Journal Article

<i>Hprt</i> mutant frequency and molecular analysis of <i>Hprt</i> mutations in Fischer 344 rats treated with thiotepa

Tao Chen, Anane Aidoo, Roberta A. Mittelstaedt, Daniel A. Casciano and Robert H. Heflich

in Carcinogenesis

Volume 20, issue 2, pages 269-277
Published in print February 1999 | ISSN: 0143-3334
Published online February 1999 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/20.2.269
Hprt mutant frequency and molecular analysis of Hprt mutations in Fischer 344 rats treated with thiotepa

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Thiotepa is a bifunctional alkylating anticancer drug that is a rodent carcinogen and a suspected human carcinogen. In order to determine the sensitivity of mutant induction in the Hprt lymphocyte assay for detecting tumorigenic doses of thiotepa, Fischer 344 rats were treated for 4 weeks with thiotepa using a procedure adapted from a carcinogenesis protocol. At various times after beginning the treatment regimen, rats were killed and the lymphocyte Hprt assay was performed on splenic lymphocytes isolated from the animals. The 6-thioguanine-resistant T lymphocyte mutant frequency increased with time during the period of thiotepa exposure and declined slightly thereafter. Significant dose-dependent increases in mutant frequency were found using concentrations of thiotepa that eventually result in lymphoproliferative tumors. Hprt mRNA from mutant lymphocytes was reverse transcribed to cDNA, amplified by PCR and examined for mutations by DNA sequencing. This analysis indicated that the major type of point mutation was G:C→T:A transversion and that 33% of the mutants contained simple or complex frameshifts. Also, a multiplex PCR performed on DNA from mutant clones that were expanded in vitro indicated that 34% of the clones had deletions in the Hprt gene. These results indicate that the induction of lymphocyte Hprt mutants is a sensitive biomarker for the carcinogenicity of thiotepa and that the types of mutations found in the lymphocyte Hprt gene reflect the kinds of DNA damage produced by thiotepa.

Keywords: CE, cloning efficiency; CM, conditioned medium; ConA, concanavalin A; DGGE, denaturing gradient gel electrophoresis; DMSO, dimethyl sulfoxide; dNTPs, deoxynucleotide triphosphates; EM, expansion medium; FBS, fetal bovine serum; GM, growth medium; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; RT, reverse transcriptase; SPBS, supplemented PBS; TGr, 6-thioguanine-resistant.

Journal Article.  8326 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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