A new T-lymphocyte cloning assay for detection of in vivo mutations in the human hypoxanthine-guanine phosphoribosyltransferase gene

Kazutsugu Uematsu, Jun Yasuda, Teruaki Sekine, Kokichi Sugano, Shoji Kudoh, Takao Sekiya

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

The X-linked hypoxanthine-guanine phosphoribosyltransferase (hprt) gene is a target of analyses of in vivo mutation frequencies in circulating T-lymphocytes. We established a novel, accessory cell-free cloning method of T lymphocytes with a hprt mutation by a combined use of recombinant interleukin-2, conditioned medium from activating T-lymphocytes and culture plates coated with anti-CD3 monoclonal antibody. Using the method, we examined mutation frequencies of the hprt gene in T-lymphocytes from six healthy individuals, nine patients with colon cancer including two patients from different families with hereditary nonpolyposis colon cancer and six cancer-free relatives of the patients. In six healthy individuals, the mean cloning efficiency and mutation frequency (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 reported 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 cancer-free individuals (11.6 ± 9.4 x 10-6). The correlation between mutation frequencies and age of the individuals was significant regardless of the presence or absence of cancers. The single-strand conformation polymorphism analyses of nested RT-PCR products of hprt mRNA were done in 33 mutant clones from five members of a family of which MF values were high. All the analyzed mutant clones show a genetic aberration in the coding region of the hprt gene. At least 28 of 33 mutants were independent. Our method provides a new versatile tool for in vivo analysis for mutations of the hprt gene.

Original languageEnglish
Pages (from-to)31-39
Number of pages9
JournalEnvironmental and Molecular Mutagenesis
Volume30
Issue number1
DOIs
Publication statusPublished - 1997 Aug 29

Keywords

  • Anti-CDB monoclonal antibody
  • Hereditary nonpolyposis colon cancer (HNPCC)
  • Hypoxanthine-guanine phosphoribosyltransferase (hprt) gene
  • In viva mutation frequency
  • Single-strand conformation polymorphism nonpolyposis (HNPCC) (SSCP) analysis

ASJC Scopus subject areas

  • Epidemiology
  • Genetics(clinical)
  • Health, Toxicology and Mutagenesis

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