A NEW T-LYMPHOCYTE CLONING ASSAY FOR DETECTION OF IN-VIVO MUTATIONS IN THE HUMAN HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE GENE

The X-linked hypoxanthine-guanine phosphoribosyltransferase (hprt) gen e is a target of analyses of in vivo mutation frequencies in circulati ng T-lymphocytes. We established a novel, accessory cell-free cloning method of T-lymphocytes with a hprt mutation by a combined use of reco mbinant interleukin-2, conditioned medium from activating T-lymphocyte s and culture plates coated with anti-CDS monoclonal antibody. Using t he method, we examined mutation frequencies of the hprt gene in T-lymp hocytes from six healthy individuals, nine patients with colon cancer including two patients from different families with hereditary nonpoly posis colon cancer and six cancer-free relatives of the patients. In s ix healthy individuals, the mean cloning efficiency and mutation frequ ency (MF) of the hprt gene in T-lymphocytes were 0.51 +/- 0.28 and 9.4 +/- 7.5 x 10(-6), respectively. These data were similar to the report ed values. The mean MFs in the nine colon cancer patients (10.6 +/- 7. 3 x 10(-6)) were not significantly different from those of the 12 canc er-free individuals (11.6 +/- 9.4 x 10(-6)). The correlation between m utation frequencies and age of the individuals was significant regardl ess of the presence or absence of cancers. The single-strand conformat ion polymorphism analyses of nested RT-PCR products of hprt mRNA were clone in 33 mutant clones from five members of a family of which MF va lues were high. All the analyzed mutant clones show a genetic aberrati on in the ceding region of the hprt gene. At least 28 of 33 mutants we re independent. Our method provides a new versatile tool For in vivo a nalysis for mutations of the hprt gene. (C) 1997 Wiley-Liss, Inc.