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. 1998 Apr 28;95(9):5275-80.
doi: 10.1073/pnas.95.9.5275.

Increased skin tumorigenesis in mice lacking pi class glutathione S-transferases

C J Henderson  1 , A G Smith, J Ure, K Brown, E J Bacon, C R Wolf
Affiliations

Increased skin tumorigenesis in mice lacking pi class glutathione S-transferases

C J Henderson et al. Proc Natl Acad Sci U S A. .

Abstract

The activity of chemical carcinogens is a complex balance between metabolic activation by cytochrome P450 monooxygenases and detoxification by enzymes such as glutathione S-transferase (GST). Regulation of these proteins may have profound effects on carcinogenic activity, although it has proved impossible to ascribe the observed effects to the activity of a single protein. GstP appears to play a very important role in carcinogenesis, although the precise nature of its involvement is unclear. We have deleted the murine GstP gene cluster and established the effects on skin tumorigenesis induced by the polycyclic aromatic hydrocarbon 7, 12-dimethylbenz anthracene and the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate. After 20 weeks, a highly significant increase in the number of papillomas was found in the GstP1/P2 null mice [GstP1/P2(-/-) mice, 179 papillomas, mean 9.94 per animal vs. GstP1/P2(+/+) mice, 55 papillomas, mean 2.89 per animal, (P < 0.001)]. This difference in tumor incidence provides direct evidence that a single gene involved in drug metabolism can have a profound effect on tumorigenicity, and demonstrates that GstP may be an important determinant in cancer susceptibility, particularly in diseases where exposure to polycyclic aromatic hydrocarbons is involved, for instance in cigarette smoke-induced lung cancer.

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Figures

Figure 1
Figure 1
Strategy for inactivation of the murine GstP gene cluster. (A) GstP1/P2 gene cluster and (B) targeted GstP1/P2 allele. Restriction endonucleases: B, BglII; E, EcoRI; Ev, EcoRV; K, KpnI; N, NheI; P, PstI. A cassette containing the en-2A splice acceptor site, internal ribosome binding site element, lacZ, neo (βGeo), and simian virus 40 polyadenylation site, replaces genomic DNA from the EcoRI site in GstP2, exon 5, to the first PstI site in the 3′ untranslated region of GstP1. Coding exons are represented by black boxes; white boxes represent the region of DNA used as 5′- and 3′-flanking probes for Southern screening. (C) Southern blot analysis of ES cell genomic DNA from targeted clones. The 19-kb KpnI fragment represents the wild-type allele, and the 11.5-kb band corresponds to the targeted allele, using the 5′-flanking probe. (D) Southern blot analysis of tail-tip genomic DNA from wild-type (+/+), heterozygous (+/−), and null (−/−) mice. The 7-kb EcoRI fragment represents the wild-type allele, whereas the 6.3-kb band corresponds to the targeted allele, using the 3′-flanking probe.
Figure 2
Figure 2
Characterization of GstP1/P2(−/−), GstP1/P2(+/−), and GstP1/P2(+/+) mice. Samples were from male (M) and female (F) wild-type (+/+), heterozygote (+/−), and null (−/−) mice. (A) Northern analysis of hepatic RNA (10 μg per lane), using a cDNA representing full-length GstP1. Sample integrity was confirmed by staining of 16S and 28S rRNA in the gel with ethidium bromide before transfer to nitrocellulose, and equivalence of loading was confirmed by using a cDNA for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (B) Western blot analysis of hepatic cytosol (10 μg protein per lane), using a polyclonal antiserum raised against the mouse GstP1–2 protein and ECL detection (Amersham). Equivalence of loading was confirmed by Coomassie blue staining of a duplicate SDS/PAGE gel. (C) Enzymatic activity of hepatic cytosol toward ethacrynic acid and CDNB was measured spectrophotometrically. Data represent mean of five animals ± SEM. (D) Western blot analysis of hepatic cytosol (10 μg protein per lane), using antisera toward GST from alpha and mu families (38, 39), and ECL detection (Amersham). Equivalence of loading was confirmed by Coomassie blue staining of a duplicate SDS/PAGE gel.
Figure 3
Figure 3
GstP expression in lung and kidney. Samples were from male (M) and female (F) wild-type (+/+), heterozygote (+/−), and null (−/−) mice. Western blot analysis of kidney and lung cytosolic samples (10 μg protein per lane), using a polyclonal antiserum raised against the mouse GstP1–2 protein (36) and ECL detection (Amersham). Equivalence of loading was confirmed by Coomassie blue staining of a duplicate SDS/PAGE gel. Activity of kidney and lung cytosolic samples toward CDNB was measured spectrophotometrically and is shown in the corresponding bar charts. Values are expressed as mean ± SEM from five determinations
Figure 4
Figure 4
Expression of GSH-dependent enzymes in GstP null mice. Samples were from male (M) and female (F) wild-type (+/+), heterozygote (+/−), and null (−/−) mice. Western blot analysis of hepatic cytosolic samples (10 μg protein per lane), with antisera to GSH synthetase and γ-glutamylcysteine synthetase (light chain). Total GSH content was determined in hepatic cytosol as detailed in Materials and Methods. Values are expressed as mean ± SEM from five determinations.
Figure 5
Figure 5
Tumor incidence of papillomas in mice treated with DMBA and TPA. Susceptibility of the mice to the tumorigenic effects of DMBA was determined by using the initiation-promotion protocol outlined in Material and Methods. After treatment, GstP1/P2(−/−) and GstP1/P2(+/+) mice were scored for papilloma numbers on a weekly basis, and statistical analysis was carried out by using the Statview software package (Abacus Concepts, Berkeley, CA). a = P < 0.0001, b = P < 0.001.
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