Identification of glutathione S-transferase p-1 as the class pi form dominantly expressed in mouse hepatic adenomas.
To clarify which of the two genes for pi class glutathione S-transferases (GSTs) (p-1 and p-2) is dominantly expressed in mouse hepatic adenomas, the relative mRNA levels were examined by means of the reverse transcription-polymerase chain reaction (RT-PCR). Hepatic adenomas were induced in male and female B6C3F1 mice by diethylnitrosamine treatment. Northern blot analysis revealed that pi class mRNA levels were decreased in adenomas of male mice, but increased in those of females, with reference to the respective surrounding non-adenoma tissues. In contrast to the marked sex difference in surrounding tissues, pi class GST mRNA levels in adenomas were almost the same in both males and females. To evaluate p-1 and p-2 mRNA levels separately, the products of RT-PCR employing primers common for both cDNAs were digested with the endonuclease BanI (specific for p-2) and then resolved by electrophoresis. The p-1 mRNA was thus found to be dominant in adenomas of both female and male mice. The p-2 mRNA levels were increased in the lesions as compared with those in the surrounding non-adenoma tissues. Recombinant p-1 and p-2 proteins were expressed in Escherichia coli. Unlike p-1, the p-2 protein did not show any significant activity towards 1-chloro-2,4-dinitrobenzene and did not bind to S-hexylglutathione-Sepharose despite immunological cross-reactivity. The dominant pi class form in adenomas could also be identified as p-1 by its binding to S-hexylglutathione-Sepharose. Single radial immunodiffusion analyses confirmed that the p-1 protein levels were in line with the mRNA findings, i.e., 1.9+/-0.3 mg/g adenoma as compared to 6.5+/-1.2 mg/g non-adenoma tissue for males and 2.2+/-0.6 mg/g as compared to 0.7+/-0.2 mg/g for females. The results thus indicated that the change of pi class forms in adenomas is caused mainly by alteration in the p-1 level and the contribution of p-2 is minimal.
Ookawa
K
K
Second Department of Biochemistry, Hirosaki University School of Medicine.
Nakano
H
H
Kakizaki
I
I
Hatayama
I
I
Kajihara-Kano
H
H
Kimura
J
J
Hayakari
M
M
Takahata
T
T
Satoh
K
K
Tsuchida
S
S
D000236:Adenoma / Q000139:chemically induced / Q000201:enzymology*; D000818:Animals; D004052:Diethylnitrosamine; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005982:Glutathione Transferase / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000139:chemically induced / Q000201:enzymology*; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase; D011994:Recombinant Proteins / Q000378:metabolism; D012727:Sex Characteristics
D012333:RNA, Messenger; D011994:Recombinant Proteins; D004052:Diethylnitrosamine; D005982:Glutathione Transferase; D012194:RNA-Directed DNA Polymerase