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Martin Tenniswood - One of the best experts on this subject based on the ideXlab platform.
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induction of gene expression during involution of the lactating mammary gland of the rat
Journal of Molecular Endocrinology, 1994Co-Authors: R S Guenette, Jocelyne G. Léger, H B Corbeil, Kim Wong, V Mezl, M Mooibroek, Martin TenniswoodAbstract:After weaning, the mammary gland ceases lactation and involutes. The wet weight of the gland decreases by 70% within 4 days of weaning. This involves significant tissue remodelling as the ducts regress and return to the resting state. The presence of apoptotic bodies in the luminal epithelial compartment 2 to 3 days after weaning provides clear evidence that a substantial proportion of the regression is attributable to the induction of active cell death (ACD) of the epithelial cells. These changes in the architecture of the gland were found to be mirrored by changes in gene expression. The steady-state level of beta-casein mRNA decreased rapidly after weaning from the high levels seen during lactation to undetectable levels by 8 days after weaning. The steady-state levels of expression of a number of genes associated with ACD, including TRPM-2, tissue transglutaminase (TGase) and poly(ADP-ribose) polymerase (PARP), increased transiently during this time-frame. The steady-state level of TRPM-2 mRNA increased 2 days after weaning, reaching a peak on day 4, and decreasing to undetectable levels by day 8 after weaning. The steady-state levels of two other mRNAs, TGase and PARP, showed very similar kinetics. In contrast, the mRNA for Hsp 27, which has been shown to be induced during prostate regression, was not significantly induced in the regressing mammary gland. In-situ hybridization demonstrated that the TRPM-2, TGase and PARP genes were expressed predominantly in the luminal epithelial cells of the ducts. These cells expressed beta-casein mRNA during lactation, and underwent ACD after weaning. While the ultrastructural changes in the mammary gland after weaning, and the induction of TRPM-2, TGase and PARP mRNAs, are reminiscent of apoptosis in the prostate, several features of the process are different. Most notably, the disruption of the secretory processes and the lack of increased expression of Hsp 27 in the regressing mammary gland suggest that there may be a number of important events in ACD that are not common to all cells.
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Molecular characterization of human TRPM-2/clusterin, a gene associated with sperm maturation, apoptosis and neurodegeneration
European Journal of Biochemistry, 1994Co-Authors: Paul Wong, J Pineault, J Lakins, D Taillefer, Gerald J. Chader, Martin TenniswoodAbstract:The TRPM-2/clusterin gene and its cognate protein has been characterized in a number of species. Although the functional role, or roles, of the TRPM-2/clusterin protein remains to be firmly established, the gene has been implicated in a variety of physiological processes, including sperm maturation, lipid transport, membrane remodelling and inhibition of the complement cascade. TRPM-2/clusterin is induced de novo during the regression of the prostate and other hormone-dependent tissues after hormone ablation, and is over-expressed in several human neurodegenerative diseases including Alzheimer's disease, epilepsy and retinitis pigmentosa. We describe the genomic structure of the human TRPM-2/clusterin gene which is organized into nine exons, ranging in size from 47 bp (exon I) to 412 bp (exon V), spanning a region of 16580 bp. Comparison with sequences registered in the databases shows that it has extensive similarity to the human protein designated as SP-40,40 or complement-lysis inhibitor (CLI), a protein that appears to block the membrane-attack complex of complement. However, the cDNA sequences reported for SP-40,40 and CLI diverge significantly in the 5′ untranslated region of the mRNA (coded for by exon I), raising the possibility that the TRPM-2/clusterin gene is present in the human genome as a small multi-gene family or that there are several alternate exon I sequences in the TRPM-2 gene. Southern analysis and fluorescent in situ hybridization suggest that the clusterin gene is a single-copy gene, and that, if alternative exon I sequences are present in the genome, they lie outside of the λ clones that have been characterized. Analysis of the promoter region of the human TRPM-2/clusterin gene shows many similarities to the rat TRPM-2/clusterin promoter including a putative control region containing several potential regulatory elements that may regulate the complex tissue-specific control of the gene which must be constitutively expressed in some tissues but is inducible in others.
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Effect of tumour progression on the androgenic regulation of the androgen receptor, TRPM-2 and YPT1 genes in the Shionogi carcinoma.
The Journal of Steroid Biochemistry and Molecular Biology, 1994Co-Authors: Paul S. Rennie, Paul Wong, Nicholas Bruchovsky, Koichiro Akakura, S. Larry Goldenberg, Navdeep Otal, Sanae Akakura, Martin TenniswoodAbstract:Progression of an androgen-dependent tumour to an androgen-independent state is characterized by the loss of apoptotic potential, a property of cells which have differentiated under the influence of androgens. In an attempt to relate progression to mechanisms of apoptotic failure, we compared the relative levels of expression of androgen receptor and TRPM-2 (clusterin) genes in androgen-dependent and -independent tumours derived from the Shionogi carcinoma. The amount of 10 kb androgen receptor mRNA in androgen-dependent and -independent cells was similar thus showing no relationship to progression. Owing to cross-hybridization of androgen receptor cDNA with non-receptor transcripts, two new androgen-repressed mRNAs (ADS31 and ADS39) were cloned. Each was found to have a 20/21 bp GC-rich region of sequence homology with the androgen receptor, implying selective conservation of a domain whose function is unknown. Sequencing results also revealed that ADS31 cDNA encodes a polypeptide identical to mouse YPT1, a ras-related GTP-binding protein. Expression of the ADS31/YPT1, ADS39 and TRPM-2 genes was sensitive to androgen withdrawal and replacement both in the parent androgen-dependent and the recurrent androgen-independent carcinomas. The uncoupling of TRPM-2 expression and apoptosis observed in androgen-independent tumour cells implies that the function of androgen receptor becomes more restricted with tumour progression. Furthermore, the fact that the expression of ADS31/YPT1 transcript becomes dominant in the advanced stages of androgen-independent growth, suggests that the mechanism of progression is subserved by duplication and possibly redundancy of alternative (signal transduction) pathways mediating tumour cell survival and growth.
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Genomic organization and expression of the rat TRPM-2 (clusterin) gene, a gene implicated in apoptosis.
The Journal of biological chemistry, 1993Co-Authors: Paul Wong, J Pineault, J Lakins, D Taillefer, Jocelyne G. Léger, C Wang, Martin TenniswoodAbstract:We describe the genomic structure of the rat TRPM-2 gene. This gene is induced de novo during the regression of the prostate and other hormone-dependent tissues after hormone ablation and plays an important role in apoptosis, or active cell death. TRPM-2 is a single copy gene, organized into nine exons, ranging in size from 47 base pairs (exon I) to 412 base pairs (exon IV), spanning a region of 13,750 base pairs. Comparison with sequences registered in the data bases shows that it has extensive homology to SGP-2, a gene that is expressed constitutively in the testis, although the sequences diverge dramatically in the 5'-untranslated region of the mRNA (coded for by exon I in TRPM-2), raising the possibility of alternative exon I usage in SGP-2. Primer extension, S1 nuclease protection, and extensive polymerase chain reaction analysis suggest that the TRPM-2 transcript from the prostate and the SGP-2 transcript from the testis are in fact identical and only contain the exon I sequence identified in the TRPM-2 genomic clone. Analysis of the promoter region of the TRPM-2 gene demonstrates that the putative control region contains several potential regulatory elements that may regulate the complex tissue-specific control of a gene which must be constitutively expressed in some tissues but repressed in others until induced during active cell death.
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developmental expression of the s35 s45 sgp 2 TRPM 2 gene in rat testis and epididymis
Molecular Reproduction and Development, 1992Co-Authors: Zahra Zakeri, Marcello Curto, Dennis Hoover, Karen Wightman, Jeff Engelhardt, Frank F. Smith, Abraham L. Kierszenbaum, Timothy Gleeson, Martin TenniswoodAbstract:Testosterone-repressed prostate message-2 (TRPM-2) was originally isolated and cloned from the regressing ventral prostate of the rat. In this tissue, and in other hormone-dependent tissues such as the mammary gland, this gene is induced in the absence of the appropriate trophic hormone. Sequence analysis of the cDNA and genomic clones of TRPM-2 have demonstrated that the coding sequence of this gene is identical to S35-S45 (also known as SGP-2 and clusterin), which is constitutively expressed by the Sertoli cells of the adult testis. Using Northern, slot blot, S1-nuclease analysis, and in situ hybridization, we have investigated the regulation of TRPM-2 expression in the testis and epididymis during development. Slot blot analysis of RNA extracted from the testis and epididymis of 7-, 14-, 28-, 35-, and 91-day-old rats demonstrates that the gene is induced to detectable levels between days 7 and 14 and that the relative level of expression does not change significantly after day 14. In situ hybridization using frozen sections of testis from day 2-, 7-, 14-, 28-, 35-, and 91-day-old rats confirms that there is little expression of TPRM-2 in the seminiferous epithelium of 7-day-old rats, but this increases considerably after 14 days, primarily in Sertoli cells but also in association with meiotic developing spermatogenic cells. However, TRPM-2 mRNA is expressed in the rete testis at 2 days of age, reaches a peak at 35 days of age, and continues to be expressed in the adult. Slot blot analysis demonstrates that TRPM-2 is also induced in the epididymis between 7 and 14 days of age, although, as has been demonstrated by in situ hybridization, TRPM-2 mRNA is detectable in the epithelial cells in the head of the epididymis but is barely detectable in the midportion or tail regions. Northern analysis suggests that the size of the TRPM-2 transcript in the testis also changes during development. In the early stages of testicular development, the TRPM-2 transcript appears to be a broad band of approximately 1.5 kb, while the transcript in the adult appears to be approximately 1.8 kb in length. S1-nuclease protection assays suggest that this increase in size is not due to differential splicing of the first exon of TRPM-2/SGP-2 and most probably reflects a difference in the polyadenylation of the mRNA in the testis at different times during development. © 1992 Wiley-Liss, Inc.
Martin E. Gleave - One of the best experts on this subject based on the ideXlab platform.
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Acquisition of Chemoresistant Phenotype by Overexpression of the Antiapoptotic Gene Testosterone-repressed Prostate Message-2 in Prostate Cancer Xenograft Models
Cancer research, 2000Co-Authors: Hideaki Miyake, Colleen C. Nelson, Paul S. Rennie, Martin E. GleaveAbstract:Testosterone-repressed prostate message-2 (TRPM-2) expression is highly up-regulated in normal and malignant prostate cells after androgen withdrawal. Although recent studies have suggested a protective role of TRPM-2 expression against apoptosis in several experimental models, the functional role of TRPM-2 in chemotherapy-induced apoptosis remains undefined. Here, we demonstrated that overexpression of TRPM-2 in human androgen-dependent LNCaP prostate cancer cells by stable transfection rendered them highly resistant to paclitaxel treatment than control LNCaP cells, with a 20-fold higher IC50 through the inhibition of apoptotic cell death. In mice bearing TRPM-2-overexpressing LNCaP tumors, tumor volume and serum prostate-specific antigen increased two to three times faster after castration and paclitaxel treatment compared with mice bearing control tumors. We then tested the efficacy of combined treatment with antisense TRPM-2 oligodeoxynucleotide (ODN) and paclitaxel in the mouse androgen-dependent Shionogi tumor model. Antisense TRPM-2 ODN treatment significantly enhanced paclitaxel chemosensitivity of Shionogi tumor cells in a dose-dependent manner, reducing the IC50 by 75%. Combined treatment of Shionogi cells with 500 nM antisense TRPM-2 ODN and 10 nM paclitaxel-induced apoptosis, either agent alone did not. Adjuvant administration of antisense TRPM-2 ODN and polymeric micellar paclitaxel after castration resulted in reduced TRPM-2 levels in vivo and a significant delay of emergence of androgen-independent recurrent Shionogi tumors compared with administration of either agent alone. Furthermore, combined treatment of mice bearing androgen-independent recurrent Shionogi tumors with antisense TRPM-2 ODN and micellar paclitaxel inhibited tumor growth compared with treatment with either agent alone. Collectively, these findings demonstrate that TRPM-2 overexpression helps confer a chemoresistant phenotype through inhibition of apoptosis, and that antisense TRPM-2 ODN may be useful in enhancing the effects of cytotoxic chemotherapy in hormone-refractory prostate cancer.
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Testosterone-repressed prostate message-2 is an antiapoptotic gene involved in progression to androgen independence in prostate cancer.
Cancer research, 2000Co-Authors: Hideaki Miyake, Colleen C. Nelson, Paul S. Rennie, Martin E. GleaveAbstract:Although initially reported as an androgen-repressed gene in the rat prostate, the functional role of testosterone-repressed prostate message-2 (TRPM-2) in apoptosis remains undefined. Inhibition of castration-induced apoptosis by calcium channel blocker treatment in androgen-dependent Shionogi tumors resulted in the prevention of TRPM-2 gene up-regulation, suggesting that TRPM-2 is not directly androgen-repressed, but is regulated by apoptotic stimuli. The overexpression of the TRPM-2 gene in human androgen-dependent LNCaP prostate cancer cells by stable transfection rendered them highly resistant to androgen ablation in vivo. We then tested the efficacy of antisense TRPM-2 oligodeoxynucleotide (ODN) therapy in the Shionogi tumor model and demonstrated that the systemic administration of antisense TRPM-2 ODNs in mice bearing Shionogi tumors after castration resulted in a more rapid onset of apoptosis and time to complete regression, as well as a significant delay of emergence of androgen-independent recurrent tumors compared to control ODN treatment. Collectively, these findings illustrate that TRPM-2 is an antiapoptotic rather than an androgen-repressed gene that confers resistance to androgen ablation and thereby helps accelerate the progression to androgen independence.
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Antisense TRPM-2 oligodeoxynucleotides chemosensitize human androgen-independent PC-3 prostate cancer cells both in vitro and in vivo.
Clinical cancer research : an official journal of the American Association for Cancer Research, 2000Co-Authors: Hideaki Miyake, Kim N. Chi, Martin E. GleaveAbstract:Although numerous chemotherapeutic regimens have been evaluated for patients with hormone-refractory prostate cancer, none has improved survival. Testosterone-repressed prostate message-2 (TRPM-2), which is highly up-regulated after androgen withdrawal and during androgen-independent progression in prostate cancer, has been shown to inhibit apoptosis induced by various kinds of stimuli. The objectives in this study were to test whether antisense (AS) oligodeoxynucleotides (ODNs) targeted against TRPM-2 enhance chemosensitivity in human androgen-independent prostate cancer PC-3 cells both in vitro and in vivo . Initially, the potency of 10 AS ODNs targeting various regions of the TRPM-2 mRNA were evaluated, and the AS ODN targeted to the TRPM-2 translation initiation site (AS ODN#2) was found to be the most potent sequence for inhibiting TRPM-2 expression in PC-3 cells. Despite significant dose-dependent and sequence-specific suppression of TRPM-2 expression, AS ODN#2 had no effect on growth of PC-3 cells both in vitro and in vivo . However, pretreatment of PC-3 cells with AS ODN#2 significantly enhanced chemosensitivity of Taxol (paclitaxel) and mitoxantrone in vitro . Characteristic apoptotic DNA laddering and cleavage of poly(ADP-ribose) polymerase were observed after combined treatment with AS ODN#2 plus paclitaxel or mitoxantrone but not with either agent alone. In vivo administration of AS ODN#2 plus either paclitaxel or mitoxantrone significantly decreased PC-3 tumor volume by 80 or 60%, respectively, compared with mismatch control ODN plus either paclitaxel or mitoxantrone. In addition, terminal deoxynucleotidyl transferase-mediated nick end labeling staining revealed increased apoptotic cells in tumors treated with AS ODN#2 plus paclitaxel or mitoxantrone. These findings confirm that TRPM-2 overexpression confers resistance to cytotoxic chemotherapy in prostate cancer cells and illustrates the potential utility of combined treatment with AS TRPM-2 ODN plus chemotherapeutic agents for patients with hormone-refractory prostate cancer.
Kotohiko Kimura - One of the best experts on this subject based on the ideXlab platform.
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effect of heat shock treatment on the production of variant testosterone repressed prostate message 2 TRPM 2 mrna in culture cells
Cell Biochemistry and Function, 1997Co-Authors: Kotohiko Kimura, Kimie Asami, Mikio YamamotoAbstract:The testosterone-repressive prostate message-2 (TRPM-2) variant mRNA lacking the exon 5 was induced in rat primary culture hepatocytes by heat shock treatment. A similar variant mRNA lacking exon 5 was also induced by heat shock treatment of the human culture cell line HepG2. On the other hand, in mouse cell line L929, heat shock treatment induced a variant TRPM-2 mRNA lacking only a small region located in exon 5. However, irrespective of the difference of mechanism of variant production, all the variant TRPM-2 mRNA species derived from each animal species encoded a putative protein constituted from the N-terminal one-third of TRPM-2 protein attached to a C-terminal TRPM-2 unrelated tail. In humans, the variant TRPM-2 species was not detected in normal tissues but was present in certain kinds of tumour cells. These results indicate that the splicing variants were induced as a direct result of heat shock treatment on cells per se and that the phenomenon of heat shock induction was observed in culture cells derived from different animal species.
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Modification of the alternative splicing process of testosterone-repressed prostate message-2 (TRPM-2) gene by protein synthesis inhibitors and heat shock treatment.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1996Co-Authors: Kotohiko Kimura, Mikio YamamotoAbstract:Abstract During the course of the study to examine the effect of cycloheximide on apoptosis-related genes, the variant rat testosterone-repressed prostate message-2 (TRPM-2) mRNA deficient of the axon 5 was found. The putative protein encoded by the variant TRPM-2 mRNA is only constituted from the N-terminal one-third portion of the ordinary TRPM-2 protein. The expression of the variant form was increased dramatically by cycloheximide treatment, while that of the ordinary form was not affected very much. The similar phenomenon was also observed by the use of other types of protein synthesis inhibitors, anisomycin and emetine. The enhancement of expression of the variant was observed in the rat treated with heat shock as well. The variant form was presumably generated by the exon skip mechanism. Systematic analyses of cycloheximide effect on the alternative splicing at various splicing junctions were performed. However, cycloheximide did not exhibit any remarkable effects on other types of alternative splicing, including exon skip in βA4-amyloid protein precursor (APP) gene, alternative donor selection in Fas antigen gene and alternative acceptor selection in catechol O -methyltransferase (COMT) gene. These results indicated that the induction of exon skip by both protein synthesis inhibition and heat shock treatment occurs in a limited number of genes, if not only in TRPM-2.
Paul S. Rennie - One of the best experts on this subject based on the ideXlab platform.
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Acquisition of Chemoresistant Phenotype by Overexpression of the Antiapoptotic Gene Testosterone-repressed Prostate Message-2 in Prostate Cancer Xenograft Models
Cancer research, 2000Co-Authors: Hideaki Miyake, Colleen C. Nelson, Paul S. Rennie, Martin E. GleaveAbstract:Testosterone-repressed prostate message-2 (TRPM-2) expression is highly up-regulated in normal and malignant prostate cells after androgen withdrawal. Although recent studies have suggested a protective role of TRPM-2 expression against apoptosis in several experimental models, the functional role of TRPM-2 in chemotherapy-induced apoptosis remains undefined. Here, we demonstrated that overexpression of TRPM-2 in human androgen-dependent LNCaP prostate cancer cells by stable transfection rendered them highly resistant to paclitaxel treatment than control LNCaP cells, with a 20-fold higher IC50 through the inhibition of apoptotic cell death. In mice bearing TRPM-2-overexpressing LNCaP tumors, tumor volume and serum prostate-specific antigen increased two to three times faster after castration and paclitaxel treatment compared with mice bearing control tumors. We then tested the efficacy of combined treatment with antisense TRPM-2 oligodeoxynucleotide (ODN) and paclitaxel in the mouse androgen-dependent Shionogi tumor model. Antisense TRPM-2 ODN treatment significantly enhanced paclitaxel chemosensitivity of Shionogi tumor cells in a dose-dependent manner, reducing the IC50 by 75%. Combined treatment of Shionogi cells with 500 nM antisense TRPM-2 ODN and 10 nM paclitaxel-induced apoptosis, either agent alone did not. Adjuvant administration of antisense TRPM-2 ODN and polymeric micellar paclitaxel after castration resulted in reduced TRPM-2 levels in vivo and a significant delay of emergence of androgen-independent recurrent Shionogi tumors compared with administration of either agent alone. Furthermore, combined treatment of mice bearing androgen-independent recurrent Shionogi tumors with antisense TRPM-2 ODN and micellar paclitaxel inhibited tumor growth compared with treatment with either agent alone. Collectively, these findings demonstrate that TRPM-2 overexpression helps confer a chemoresistant phenotype through inhibition of apoptosis, and that antisense TRPM-2 ODN may be useful in enhancing the effects of cytotoxic chemotherapy in hormone-refractory prostate cancer.
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Testosterone-repressed prostate message-2 is an antiapoptotic gene involved in progression to androgen independence in prostate cancer.
Cancer research, 2000Co-Authors: Hideaki Miyake, Colleen C. Nelson, Paul S. Rennie, Martin E. GleaveAbstract:Although initially reported as an androgen-repressed gene in the rat prostate, the functional role of testosterone-repressed prostate message-2 (TRPM-2) in apoptosis remains undefined. Inhibition of castration-induced apoptosis by calcium channel blocker treatment in androgen-dependent Shionogi tumors resulted in the prevention of TRPM-2 gene up-regulation, suggesting that TRPM-2 is not directly androgen-repressed, but is regulated by apoptotic stimuli. The overexpression of the TRPM-2 gene in human androgen-dependent LNCaP prostate cancer cells by stable transfection rendered them highly resistant to androgen ablation in vivo. We then tested the efficacy of antisense TRPM-2 oligodeoxynucleotide (ODN) therapy in the Shionogi tumor model and demonstrated that the systemic administration of antisense TRPM-2 ODNs in mice bearing Shionogi tumors after castration resulted in a more rapid onset of apoptosis and time to complete regression, as well as a significant delay of emergence of androgen-independent recurrent tumors compared to control ODN treatment. Collectively, these findings illustrate that TRPM-2 is an antiapoptotic rather than an androgen-repressed gene that confers resistance to androgen ablation and thereby helps accelerate the progression to androgen independence.
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Effect of tumour progression on the androgenic regulation of the androgen receptor, TRPM-2 and YPT1 genes in the Shionogi carcinoma.
The Journal of Steroid Biochemistry and Molecular Biology, 1994Co-Authors: Paul S. Rennie, Paul Wong, Nicholas Bruchovsky, Koichiro Akakura, S. Larry Goldenberg, Navdeep Otal, Sanae Akakura, Martin TenniswoodAbstract:Progression of an androgen-dependent tumour to an androgen-independent state is characterized by the loss of apoptotic potential, a property of cells which have differentiated under the influence of androgens. In an attempt to relate progression to mechanisms of apoptotic failure, we compared the relative levels of expression of androgen receptor and TRPM-2 (clusterin) genes in androgen-dependent and -independent tumours derived from the Shionogi carcinoma. The amount of 10 kb androgen receptor mRNA in androgen-dependent and -independent cells was similar thus showing no relationship to progression. Owing to cross-hybridization of androgen receptor cDNA with non-receptor transcripts, two new androgen-repressed mRNAs (ADS31 and ADS39) were cloned. Each was found to have a 20/21 bp GC-rich region of sequence homology with the androgen receptor, implying selective conservation of a domain whose function is unknown. Sequencing results also revealed that ADS31 cDNA encodes a polypeptide identical to mouse YPT1, a ras-related GTP-binding protein. Expression of the ADS31/YPT1, ADS39 and TRPM-2 genes was sensitive to androgen withdrawal and replacement both in the parent androgen-dependent and the recurrent androgen-independent carcinomas. The uncoupling of TRPM-2 expression and apoptosis observed in androgen-independent tumour cells implies that the function of androgen receptor becomes more restricted with tumour progression. Furthermore, the fact that the expression of ADS31/YPT1 transcript becomes dominant in the advanced stages of androgen-independent growth, suggests that the mechanism of progression is subserved by duplication and possibly redundancy of alternative (signal transduction) pathways mediating tumour cell survival and growth.
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Relationship between Variant Forms of Estrogen Receptor RNA and an Apoptosis-Related RNA, TRPM-2, with Survival in Patients with Breast Cancer
Cancer, 1993Co-Authors: Paul S. Rennie, Nasrin R. Mawji, Andrew J. Coldman, William Godolphin, Edward C. Jones, Juergen R. Vielkind, Nicholas BruchovskyAbstract:Background. Although smaller variant forms of estrogen receptor (ER) messenger RNA (mRNA) have been detected in breast tumors, neither their prevalence nor their prognostic significance have been evaluated. Similarly, TRPM-2 mRNA, the product of a gene induced principally during the onset of apoptosis, is present in mouse and human breast cancer cell lines, but whether it also occurs in primary breast tumors and is related to disease outcome is unknown. Methods. The relative expression and transcript size of ER mRNA and TRPM-2 mRNA in 126 primary breast tumors were measured by Northern analysis and compared with tumor grade, hormone receptor status, extent of tumor necrosis, and survival. Results. In ER-positive tumors, 64% of the tumors had only the normal 6.5 kb ER mRNA, an additional 9% had the normal plus smaller ER mRNA, and 2% had variant forms. Only 8% of ER-negative tumors had ER mRNA transcripts. There were significant relationships between the occurrence of ER mRNA and low tumor grade, ER-positive receptor status, and better survival. In contrast, TRPM-2 mRNA was found in only 17% of breast tumors, none of which could be grouped with respect to grade, hormone receptor status, or survival. Conclusions. The presence of smaller variant forms of ER mRNA either alone or in association with the normal ER transcript is not indicative of an unfavorable prognosis, whereas TRPM-2 mRNA occurs in many primary breast tumors, but has no apparent relationship to survival.
Dominik Oliver - One of the best experts on this subject based on the ideXlab platform.
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direct modulation of TRPM4 and TRPM3 channels by the phospholipase c inhibitor u73122
British Journal of Pharmacology, 2016Co-Authors: Michael G Leitner, Marc Behrendt, Marlen Dierich, Sandeep Dembla, Bettina U Wilke, Maik Konrad, Johannes Oberwinkler, Moritz Lindner, Niklas Michel, Dominik OliverAbstract:Background and Purpose Signalling through phospholipase C (PLC) controls many cellular processes. Much information on the relevance of this important pathway has been derived from pharmacological inhibition of the enzymatic activity of PLC. We found that the most frequently employed PLC inhibitor, U73122, activates endogenous ionic currents in widely used cell lines. Given the extensive use of U73122 in research, we set out to identify these U73122-sensitive ion channels. Experimental Approach We performed detailed biophysical analysis of the U73122-induced currents in frequently used cell lines. Key Results At concentrations required to inhibit PLC, U73122 modulated the activity of transient receptor potential melastatin (TRPM) channels through covalent modification. U73122 was shown to be a potent agonist of ubiquitously expressed TRPM4 channels and activated endogenous TRPM4 channels in CHO cells independently of PLC and of the downstream second messengers PI(4,5)P2 and Ca2+. U73122 also potentiated Ca2+-dependent TRPM4 currents in human Jurkat T-cells, endogenous TRPM4 in HEK293T cells and recombinant human TRPM4. In contrast to TRPM4, TRPM3 channels were inhibited whereas the closely related TRPM5 channels were insensitive to U73122, showing that U73122 exhibits high specificity within the TRPM channel family. Conclusions and Implications Given the widespread expression of TRPM4 and TRPM3 channels, these actions of U73122 must be considered when interpreting its effects on cell function. U73122 may also be useful for identifying and characterizing TRPM channels in native tissue, thus facilitating the analysis of their physiology.
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direct modulation of TRPM4 and TRPM3 channels by the phospholipase c inhibitor u73122
British Journal of Pharmacology, 2016Co-Authors: Michael G Leitner, Marc Behrendt, Marlen Dierich, Sandeep Dembla, Bettina U Wilke, Maik Konrad, Johannes Oberwinkler, Moritz Lindner, Niklas Michel, Dominik OliverAbstract:Background and Purpose Signalling through phospholipase C (PLC) controls many cellular processes. Much information on the relevance of this important pathway has been derived from pharmacological inhibition of the enzymatic activity of PLC. We found that the most frequently employed PLC inhibitor, U73122, activates endogenous ionic currents in widely used cell lines. Given the extensive use of U73122 in research, we set out to identify these U73122-sensitive ion channels. Experimental Approach We performed detailed biophysical analysis of the U73122-induced currents in frequently used cell lines. Key Results At concentrations required to inhibit PLC, U73122 modulated the activity of transient receptor potential melastatin (TRPM) channels through covalent modification. U73122 was shown to be a potent agonist of ubiquitously expressed TRPM4 channels and activated endogenous TRPM4 channels in CHO cells independently of PLC and of the downstream second messengers PI(4,5)P2 and Ca2+. U73122 also potentiated Ca2+-dependent TRPM4 currents in human Jurkat T-cells, endogenous TRPM4 in HEK293T cells and recombinant human TRPM4. In contrast to TRPM4, TRPM3 channels were inhibited whereas the closely related TRPM5 channels were insensitive to U73122, showing that U73122 exhibits high specificity within the TRPM channel family. Conclusions and Implications Given the widespread expression of TRPM4 and TRPM3 channels, these actions of U73122 must be considered when interpreting its effects on cell function. U73122 may also be useful for identifying and characterizing TRPM channels in native tissue, thus facilitating the analysis of their physiology.
