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.

Abstract

The chloroethylnitrosourea (CNU) alkylating agents are commonly used for cancer chemotherapy, but their usefulness is limited by severe bone marrow toxicity that causes the cumulative depletion of all hematopoietic lineages (pancytopenia). Bone marrow CNU sensitivity is probably due to the inefficient repair of CNU-induced DNA damage; relative to other tissues, bone marrow cells express extremely low levels of the 06-methylguanine DNA methyltransferase (MGMT) protein that repairs cytotoxic 06-chloroethylguanine DNA lesions. Using a simplified recombinant retroviral vector expressing the human MGMT gene under control of the phosphoglycerate kinase promoter (PGK-MGMT) we increased the capacity of murine bone marrow-derived cells to repair CNU-induced DNA damage. Stable reconstitution of mouse bone marrow with genetically modified, MGMTexpressing hematopoietic stem cells conferred considerable resistance to the cytotoxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a CNU commonly used for chemotherapy. Bone marrow harvested from mice transplanted with PGK-MGMT-transduced cells showed extensive in vitro BCNU resistance. Moreover, MGMT expression in mouse bone marrow conferred in vivo resistance to BCNU-induced pancytopenia and significantly reduced BCNU-induced mortality due to bone marrow hypoplasia. These data demonstrate that increased DNA alkylation repair in primitive hematopoietic stem cells confers multilineage protection from the myelosuppressive effects of BCNU and suggest a possible approach to protecting cancer patients from CNU chemotherapy-related toxicity.The chloroethylnitrosourea (CNU) chemotherapeutic alkylating agents have been used to treat certain kinds of cancer for over two decades (1), and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, carmoustine) is typical of this class of chemotherapeutic drug. BCNU is particularly effective in treating childhood and adult glial tumors (2, 3) and has been used in high-dose chemotherapy for lymphomas, breast, lung, and gastrointestinal cancers (1,4). Unfortunately, the clinical success of the CNUs has been limited by their severe bone marrow and lung toxicities. Myelosuppression is characteristically delayed following CNU treatment and can lead to severe, prolonged, and cumulative pancytopenia (3). The extreme sensitivity of bone marrow to CNUs may be explained by the inability of this tissue to repair CNU-induced cytotoxic DNA damage efficiently (5).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.CNU alkylation produces numerous DNA base modifications plus DNA inter-and intrastrand crosslinks. Interstrand crosslinks are particularly cytotoxic because they grossly interfere with DNA replication; their formation can be initiated by a chloroethyl group at the 06 position of guanine which slowly rearranges to form an ethyl bridge between NI of guanine and N3 of cytosine in the opposite ...

A pilot study of dose-intensified procarbazine, CCNU, vincristine for poor prognosis brain tumors utilizing fibronectin-assisted, retroviral-mediated modification of CD34+ peripheral blood cells with O6-methylguanine DNA methyltransferase

“…20,21 The neomycin-resistance gene is downstream and expressed under the control of the PGK promoter. The MSCVpgkNeoMGMT plasmid was transfected into ecotropic GP þ E86 retroviral packaging cell line and supernatant was used to transduce the amphotropic packaging cell line GP-env-AM12.…”

Protection of committed murine haemopoietic progenitors against BCNU toxicity does not predict protection of primitive, multipotent spleen colony-forming cells – implications for chemoprotective gene therapy

“…ATase-mediated protection of lineage-specific or bipotent bone marrow progenitors, both in vitro and in vivo, against the toxicity of a range of O 6 -alkylating agents has been demonstrated. [40][41][42][43][44][45]59 Some of these studies have made use of mutant ATases, which exhibit various degrees of resistance to the tumour sensitiser O 6 -beG, [46][47][48][49][50] and have shown that haemopoietic cell protection is maintained under conditions where an otherwise resistant tumour might be expected to be sensitised to treatment. Despite the fact that this strategy is often referred to as stem cell protection, little direct qualitative evaluation of any protective effects that may occur in primitive haemopoietic cells has been undertaken.…”

“…38,39 An approach to circumventing the dose limiting toxicity of O 6 -alkylating agents, whilst simultaneously sensitising tumours via O 6 -beG, would be to enhance the repair capacity of bone marrow for O 6 -alkG via expression of a mutant version of human ATase (hAT) that is both refractory to O 6 -beG inactivation and can efficiently repair O 6 -alkG in DNA. Increasing the expression of hAT [40][41][42][43][44][45] or O 6 -beG-resistant mutants of hAT, [46][47][48][49][50] either in murine bone marrow or in human primary haemopoetic cells, has been shown to afford some degree of protection against O 6 -alkylating agentinduced toxicity in the bipotent granulocyte-macrophage progenitor population. These observations have stimulated interest in a clinical trial to protect bone marrow against BCNUinduced myelosuppression, even though evidence of enhanced resistance to BCNU in multipotent haemopoietic progenitors has not yet been reported.…”

Gene therapy approaches for stem cell protection

“…8,9 Antioxidant approaches toward enhancing stem cell protection include transfection of the transgenes for manganese superoxide dismutase (MnSOD), catalase, glutathione, peroxidase, metallothione and others. 10,11 While there is some protective effect of antioxidant transgene expression shown with respect to clonogenic survival of cells in culture, 12,13 no assays of stem cell competitive repopulation capacity have been reported. In contrast, when the microenvironment is induced to overexpress antioxidants by gene therapy, the homing capacity for engrafting stem cells, and also survival of residual stem cells, is enhanced.…”

“…In contrast, when the microenvironment is induced to overexpress antioxidants by gene therapy, the homing capacity for engrafting stem cells, and also survival of residual stem cells, is enhanced. 12 Gene therapy approaches toward directly protecting stem cells represent an attractive strategy; 10,11,14 however, there is also evidence that gene therapy-mediated protection of the microenvironment may also be of value. Expression of the MnSOD transgene by plasmid liposome transfection of the recipient has been shown to enhance capacity to reconstitute the irradiated esophagus and other tissues with donor bone marrow origin cells.…”