Semiquantitative Chemiluminescent Detection of UV-B-Induced Point Mutations in the p53 Tumor-Suppressor Gene

The technique of allele-specif ic PCR (AS-PCR) enables the detection of a smal l number of mutant alleles in a large numbe r of wild-type (WT) alleles. We used the AS PCR technique and Southern blotting, using a nonradioactive labeled probe to analyz e the formation of point mutations in the tu mor-suppressor gene p53 of primary ke r atinocytes after UV-B irradiation. Thes e permanent mutations resulting from CC dimers occur at distinct “hot-spots”, one o f which is affected in the human keratinocyt e cell line HaCaT. This enabled us to estab lish the method with a defined positive con trol template, which also allowed semiquan titative determination of the mutation frequency. This, and the determination o f the detection limit, was done with the use o f serial dilutions of WT genomic DNA from primary keratinocytes with mutant genomi c HaCaT DNA in the AS-PCR assay . Nonradioactive methods have man y advantages over their radioactive cou n terparts, including increased safety an d the use of labeled probes that are stabl e over a long period of time. One parti c ular example in which a sensitive tec h nique is required is the detection of m u tations in cultured cells. In contrast t o homogeneously mutated populations o f cells, such as in tumor tissue, mutation s in cultured cells require a sensitive d e tection method because they occur i n frequently, and only a relatively few cells are affected. Also, the method fo r detecting genomic mutants has to di s tinguish between mutated and wild type (WT) alleles. The principle of a l lele-specific polymerase chain reactio n (AS-PCR) is based on a 3 ′ OH mi s match of one primer with the WT DNA (5,7). The primer can hybridize wit h the WT template strand, but it is elo n gated less efficiently by several order s of magnitude compared with a perfec t ly matched primer on mutant DNA an d depending on the bases involved (5,8) . This results in poor amplification o f WT alleles, whereas the mutant allele s form a visible PCR product. The aim o f our study is to detect a CC →TT ta n dem transition in the tumor-suppresso r gene p5 3, codons 281/282 on exon 8 . Point mutations in the p5 3gene wer e demonstrated in many tumors (9). Th e UV-B-induced CC →TT tandem trans i tions might play an important role, e s pecially in skin cancer developmen t (3).These mutations at dipyrimidin e sites are known to be specificall y formed following UV-B irradiation (2) . The p5 3gene has several “hot spots ” (4) in which the CC →TT mutations o c cur at certain codons, including codon s 281/282. In addition, the spontaneousl y immortalized keratinocyte cell line H a CaT (1) includes the same mutation (6 ) and, thus, can serve as a positive co n trol. We show that the use of serial dil u tions of mutant DNA makes a sem i quantitative AS-PCR possible. Thi s allows determination of the detectio n limit and the generated mutation fr e quency in UV-B-treated cultured cells . Neonatal human foreskin keratin o cytes were grown in Keratinocyt e Growth Medium plus 0.15 mM C a ++