A new T-lymphocyte cloning assay for detection of in vivo mutations in the human hypoxanthine-guanine phosphoribosyltransferase gene.
The X-linked hypoxanthine-guanine phosphoribosyl transferase (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.
Uematsu
K
K
Oncogene Division, National Cancer Center Research Institute, Tokyo, Japan.
Yasuda
J
J
Sekine
T
T
Sugano
K
K
Kudoh
S
S
Sekiya
T
T
D000328:Adult; D003001:Cloning, Molecular / Q000379:methods*; D003110:Colonic Neoplasms / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D012333:RNA, Messenger / Q000032:analysis; D012194:RNA-Directed DNA Polymerase; D013601:T-Lymphocytes / Q000737:chemistry / Q000201:enzymology* / Q000473:pathology; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D007041:Hypoxanthine Phosphoribosyltransferase; D012194:RNA-Directed DNA Polymerase