Characterization of SV40-transformed xeroderma pigmentosum cell lines for their usability in HPRT mutation studies.
We have characterized six SV40-transformed xeroderma pigmentosum cell lines (XP20S and XP12BE derived from female donors, XP12RO-SV, XP3BR/12SV, XP4PA-SV and XP8CAC-SV from male donors) for their usability in HPRT mutation studies. All cell lines exhibit hypersensitivity, compared with MRC5CV1 cells, towards the cytotoxic action of UV-irradiation. They were all shown to be heteronuclear and hyperdiploid with pronounced variability in chromosome number. Fluorescence in situ hybridization (FISH) with an X-chromosomal library (X-chromosome painting) and BrdUrd-labelling of late-replicating X-chromosomes demonstrated the presence of variable numbers of X-chromosomes and additional X-chromosomal material and suggested the presence of more than one genetically active HPRT allele in the majority of cells of five cell lines. The cell line XP8CAC-SV (complementation group C) seemed to be most suitable for HPRT mutation studies due to its near-diploid karyotype with only one X-chromosome in the majority of cells. From this cell line, a clonal subline was established (XP8CAC-SV-C1) which revealed the same UV-hypersensitivity as the parental cell line and a near-diploid karyotype with one X-chromosome in 94% of the metaphases. Molecular analysis of the HPRT gene gave a normal PCR amplification pattern for all exons and the normal wild-type sequence of the cDNA. HPRT tests with (+)-anti-benzo[a]pyrene-7,8-diol-9,10-oxide [(+)-anti-BPDE] showed a reproducible, concentration related increase in mutant frequencies. Compared with results with MRC5CV1 cells, the obtained data indicate spontaneous and (+)-anti-BPDE-induced hypermutability of the XP line. XP8CAC-SV-C1 thus represents a permanent XP cell line with characteristic cellular XP features which is convenient for studying the influence of deficient excision repair on HPRT mutant frequencies and mutation spectra.
Merk
O
O
Universität Ulm, Abteilung Medizinische Genetik, Germany.
Speit
G
G
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000633:toxicity; D002460:Cell Line; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000528:radiation effects; D002472:Cell Transformation, Viral; D005260:Female; D005347:Fibroblasts; D005816:Genetic Complementation Test; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D016296:Mutagenesis; D009152:Mutagenicity Tests / Q000379:methods*; D016133:Polymerase Chain Reaction; D012729:Sex Chromosome Aberrations; D013539:Simian virus 40 / Q000235:genetics*; D012867:Skin / Q000166:cytology / Q000528:radiation effects; D014178:Translocation, Genetic; D014466:Ultraviolet Rays; D014960:X Chromosome / Q000528:radiation effects*; D014983:Xeroderma Pigmentosum
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D007041:Hypoxanthine Phosphoribosyltransferase