Engineered resistance to camptothecin and antifolates by retroviral coexpression of tyrosyl DNA phosphodiesterase-I and thymidylate synthase.

Gene transfer of cDNA sequences that confer drug resistance can be used (1) to protect hematopoietic cells against the toxic effects of chemotherapy, (2) for in vivo enrichment of genetically engineered cells and (3) to protect cytotoxic T lymphocytes in drug-resistant immunotherapy approaches for the treatment of cancer. We have previously developed strategies to confer resistance to agents targeting thymidylate synthase (TS) and have now expanded our drug resistance strategies to include retroviral expression of tyrosyl-DNA phosphodiesterase (TDP-I), an enzyme recently implicated in the repair of topoisomerase-I (Top-I)/DNA lesions induced by camptothecin (CPT). The combination of TS and Top-I inhibition has been shown to be an effective treatment for several types of cancer. Retroviral vectors were generated that individually encoded TS and TDP-I or that coexpressed both enzymes. Murine fibroblast and Chinese hamster lung transfectants were generated with the vectors and resistance to TS- and Top-I-directed inhibitors was tested. Murine bone marrow progenitor cells were also transduced using recombinant retroviruses encoding TS and TDP-I and the degree of drug resistance conferred to gene-modified cells was tested. Enforced expression of TDP-I increased TDP-I activity in gene-modified cells and conferred up to threefold resistance to CPT. The degree of resistance was dependent on the duration of drug treatment. Simultaneous expression of the TS gene encoding E. coli TS optimized for expression in mammalian cells (optecTS) and TDP-I conferred extremely high-level resistance to concurrent treatment with the TS-inhibitor BW1843U89 and CPT. Furthermore, by direct analysis of DNA fragmentation using the comet assay, substantial protection was conferred (fourfold) against DNA fragmentation associated with combination drug treatments by dual enzyme expression compared to non-modified cells. Hematopoietic progenitor assays of murine bone marrow cells transduced with retroviral vectors encoding TS and TDP-I showed that bone marrow cells could be protected from the cytotoxic effects of TS and Top-I inhibition. Enforced expression of optecTS and TDP-I conferred antifolate and CPT resistance to genetically modified cells. Additionally, this work further illustrated a role for TDP-I in the repair of dead-end Top-I complexes and implied that TDP-I expression analysis may aid in predicting the therapeutic effectiveness of the CPT class of compounds.