A novel variant of glutamine: fructose-6-phosphate amidotransferase-1 (GFAT1) mRNA is selectively expressed in striated muscle.

Glutamine:fructose-6-phosphate amidotransferase(GFAT) is the rate-limiting enzyme of the hexosamine synthesis pathway. Products of this pathway have been implicated in insulin resistance and glucose toxicity. GFAT1 is ubiquitous, whereas GFAT2 is expressed mainly in the central nervous system. In the course of developing a competitive reverse transcriptase-polymerase chain reaction assay, we noted that GFAT1 cDNA from muscle but not from other tissues migrated as a doublet. Subsequent cloning and sequencing revealed two GFAT1 mRNAs in both mouse and human skeletal muscles. The novel GFAT1 mRNA (GFAT1Alt [muscle selective variant of GFAT1]) is likely a splice variant. It is identical to GFAT1 except for a 48 or 54 bp insert in the mouse and human, respectively, at nucleotide position 686 of the coding sequence, resulting in a 16 or 18 amino acid insert at position 229 of the protein. GFAT1Alt is the predominant GFAT1 mRNA in mouse hindlimb muscle, is weakly expressed in the heart, and is undetectable in the brain, liver, kidney, lung, intestine, spleen, and 3T3-L1 adipocytes. In humans, it is strongly expressed in skeletal muscle but not in the brain. GFAT1 and GFAT1Alt expressed by recombinant adenovirus infection in COS-7 cells displayed robust enzyme activity and kinetic differences. The apparent K(m) of GFAT1Alt for fructose-6-phosphate was approximately twofold higher than that of GFAT1, whereas K(i) for UDP-N-acetylglucosamine was approximately fivefold lower. Muscle insulin resistance is a hallmark and predictor of type 2 diabetes. Variations in the expression of GFAT isoforms in muscle may contribute to predisposition to insulin resistance.

Exogenous 17beta-estradiol blocks alpha and mu but not pi class glutathione S-transferase immunoreactivity in epithelium of Syrian hamster vas deferens.

Members of the glutathione S-transferase (GST) family of detoxification enzymes play a role in chemotherapy resistance in certain cancers but have not been directly implicated as agents whose absence may predispose tissues to hormonally induced tumorigenesis. Here we report the development of a polyclonal antiserum to a hamster mu class GST, and immunohistochemical analysis of alpha, mu, and pi class GSTs to study the effects of hormone treatment on their expression in reproductive tract tissues of male golden Syrian hamsters. These animals develop leiomyosarcomas in the vas deferens after treatment with testosterone propionate (TP) and 17beta-estradiol (E2). High levels of all three GST classes were detected throughout the reproductive tract epithelium of control animals. In 100% of the experimental animals, 4 weeks of treatment either with E2 alone, or with E2 plus TP promoted a complete loss of immunostaining for alpha and mu class GSTs, but not for pi class GSTs, only in the epithelial lining of the vas deferens. In contrast, treatment with TP alone resulted in moderate hyperplasia of smooth muscle in the proximal vas deferens, with no cellular atypia and no changes in immunoreactivity of any of the GST classes. The consistent and site-specific nature of these results strongly suggests a functional role for GSTs in hormonally induced carcinogenic process. (J Histochem Cytochem 47:91-98, 1999)

Glutathione S-transferase in hormonal carcinogenesis.

Treatment with testosterone propionate (TP) and diethylstilbestrol (DES) or TP and estradiol (E2) for 8-9 months causes development of leiomyosarcomas in the vas deferens or uterus of Golden Syrian hamsters at a frequency of 100%. In males, treatment with estrogens alone results in renal tumors, fatal within 6 months. No leiomyosarcomas have been detected after treatment with estrogens alone, perhaps due to this high mortality rate. In tissue culture, treatment with the glucocorticoid (GC) triamcinolone acetonide (TA) results in an increased expression of a mu-class glutathione S-transferase (GST) (hGSTYBX). We have characterized this induction as a secondary response, i.e. requiring new protein synthesis. Here we describe homologies to known transcription factor-binding sites in the hGSTYBX promoter which may be involved in the induction event. hGSTYBX is a member of a superfamily of detoxification enzymes, induced by genotoxic compounds and reactive oxygen species (ROS). We also describe the effects of several known inducers of other GST family members on hGSTYBX. While implicated in many chemotherapeutic-resistant tumors, GST enzymes have not yet been characterized as a functional agent in hormonal carcinogenesis. This latter possibility is the focus of our investigations. To study the effects of hormone treatment on GST levels in vivo, we have developed a polyclonal antibody to hGSTYBX, and conducted immunohistochemistry on tissues from control and treated animals. Treatment with TP and E2 causes a loss of hGSTYBX expression in the epithelium of the vas deferens. We hypothesize that the loss of this protective enzyme leaves the cells vulnerable to the genotoxic effects of estrogen or estrogenic metabolites.

Novel retroviral sequences are expressed in the epididymis and uterus of Syrian hamsters.

Combined androgen and oestrogen treatment of male or female Syrian hamsters results via an unknown mechanism in the formation of leiomyosarcomas in the reproductive tract. We have examined the possibility that retroviral gene expression may play a role in tumorigenesis. Evidence of virus-like particles in epididymis and seminal fluid is shown in electron micrographs. We identified expressed retroviral sequences by using RT-PCR to amplify a conserved retroviral reverse transcriptase coding region in RNA isolated from epididymis, testis, clarified seminal fluid and uterus. Phylogenetic analysis allowed us to classify the sequences into two distinct groups: (1) mammalian type-C viruses, having similarity to Moloney murine leukaemia virus, feline leukaemia virus and gibbon ape leukaemia virus amongst others; (2) a mixed ABCD group containing, for example, Chinese hamster and murine intracisternal A-particle virus sequences, mouse mammary tumour virus and human and simian retroviral sequences. The presence of putative full-length retrovirus related to mammalian type-C viruses in the epididymis and uterus was confirmed by Northern blot analysis. However, steroid treatment did not alter retroviral RNA levels in the epididymis or in a uterine tumour relative to untreated uterus. In summary, Syrian hamster reproductive tissues were found to express unique retroviral sequences; however, their role, if any, in hormonal carcinogenesis remains unresolved.