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Retroviral expression of a mutant (GLY‐533) human DNA topoisomerase I cDNA confers a dominant form of camptothecin resistance
Author(s) -
Yanase Kae,
Sugimoto Yoshikazu,
Andoh Toshiwo,
Tsuruo Takashi
Publication year - 1999
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/(sici)1097-0215(19990331)81:1<134::aid-ijc22>3.0.co;2-y
Subject(s) - camptothecin , mutant , microbiology and biotechnology , biology , topoisomerase , complementary dna , cell culture , dna , gene , biochemistry , genetics
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 S3 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. Int. J. Cancer 81:134–140, 1999. © 1999 Wiley‐Liss, Inc.