Mutation specificity of 8-methoxypsoralen plus two doses of UVA irradiation in the hprt gene in diploid human fibroblasts

Abstract

To investigate which specific kinds of base changes are induced by psoralen adducts in the genomic DNA of diploid human fibroblasts, cells were exposed to 8-methoxypsoralen (8-MOP) at 2-12 microM followed by one dose of UVA (365 nm) irradiation (PUVA-I treatment) or two doses of UVA (PUVA-II treatment). While PUVA-I treatment produced little effect on the induction of cytotoxicity, PUVA-II treatment significantly reduced the fibroblasts' colony-forming ability and resulted in about 10-fold increases in mutation frequency at the D0 dose. Mutations in the hypoxanthine (guanine) phosphoribosyltransferase (hprt) gene of 36 independent PUVA-II mutants were characterized by direct sequencing of cDNA amplified by the polymerase chain reaction (PCR). Seventeen mutants contained single base substitutions and the other 19 mutants either lacked one or more exons, or had deleted or gained nucleotides in the exon boundaries in their cDNA. The intron--exon boundaries of 10 of these 19 putative splicing mutants were further characterized by direct sequencing of the PCR-amplified hprt gene. The results showed that nine contained single base substitutions at the consensus splicing donor and acceptor sites. One splicing mutant possessed two base substitutions located at exon 8, whereas its splicing sites were intact. Most of the base substitutions occurred at T-A base pairs (24/29). The majority of T.A changes occurred at thymine of 5'TA and 5'ATA on the non-transcribed strand. Four of the five G.C base substitutions were located at guanines of 5'TG sites adjacent 3' to AT or TA sequences. In addition, the occurrence of a specific type of mutation was highly correlated to the 5' flanking bases of TA sites. The mutagenesis of 13 of the 16 mutational events at 5'TA sites on the non-transcribed strand can be explained by the preferential incisions of the photoadducts on the transcribed strand followed by misalignment--realignment during translesion repair synthesis of the bulky lesions on the non-transcribed strand.

Signature p53 mutation at DNA cross-linking sites in 8-methoxypsoralen and ultraviolet A (PUVA)-induced murine skin cancers.

“…Several studies have shown that PUVA is a potent mutagen and a carcinogen (7)(8)(9)(10)(11)(12)(13). In vitro studies have revealed that PUVA induces mutations in mammalian cells predominantly at 5'-TA sequences, which are sites for formation of both monoadducts and interstrand DNA cross-links (7,8,13). Although it is known that PUVA-induced DNA cross-links and monoadducts play a direct role in mutagenesis, their role in tumorigenesis is unclear.…”

“…These results suggest that the increased risk of developing skin cancer in PUVA-treated patients is quite complex and is dependent on patient's genetic background, the number and intensity of PUVA treatments, and prior exposure to other carcinogens. Nonetheless, although the mutagenic and carcinogenic effects of PUVA treatment have been well established (7)(8)(9)(10)(11)(12)(13), the molecular mechanisms by which PUVA induces skin cancer are obscure.…”

“…While UV radiation induces primarily cyclobutane-type pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts (23), PUVA induces monoadducts and interstrand diadducts (cross-links) in cellular DNA (7,24,25). Since photoactivated psoralen crosslinks occur exclusively at 5'-TA and 5'-TAT sites (7,8,25,26), it i<quite possible that PUVA induces signature mutations at thes' sites in the p53 tumor suppressor gene leading to the development of skin cancer. If so, it may be possible to usep53 gene mutations as a molecular marker and to determine whether skin cancers arising in PUVA-treated patients have a UV etiology or a PUVA etiology.…”

Strand Specificity of Mutagenic Bypass Replication of DNA Containing Psoralen Monoadducts in a Human Cell Extract

“…Relative contributions of MAs versus biadducts. Most mutations observed in studies of PUVA-induced mutagenesis in mammalian cells are substitutions at T ⅐ A base pairs (4,25,34,53; for a review, see reference 33). In the present study, PUVA treatment resulted in a substantial increase in T ⅐ A3C ⅐ G replication errors (Table 2).…”

Biological Consequences of 8-Methoxypsoralen-Photoinduced Lesions: Sequence-Specificity of Mutations and Preponderance of T to C and T to A Mutations

“…A single psoralen plus UVA treatment (PUVA-I) yields a combination of MA and ICL psoralen-adducts. The PUVA-I followed by a second UVA irradiation (PUVA-II), however, generates more homogeneous photolesions by converting most, if not all, of the MA to ICL psoralen-adducts (Yang et al, 1994). The PUVA-II treatment has been shown to have equal cytotoxicity and mutagenicity at the hypoxanthine-guanine phosphoribosyl transferase locus in both human lymphoblasts and fibroblasts relative to PUVA-I treatment (Chiou and Yang, 1995;Laquerbe et al, 1995).…”