Analysis of HPRT and supF mutations caused by pierisin-1, a guanine specific ADP-ribosylating toxin derived from the cabbage butterfly.
Pierisin-1, an ADP-ribosylating toxin derived from the cabbage butterfly, Pieris rapae, induces apoptosis in various mammalian cell lines. We recently reported that the target for ADP ribosylation by pierisin-1 is the 2'-deoxyguanosine residue in DNA. To examine whether pierisin-1 would induce mutations in mammalian cell genes, we conducted a mutational analysis for the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in pierisin-1-treated Chinese hamster lung (CHL) cells. N(2)-(ADP-ribos-1-yl)-2'-deoxyguanosine was detected by the (32)P-postlabeling method in CHL cells after treatment with pierisin-1 at doses of 2-32 ng/mL; adduct levels were 1.1-12.0 per 10(6) nucleotides. Pierisin-1 induced mutations in the HPRT gene dose-dependently, and the frequency was 38 times higher than the control, at a dose of 32 ng/mL. To confirm that mono(ADP-ribosyl)ated dG itself leads to mutations, the pierisin-1-treated DNA of plasmid pMY189 bearing the supF gene was used for mutational analysis. The mutation frequency of the supF gene treated with 2-8 micro g/mL of pierisin-1 was 17-40-fold the control value. Mutation spectrum analysis showed that single base substitutions dominated in both HPRT and supF genes. Among these, transversions were predominant, and more than 70% of the base substitutions occurred at G:C base pairs in both genes. The most frequent mutations were G:C to C:G, followed by G:C to T:A in HPRT gene, whereas G:C to T:A transversions dominated in the supF gene. Our results indicate that pierisin-1 produced N(2)-(ADP-ribos-1-yl)-2'-deoxyguanosine and this guanine-adduct could lead to mutations in the HPRT and supF genes. These findings could provide very useful information for understanding the biological significance of pierisin-1.
Totsuka
Yukari
Y
Cancer Prevention Basic Research Project, National Cancer Center Research Institute, 1-1 Tsukiji 5-Chome, Chuo-ku, Tokyo 104-0045, Japan.
Kawanishi
Masanobu
M
Nishigaki
Rena
R
Matsukawa
Kazuki
K
Yagi
Takashi
T
Takamura-Enya
Takeji
T
Watanabe
Masahiko
M
Sugimura
Takashi
T
Wakabayashi
Keiji
K
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002080:Butterflies; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D016153:Genes, Suppressor; D006147:Guanine / Q000378:metabolism; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D019476:Insect Proteins / Q000633:toxicity*; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests; D009154:Mutation; D012343:RNA, Transfer / Q000235:genetics*
D019476:Insect Proteins; C488125:N(2)-(ADP-ribos-1-yl)-2'-deoxyguanosine; C113557:pierisin protein, insect; C096275:supF tRNA; D000246:Adenosine Diphosphate Ribose; D006147:Guanine; D012343:RNA, Transfer; D036002:ADP Ribose Transferases; D007041:Hypoxanthine Phosphoribosyltransferase; D003849:Deoxyguanosine