and 5-fluorouracil (5-FU) alone and in combination. We also attempt to design a treatment strategy for the potential in vivo selection of cN-I-modified cells by administering 5-FU to mice prior to 2-CdA treatment. Results: Our results show that cN-I can be transferred by recombinant retroviruses, and that enforced expression of cN-I protects murine fibroblast and hematopoietic progenitor cells from the cytotoxic effects of 2-CdA and/or 5-FU. Furthermore, we show that the combined administration of 5-FU and 2-CdA potentiates hematopoietic stem cell toxicity. However, the treatment also results in severe myelosuppression. Conclusion: These results show that while cN-I provides both protective and selective benefits to gene-modified cells in vitro, selection requires a treatment strategy that is likely too toxic to consider cN-I as an in vivo selectable marker
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References
Allay JA, Dumenco LL, Koc ON, Liu L, Gerson SL (1995) Retroviral transduction and expression of the human alkyltransferase cDNA provides nitrosourea resistance to hematopoietic cells. Blood 85(11):3342–3351
Allay JA, Galipeau J, Blakley RL, Sorrentino BP (1998) Retroviral vectors containing a variant dihydrofolate reductase gene for drug protection and in vivo selection of hematopoietic cells. Stem Cells 16(Suppl. 1):223–233
Allay JA, Spencer HT, Wilkinson SL, Belt JA, Blakley RL, Sorrentino BP (1997) Sensitization of hematopoietic stem and progenitor cells to trimetrexate using nucleoside transport inhibitors. Blood 90(9):3546–3554
Beausejour CM, Eliopoulos N, Momparler L, Le NL, Momparler RL (2001) Selection of drug-resistant transduced cells with cytosine nucleoside analogs using the human cytidine deaminase gene. Cancer Gene Ther 8(9):669–676
Blau CA, Neff T, Papayannopoulou T (1996) The hematological effects of folate analogs: implications for using the dihydrofolate reductase gene for in vivo selection. Hum Gene Ther 7(17):2069–2078
Bodine DM, McDonagh KT, Seidel NE, Nienhuis AW (1991) Survival and retrovirus infection of murine hematopoietic stem cells in vitro: effects of 5-FU and method of infection. Exp Hematol 19(3):206–212
Bowman JE, Reese JS, Lingas KT, Gerson SL (2003) Myeloablation is not required to select and maintain expression of the drug-resistance gene, mutant MGMT, in primary and secondary recipients. Mol Ther 8(1):42–50
Capiaux GM, Budak-Alpdogan T, Alpdogan O, Bornmann W, Takebe N, Banerjee D, Maley F, Bertino JR (2004) 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(12):767–773
Carson DA, Kaye J, Wasson DB (1981) The potential importance of soluble deoxynucleotidase activity in mediating deoxyadenosine toxicity in human lymphoblasts. J Immunol 126(1):348–352
Chow KU, Boehrer S, Bojunga J, Stieler M, Rummel MJ, Fauth F, Schneider B, Martin H, Hoelzer D, Weidmann E, Mitrou PS (2002) Induction of apoptosis by cladribine (2-CdA), gemcitabine and other chemotherapeutic drugs on CD34+/CD38+ and CD34+/CD38- hematopoietic progenitor cells: selective effects of doxorubicin and 2-CdA with protection of immature cells. Leuk Lymphoma 43(2):377–384
Eliopoulos N, Al-Khaldi A, Beausejour CM, Momparler RL, Momparler LF, Galipeau J (2002) Human cytidine deaminase as an ex vivo drug selectable marker in gene-modified primary bone marrow stromal cells. Gene Ther 9(7):452–462
Galmarini CM, Graham K, Thomas X, Calvo F, Rousselot P, El Jafaari A, Cros E, Mackey JR, Dumontet C (2001a) Expression of high Km 5′-nucleotidase in leukemic blasts is an independent prognostic factor in adults with acute myeloid leukemia. Blood 98(6):1922–1926
Galmarini CM, Mackey JR, Dumontet C (2001b) Nucleoside analogues: mechanisms of drug resistance and reversal strategies. Leukemia 15(6):875–890
Gora-Tybor J, Robak T (1993) The influence of 2-chlorodeoxyadenosine alone and in combination with cyclophosphamide or methotrexate on normal hematopoiesis in mice. Acta Haematol Pol 24(3):281–287
Greyz N, Saven A (2004) Cladribine: from the bench to the bedside—focus on hairy cell leukemia. Expert Rev Anticancer Ther 4(5):745–757
Guchelaar HJ, Vermes I, Koopmans RP, Reutelingsperger CP, Haanen C (1998) Apoptosis- and necrosis-inducing potential of cladribine, cytarabine, cisplatin, and 5-fluorouracil in vitro: a quantitative pharmacodynamic model. Cancer Chemother Pharmacol 42(1):77–83
Harrison DE, Lerner CP (1991) Most primitive hematopoietic stem cells are stimulated to cycle rapidly after treatment with 5-fluorouracil. Blood 78(5):1237–1240
Hunsucker SA, Spychala J, Mitchell BS (2001) Human cytosolic 5′-nucleotidase I: characterization and role in nucleoside analog resistance. J Biol Chem 276(13):10498–10504
Johnson SA (2001) Nucleoside analogues in the treatment of haematological malignancies. Expert Opin Pharmacother 2(6):929–943
Kawasaki H, Carrera CJ, Piro LD, Saven A, Kipps TJ, Carson DA (1993) Relationship of deoxycytidine kinase and cytoplasmic 5′-nucleotidase to the chemotherapeutic efficacy of 2-chlorodeoxyadenosine. Blood 81(3):597–601
Klopfer A, Hasenjager A, Belka C, Schulze-Osthoff K, Dorken B, Daniel PT (2004) Adenine deoxynucleotides fludarabine and cladribine induce apoptosis in a CD95/Fas receptor, FADD and caspase-8-independent manner by activation of the mitochondrial cell death pathway. Oncogene 23(58):9408–9418
Licht T, Goldenberg SK, Vieira WD, Gottesman MM, Pastan I (2000) Drug selection of MDR1-transduced hematopoietic cells ex vivo increases transgene expression and chemoresistance in reconstituted bone marrow in mice. Gene Ther 7(4):348–358
Longley DB, Harkin DP, Johnston PG (2003) 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer 3(5):330–338
Lotfi K, Juliusson G, Albertioni F (2003) Pharmacological basis for cladribine resistance. Leuk Lymphoma 44(10):1705–1712
Magro PG, Russo AJ, Li WW, Banerjee D, Bertino JR (2004) p14ARF expression increases dihydrofolate reductase degradation and paradoxically results in resistance to folate antagonists in cells with nonfunctional p53. Cancer Res 64(12):4338–4345
Marzo I, Perez-Galan P, Giraldo P, Rubio-Felix D, Anel A, Naval J (2001) Cladribine induces apoptosis in human leukaemia cells by caspase-dependent and -independent pathways acting on mitochondria. Biochem J 359(Pt 3):537–546
McMillin DW, Landazuri N, Gangadharan B, Hewes B, Archer DR, Spencer HT, Le Doux JM (2005) Highly efficient transduction of repopulating bone marrow cells using rapidly concentrated polymer-complexed retrovirus. Biochem Biophys Res Commun 330(3):768–775
Persons DA, Allay JA, Bonifacino A, Lu T, Agricola B, Metzger ME, Donahue RE, Dunbar CE, Sorrentino BP (2004) Transient in vivo selection of transduced peripheral blood cells using antifolate drug selection in rhesus macaques that received transplants with hematopoietic stem cells expressing dihydrofolate reductase vectors. Blood 103(3):796–803
Randall TD, Weissman IL (1997) Phenotypic and functional changes induced at the clonal level in hematopoietic stem cells after 5-fluorouracil treatment. Blood 89(10):3596–3606
Rots MG, Pieters R, Kaspers GJ, van Zantwijk CH, Noordhuis P, Mauritz R, Veerman AJ, Jansen G, Peters GJ (1999) Differential methotrexate resistance in childhood T- versus common/preB-acute lymphoblastic leukemia can be measured by an in situ thymidylate synthase inhibition assay, but not by the MTT assay. Blood 93(3):1067–1074
Sawai N, Persons DA, Zhou S, Lu T, Sorrentino BP (2003) Reduction in hematopoietic stem cell numbers with in vivo drug selection can be partially abrogated by HOXB4 gene expression. Mol Ther 8(3):376–384
Schiedlmeier B, Wermann K, Kuhlcke K, Eckert HG, Baum C, Fruehauf S, Zeller WJ (2000) Human multidrug resistance-1 gene transfer to long-term repopulating human mobilized peripheral blood progenitor cells. Bone Marrow Transplant 25(Suppl. 2):S118–124
Spencer HT, Sleep SE, Rehg JE, Blakley RL, Sorrentino BP (1996) A gene transfer strategy for making bone marrow cells resistant to trimetrexate. Blood 87(6):2579–2587
Zhao SC, Li MX, Banerjee D, Schweitzer BI, Mineishi S, Gilboa E, Bertino JR (1994) Long-term protection of recipient mice from lethal doses of methotrexate by marrow infected with a double-copy vector retrovirus containing a mutant dihydrofolate reductase. Cancer Gene Ther 1(1):27–33
Zielske SP, Reese JS, Lingas KT, Donze JR, Gerson SL (2003) In vivo selection of MGMT(P140K) lentivirus-transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning. J Clin Invest 112(10):1561–1570
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Tasha Gray, Erin L. Morrey contributed equally to this work
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Gray, T., Morrey, E.L., Gangadharan, B. et al. Enforced expression of cytosolic 5′-nucleotidase I confers resistance to nucleoside analogues in vitro but systemic chemotherapy toxicity precludes in vivo selection. Cancer Chemother Pharmacol 58, 117–128 (2006). https://doi.org/10.1007/s00280-005-0156-7
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DOI: https://doi.org/10.1007/s00280-005-0156-7