Frameshift mutation in PRKDC, the gene for DNA-PKcs, in the DNA repair-defective, human, glioma-derived cell line M059J

The glioma-derived cell line M059J is hypersensitive to ionizing radiation, lacks DNA-PK activity, and fails to express protein for the catalytic subu nit, DNA-PKcs, while a sister cell line, M059K, derived from the same tumor , has normal DNA-PK activity. Both cell lines are near pentaploid and have multiple copies of chromosome 8, the chromosome on which the DNA-PKcs gene, PRKDC, is located. Sequence analysis of PCR-amplified exons revealed the l oss in M059J cells of a single "A" nucleotide in exon 32, corresponding to the first nucleotide of codon 1351 (ACC, Thr) of PRKDC. Loss of the "A" nuc leotide would terminate the DNA-PKcs reading frame early in exon 33, DNA fr om M059K cells had only the wild-type sequence. An analysis of sequences su rrounding PRKDC ex-on 32 from 87 unrelated individuals revealed no polymorp hic nucleotides except for a triplet repeat near the 3' end of this exon; n o individual had a frameshift mutation in exon 32. No other sequence differ ences in PRKDC between M059J and M059K cells were observed in similar to 15 ,000 bp of genomic sequence including the sequences of exons 5 through 38 a nd surrounding intron sequence, suggesting a possible reduction to homozygo sity at this locus prior to acquisition of the mutation leading to the M059 J cell line. (C) 2001 by Radiation Research Society.