Retroviral expression of a mutant (Gly-533) human DNA topoisomerase I cDNA confers a dominant form of camptothecin resistance

In previous studies, we isolated a mutant DNA topoisomerase I cDNA from a camptothecin (CPT)-resistant human T-lymphoblastic leukemia cell line, CPT- K5, and demonstrated that an amino acid change from Asp to Gly at residue 533 is responsible for the CPT resistance of the enzyme. In the present study, we have constructed a bicistronic retroviral vector, Ha-TM1-IRES-neo, that carries the mutant (Gly-533) TOP1 cDNA (TM1) and a neomycin-resistance gene to examine the effect of mutant DNA topoisomerase I (topo I) expression on CPT resistance of cells. HeLa 53 cells were transduced with Ha-TM1-IRES-neo, and the transduced cells were selected with G418. Two independently isolated populations of the G418-resistant cells and 2 clones showed 1.7- to 1.8-fold higher resistance to CPT than the control cells. Integration and expression of the exogenous TOP1 were confirmed by genomic and RT-PCR analyses. The topo I enzyme (mixture of mutant and wild-type) expressed in the transduced cells showed 3-fold resistance to CPT in cleavable-complex-formation assay and DNA- relaxation assay. Mutant topo I activity in the transduced cells was as much as 10% that of the endogenous enzyme. Our results clearly show that expression of Gly-533 topo I confers a dominant form of CPT resistance in cells expressing wild-type topo I. The mutant TOP1 could be used for the protection of normal bone marrow cells of cancer patients from the severe hematotoxicity of CPT-derivative anti-tumor agents.