Nucleoside and Folate Analog Resistance

Several other drug resistance genes have been evaluated for in vivo selection or chemoprotection, including cytidine deaminase (CDA), and dihydrofolate reductase (DHFR). CDA belongs to a class of enzymes involved in pyrimidine metabolism and salvage pathways. CDA can also inactivate cytosine nucleoside analogs, (e.g., cytara-bine), which are used as antineoplastic agents. Transduction of murine bone marrow cells with CDA was shown to increase hematopoietic CDA expression levels in recipient animals, but showed little evidence of drug protection (131). The selection stringency for CDA-transduced bone marrow cells ex vivo is also dependent on cell density, indicating that release of CDA into the media may inactivate these drugs, reucing the toxicity of untransduced cells (132).

DHFR converts folate into tetrahydrofolate, a cofactor required for thymidylate and purine biosynthesis. Folate analogs such as methotrexate (MTX) and trimetrexate (TMTX) bind to DHFR with greater affinity, thereby inhibiting DNA synthesis (see Fig. 5). Specific DHFR mutations have been identified that are resistant to these antifolates (133,134). Early experiments showed that murine bone marrow transduced with DHFR mutants efficiently protected irradiated recipients from methotrexate-induced marrow toxicity (133,135). Allay et al. demonstrated that nucleoside transport inhibitors, such as nitrobenzylmercaptopurineriboside phosphate (NBMPR-P), increased the sensitivity of primitive hematopoietic cells to folate analogs (136). Subsequent murine transplant experiments, using both TMTX and NBMPR-P for in vivo selection, resulted in a significant expansion of DHFR-transduced progenitors (137). However, only transient and limiting levels of DHFR-mediated enrichment was observed in nonhuman primate

Fig. 5. The role of DHFR in pyrimidine biosynthesis. Antifolates (e.g., MTX) competitively bind to DHFR, blocking tetrahydrofolate synthesis. DHFR mutants (DHFR*) are resistant to antifolate inhibition. Nucleoside transporters allow cells to salvage nucleosides. NBMPR-P inhibits nucleoside transporters, further sensitizing untransduced cells to antifolate drugs.

Fig. 5. The role of DHFR in pyrimidine biosynthesis. Antifolates (e.g., MTX) competitively bind to DHFR, blocking tetrahydrofolate synthesis. DHFR mutants (DHFR*) are resistant to antifolate inhibition. Nucleoside transporters allow cells to salvage nucleosides. NBMPR-P inhibits nucleoside transporters, further sensitizing untransduced cells to antifolate drugs.

models in vivo, following combined TMTX and NBMPR-P treatment (138). Increased toxicity was also evident in this model. Further, pretreatment with cytokines failed to significantly increase the selection stringency for long-term repopulating cells transduced with DHFR. Thus, DHFR and CDA may reduce treatment-related toxicities, but may not provide additional protection to transduced HSCs.

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