Expression Of GSTA4 Research Articles

Abstract 4210: Gsta4 modifies susceptibility to skin tumor development in mice and humans

Abstract The incidence of non-melanoma skin cancer (NMSC) is equivalent to that of all other human cancers combined, and genetic factors contribute to risk of the disease. Using genetic crosses of skin tumor promotion sensitive DBA/2 mice with relatively resistant C57BL/6 mice, loci that modify susceptibility to tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) have been mapped to chromosomes (chr) 1, 2, 9, and 19. Here, we show that Glutathione S-transferase alpha 4 (Gsta4), which maps to skin tumor promotion susceptibility locus Psl1.2 on chr 9, was expressed at significantly different levels in the epidermis of C57BL/6 versus DBA/2 mice following treatment with diverse promoting agents. Gsta4 expression was dramatically upregulated in the epidermis of C57BL/6 mice, but not DBA/2 mice, following treatment with TPA, okadaic acid, chrysarobin, or ultraviolet light. Gsta4 deficient mice were more susceptible to skin tumor development in the two-stage skin carcinogenesis protocol providing compelling evidence that Gsta4 underlies the effect of Psl1.2 on susceptibility to skin tumor promotion by TPA. A number of polymorphisms were detected in the putative promoter region of Gsta4 in C57BL/6 versus DBA/2 mice, and ongoing studies are addressing the genetic basis of strain-specific expression of Gsta4 during tumor promotion. Finally, inheritance of polymorphisms in GSTA4 was associated with risk of NMSC in human populations, further supporting Gsta4/GSTA4 as a gene that modifies susceptibility to skin tumor development. Supported by NIH grant ES016623. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4210.

Abstract B155: Effects of bamboo extract on breast cancer

Abstract Breast cancer is the most common cancer among women in the United States, and worldwide over one million new cases are diagnosed each year. Preliminary studies by Panee and colleagues demonstrated that 0.5% (w/w) dietary supplementation with an ethanol/water extract from bamboo Phyllostachys edulis inhibited the incidence of 7,12-dimethylbenz[a]anthracene (DMBA, a carcinogen)-induced mammary tumors in female Sprague-Dawley rats by 44% and dramatically reduced tumor multiplicity. This project has aimed to delineate the anti-breast cancer mechanisms of this bamboo extract (BEX) in the aforementioned rat model. We hypothesized that BEX may alter the gene expression of key proteins involved in estrogen- and/or growth factor-signaling pathways and xenobiotic biotransforming enzymes in the mammary tumors. To test this hypothesis, we extracted RNA from individual mammary tumors that were harvested from each rat. Then, we synthesized complementary DNA (cDNA) and combined the cDNA samples according to each rat's dietary group— BEX-supplemented rat diet or control rat diet. Using quantitative real-time polymerase chain reaction, we measured expression levels of target genes that included the following: estrogen receptors alpha and beta (ER-α and -β), human epidermal growth factor receptor 2 (HER2), and all glutathioine-s-transferase (GST) isoforms. Comparison between study groups revealed that BEX-supplementation significantly downregulated the gene expression of ER-α, ER-β, and HER2 in the mammary tumors by 65%, 70%, and 67% respectively (p < 0.0001). Additionally, the mammary tumors expressed all GST isoforms, whereby GSTa4 and GSTp1 resulted in high abundant values. Interestingly, BEX supplementation also significantly downregulated the gene expression of GSTa4 and GSTp1 by 32% and 31% respectively (p < 0.005). Our observations have indicated that the downregulation of ER-α, ER-β, and HER2 gene expression in the mammary tumors may contribute to the anti-breast cancer effect of BEX, and that BEX may also reduce chemoresistance in the mammary tumors by inhibiting the gene expression of xenobiotic biotransforming enzyme GST. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B155.

Protective role of glutathione S-transferase A4 induced in copper/zinc-superoxide dismutase knockout mice

Copper/zinc-superoxide dismutase (SOD1) plays a protective role in cells by catalyzing the conversion of the superoxide anion into molecular oxygen and hydrogen peroxide. Although SOD1 knockout (KO) mice exhibit a reduced life span and an elevated incidence of dysfunctions in old age, young SOD1 KO mice grow normally and exhibit no abnormalities. This fact leads to the hypothesis that other antioxidative proteins prevent oxidative stress, compensating for SOD1. Differently expressed genes in 3-week-old SOD1 KO and littermate wild-type mice were explored. A gene remarkably elevated in SOD1 KO mouse kidneys was identified as the glutathione S-transferase Alpha 4 gene ( Gsta4), which encodes the GSTA4 subunit. The GSTA4 protein level and activity were also significantly increased in SOD1 KO mouse kidneys. The administration of an iron complex, a free radical generator, induced GSTA4 expression in wild-type mouse kidneys. Iron deposition detected in SOD1 KO mouse kidney is thought to be an inducer of GSTA4. In addition, overexpression of mouse GSTA4 cDNA in human embryonic kidney cells decreased cell death caused by both 4-hydroxynonenal and hydrogen peroxide. These findings suggest that compensatory induced GSTA4 plays a protective role against oxidative stress in young SOD1 KO mouse kidneys.

Expression level and DNA methylation status of glutathione‐S‐transferase genes in normal murine prostate and TRAMP tumors

Glutathione-S-transferase (Gst) genes are downregulated in human prostate cancer, and GSTP1 silencing is mediated by promoter DNA hypermethylation in this malignancy. We examined Gst gene expression and Gst promoter DNA methylation in normal murine prostates and Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) tumors. Primary and metastatic tumors were obtained from TRAMP mice, and normal prostates were obtained from strain-matched WT mice (n = 15/group). Quantitative real-time RT-PCR was used to measure GstA4, GstK1, GstM1, GstO1, and GstP1 mRNA expression, and Western blotting and immunohistochemical staining was used to measure GstM1 and GstP1 protein expression. MassARRAY Quantitative Methylation Analysis was used to measure DNA methylation of the 5' CpG islands of GstA4, GstK1, GstM1, GstO1, and GstP1. TRAMP-C2 cells were treated with the epigenetic remodeling drugs decitabine and trichostatin A (TSA) alone and in combination, and Gst gene expression was measured. Of the genes analyzed, GstM1 and GstP1 were expressed at highest levels in normal prostate. All five Gst genes showed greatly reduced expression in primary tumors compared to normal prostate, but not in tumor metastases. Gst promoter methylation was unchanged in TRAMP tumors compared to normal prostate. Combined decitabine + TSA treatment significantly enhanced the expression of 4/5 Gst genes in TRAMP-C2 cells. Gst genes are extensively downregulated in primary but not metastatic TRAMP tumors. Promoter DNA hypermethylation does not appear to drive Gst gene repression in TRAMP primary tumors; however, pharmacological studies using TRAMP cells suggest the involvement of epigenetic mechanisms in Gst gene repression.

Specific expression of Gsta4 in mouse cochlear melanocytes: a novel role for hearing and melanocyte differentiation

Mammalian pigment cells produce melanin as the main pigment. Melanocytes, one of the two types of mammalian pigment cells, differentiate from the neural crest and migrate to a variety of organs during development. Melanocytes exist not only in the skin but also in other sites such as the cochlea where they are essential for hearing. Mitf(mi-bw) is one of the known recessive alleles of the mouse microphthalmia-associated transcription factor (Mitf) locus, which is essential for the development of pigment cells. Homozygous Mitf(mi-bw)/Mitf(mi-bw) mice have a completely white coat with black eyes and are deaf due to the lack of melanocytes. By comparing gene expression profiles in cochleae of wild-type and Mitf(mi-bw)/Mitf(mi-bw) mice, we now demonstrate the specific expression of glutathione S-transferase alpha 4 (Gsta4) in the stria vascularis. Gsta4 encodes one of the cytosolic glutathione S-transferases (GSTs) which participate in detoxification processes of many tissues. This gene is specifically expressed in intermediate cells of the stria vascularis, suggesting a novel function for cochlear melanocytes. Moreover, among mammalian pigment cells, expression of Gsta4 was restricted to cochlear melanocytes, suggesting that melanocytes in various tissues differentiate from one another depending on their location.

The effect of different zwitterionic buffers and PBS used for out-of-incubator procedures during standard in vitro embryo production on development, morphology and gene expression of bovine embryos

The effect of the zwitterionic buffers HEPES, TES and MOPS and of PBS used for out-of-incubator procedures during standard in vitro embryo production on bovine oocytes and embryo development, morphology and on the expression patterns of eight selected genes: Fgf-4, Lama1, Ube2a, Gsta4, Il6, Sod1, Prss11 and Hspb1, was evaluated. All buffers were prepared at a concentration of 10 mM in TALP medium, with the exception of PBS. The total time of oocyte/embryo exposure to each buffer was ∼41 min. The cleavage rates and number of embryos that developed to ≥8 cells at day 4 were no different among the buffers tested, however, more blastocysts developed at day 7, 8 and 9 in HEPES and MOPS treatments than in PBS and TES ( P < 0.05). No difference between buffers in total and apoptotic cell number was found. Except for Hspb1 and Ube2a genes, the levels of expression of the six remaining transcripts were higher in in vivo than in in vitro embryos irrespective of buffer used ( P < 0.05). In addition, higher expression of Hspb1 and lower expression of Ube2a and Lama1 were observed in PBS and TES than in MOPS and HEPES treatments ( P < 0.05). Expression of Fgf-4 and Gsta4 in the in vitro embryos was lower in PBS than in the remaining three buffers ( P < 0.05) and the level of expression of the Il6 gene was not affected by any buffer tested but was lower in in vitro than in in vivo derived embryos. Expression of both Sod1 and Prss11 genes in MOPS were at the level of the in vivo embryos. These results showed that the choice of buffer and short exposure time of ∼41 min, affects mRNA expression of in vitro produced bovine embryos.

Increased oxidative stress and antioxidant expression in mouse keratinocytes following exposure to paraquat

Paraquat (1,1′-dimethyl-4,4′-bipyridinium) is a widely used herbicide known to induce skin toxicity. This is thought to be due to oxidative stress resulting from the generation of cytotoxic reactive oxygen intermediates (ROI) during paraquat redox cycling. The skin contains a diverse array of antioxidant enzymes which protect against oxidative stress including superoxide dismutase (SOD), catalase, glutathione peroxidase-1 (GPx-1), heme oxygenase-1 (HO-1), metallothionein-2 (MT-2), and glutathione- S-transferases (GST). In the present studies we compared paraquat redox cycling in primary cultures of undifferentiated and differentiated mouse keratinocytes and determined if this was associated with oxidative stress and altered expression of antioxidant enzymes. We found that paraquat readily undergoes redox cycling in both undifferentiated and differentiated keratinocytes, generating superoxide anion and hydrogen peroxide as well as increased protein oxidation which was greater in differentiated cells. Paraquat treatment also resulted in increased expression of HO-1, Cu,Zn-SOD, catalase, GSTP1, GSTA3 and GSTA4. However, no major differences in expression of these enzymes were evident between undifferentiated and differentiated cells. In contrast, expression of GSTA1-2 was significantly greater in differentiated relative to undifferentiated cells after paraquat treatment. No changes in expression of MT-2, Mn-SOD, GPx-1, GSTM1 or the microsomal GST's mGST1, mGST2 and mGST3, were observed in response to paraquat. These data demonstrate that paraquat induces oxidative stress in keratinocytes leading to increased expression of antioxidant genes. These intracellular proteins may be important in protecting the skin from paraquat-mediated cytotoxicity.

Gene expression profile of pemetrexed and oxaliplatin response in a phase II induction trial in locally advanced head and neck squamous cell carcinoma

6039 Background: To assess the efficacy and identify a biomarker of response, a phase II trial of pemetrexed (PT) and oxaliplatin (OX) as an induction regimen in locally advanced head and neck squamous cell carcinoma (HNSCC) was conducted. We hypothesized that the tumors with increased DNA repair and folate biosynthesis capacities would have resistance to the regimen. Methods: Patients (pts) were given PT 500 mg/m2 and OX 85 mg/m2 Q 2 weeks for 4 cycles. Response was assessed at the end of treatment by CT scan. Frozen and formalin-fixed paraffin-embedded tumors were collected for DNA microarray analysis. The gene expression data were queried using significance analysis of microarrays (SAM) for comparison of stable disease (SD) versus partial response (PR). Results: 15 pts have been enrolled (Male/female: 14/1, Median age: 57.3). ECOG PS: 0 (6), 1 (8) and 2 (1). Primary sites: oropharynx (5), oral cavity (6), larynx (2), hypopharynx (1) and unknown primary (1). Fourteen of the 15 pts were evaluable for response: 0 CR, 10 PR, 4 SD and 0 PD. Toxicity was minimal with grade (G) 3 toxicities in 2 pts for diarrhea and elevated AST. The most common toxicity was nausea (G1–2). Eight tumors (5 PR and 3 SD) and 3 adjacent normal mucosal epithelia were collected and evaluated using DNA microarray. SAM yielded 77 differentially expressed genes between PR vs. SD. The genes most significantly associated with PR were higher expression of TS, DHFR, CDKN2C and CDKN2A, and lower expression of GSTA1 and GSTA4. Because of the association of upregulated CDKN2A, DHFR and HPV infection, the data were queried with the 91-gene HPV signature and presence of the signature was associated with response. The HPV status was also confirmed with HPV E6 and E7 PCR. Conclusions: The combination of PT/OX is active in locally advanced HNSCC patients. Higher expression of genes involving folate biosynthesis and HPV infection may associate with better response to this regimen while no known DNA repair gene expressions were found to associate with the response. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Lilly Oncology

Glutathione‐S‐transferase expression in the brain: possible role in ethanol preference and longevity

Identification of genes that are differentially expressed in rats bidirectionally selected for alcohol preference might reveal biological mechanisms underlying alcoholism or related phenotypes. Microarray analysis from medial prefrontal cortex (mPFC), a key brain region for drug reward, indicated increased expression of glutathione-S-transferases of the alpha (Gsta4) and mu (Gstm1-5) classes in ethanol-preferring AA rats compared with nonpreferring ANA rats. Real-time RT polymerase chain reaction (RT-PCR) analysis demonstrated approximately 2-fold higher Gsta4 transcript levels in several brain regions of ethanol-naive AA compared with ANA rats. Differences in mRNA levels were accompanied by differential levels of GSTA4 protein. We identified a novel haplotype variant in the rat Gsta4 gene, defined here as var3. Allele frequencies of var3 were markedly different between AA and ANA rats, 52% and 100%, respectively. Gsta4 expression was strongly correlated with the gene dose of var3, with approximately 60% of the variance in expression accounted for by genotype at this locus. The contribution of glutathione S-transferase expression to the ethanol-preferring phenotype is presently unclear. It could, however, underlie observed differences in life span between AA and ANA lines, prompting a utility of this animal model in aging research.

Glutathione S-transferase isoenzymatic response to aging in rat cerebral cortex and cerebellum

Aging is associated with increased oxidant generation. One mechanism involved in the defense of oxidative products is the family of glutathione transferases (GST). We have analyzed the activity, distribution and expression of GSTP1 and GSTA4 isoenzymes in the cerebral cortex and cerebellum of young, adult and aged rats. The total GST activity, measured with the universal substrate 1-chloro-2,4-dinitrobenzene (CDNB), increased only with the maturation process; however GSTA4 activity, using the specific substrate 4-hydroxynonenal (HNE), did show an age-dependent increase in both brain regions. Cellular location of GSTA4 in astrocytes was not changed except for young cerebral cortex and adult/aged cerebellum that also showed immunoreactivity in layer III pyramidal neurons and Bergman radial glia, respectively. Distribution of GSTP1 was similar among groups and only an increased number of positive oligodendrocytes was found in the Purkinje and granular layer of adult/aged cerebellum. The GSTA4 and GSTP1 expression increased from young to adult/aged brain and GSTA4 even augmented in the aged cerebral cortex. These results suggest a GST isoenzymatic response with aging, but above all with the maturation process.

Differential effects of iron overload on GST isoform expression in mouse liver and kidney and correlation between GSTA4 induction and overproduction of free radicles

We have investigated the effect of iron overload on the expression of mouse GSTA1, A4, M1, and P1 in liver, the main iron storage site during iron overload, and in kidney. In iron-overloaded animals, mRNA and protein levels of GSTA1, A4, and M1 were increased in liver. In kidney, GSTA4 protein level was also increased while, unexpectedly, GSTA1 and M1 expression was strongly decreased. We showed, by immunohistochemistry, that GSTA4 was more abundant in hepatocytes of periportal areas and in convoluted proximal tubular cells in normal liver and kidney, respectively. In iron-overloaded mice, GSTA4 staining was more intense in cells that preferentially accumulated iron, and conjugation of 4-hydroxynonenal, a specific substrate of GSTA4, was enhanced in both organs. Moreover an acute exposure of primary cultures of mouse hepatocytes to iron-citrate strongly induced oxidative stress and cellular injury and resulted in an increase in GSTA4 expression, while cotreatment with iron-citrate and either desferrioxamine or vitamin E prevented both toxicity and GSTA4 induction. These data demonstrate that GSTA1 and M1 are differentially regulated in liver and kidney while GSTA4 is induced in both organs during iron overload. Moreover, they support the view that iron-induction of GSTA4 is related to an overproduction of free radicals.