ABSTRACT
BACKGROUND: PEST-domain-enriched tyrosine phosphatase (PEP) is a protein tyrosine phosphatase exclusively expressed in hematopoietic cells. It is a potent negative regulator of T-cell receptor signalling that acts on receptor-coupled protein tyrosine kinases. PEST-domain-enriched tyrosine phosphatase is also expressed in mast cell and is positively regulated by glucocorticoids, but its function is unknown. In this communication, the function of PEP is analysed in mast cells. METHODS: Signal transduction cascades following IgE receptor cross-linking were compared in bone marrow-derived mast cells (BMMC) from PEP(-/-) and PEP(+/+) mice. Furthermore, antigen-induced passive systemic anaphylaxis (PSA) was analysed in PEP(+/+) and PEP(-/-) mice. RESULTS: Bone marrow-derived mast cells from PEP(-/-) mice showed impaired PLCγ1 phosphorylation and Ca(2+) mobilization. Additionally, mice deficient in PEP showed impaired mast cell degranulation and were less susceptible to PSA. Treatment of wild-type BMMC or mice with an Au(I)-phosphine complex that selectively inhibits PEP activity produced defects in Ca(2+) signalling pathway and reduced anaphylaxis similar to that caused by the deletion of the PEP gene. Glucocorticoid that negatively regulates a wide range of mast cell action increased PEP expression and only partially inhibited anaphylaxis. However, glucocorticoid potently inhibited anaphylaxis when combined with the PEP inhibitor. CONCLUSIONS: PEST-domain-enriched tyrosine phosphatase is an important positive regulator of anaphylaxis. Pharmacological inhibition of its activity together with glucocorticoid administration provide an effective rescue for PSA in mice.
Subject(s)
Anaphylaxis/immunology , Anaphylaxis/metabolism , Glucocorticoids/metabolism , Immunologic Factors/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 12/metabolism , Anaphylaxis/genetics , Animals , Calcium Signaling , Cell Degranulation/drug effects , Cell Degranulation/immunology , Gene Expression Regulation/drug effects , Gene Expression Regulation/immunology , Glucocorticoids/pharmacology , Immunologic Factors/pharmacology , MAP Kinase Signaling System , Mast Cells/drug effects , Mast Cells/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Phospholipase C gamma/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 12/geneticsABSTRACT
BAG-1L (Bcl-2-associated anthanogene 1) has been found to interact with androgen receptor (AR), and has been suggested to be involved in the development of prostate cancer. In order to determine the presence of genetic and/or expression alterations of BAG-1L in prostate cancer, we analysed human prostate cancer cell lines and xenografts as well as patient samples of untreated, hormone-naïve, and hormone-refractory prostate carcinomas for sequence variations using denaturing high-performance liquid chromatography (DHPLC), for gene copy number using fluorescence in situ hybridization (FISH), and for expression using both quantitative RT-PCR and immunostaining. Only one sequence variation was found in all 37 cell lines and xenografts analysed. BAG-1 gene amplification was detected in two xenografts. In addition, gene amplification was found in 6 of 81 (7.4%) hormone-refractory clinical tumours, whereas no amplification was found in any of the 130 untreated tumours analysed. Additionally, gain of the BAG-1 gene was observed in 27.2% of the hormone-refractory tumours and in 18.5% of the untreated carcinomas. In a set of 263 patient samples, BAG-1L protein expression was significantly higher in hormone-refractory tumours than in primary tumours (p = 0.002). Altogether, these data suggest that amplification and overexpression of BAG-1L may be involved in the progression of prostate cancer.
Subject(s)
DNA-Binding Proteins/metabolism , Neoplasm Proteins/metabolism , Prostatic Neoplasms/metabolism , Transcription Factors/metabolism , Antineoplastic Agents, Hormonal/therapeutic use , Cell Line, Tumor , Chromatography, High Pressure Liquid/methods , DNA Mutational Analysis/methods , DNA-Binding Proteins/genetics , Follow-Up Studies , Gene Expression Regulation, Neoplastic , Humans , Male , Neoplasm Proteins/genetics , Neoplasm Transplantation , Orchiectomy , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Prostatic Neoplasms/therapy , RNA, Messenger/genetics , RNA, Neoplasm/genetics , Receptors, Androgen/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Transcription Factors/genetics , Transplantation, Heterologous , Treatment FailureABSTRACT
Aplidin is an antitumor agent in phase II clinical trials that induces apoptosis through the sustained activation of Jun N-terminal kinase (JNK). We report that Aplidin alters glutathione homeostasis increasing the ratio of oxidized to reduced forms (GSSG/GSH). Aplidin generates reactive oxygen species and disrupts the mitochondrial membrane potential. Exogenous GSH inhibits these effects and also JNK activation and cell death. We found two mechanisms by which Aplidin activates JNK: rapid activation of Rac1 small GTPase and downregulation of MKP-1 phosphatase. Rac1 activation was diminished by GSH and enhanced by L-buthionine (SR)-sulfoximine, which inhibits GSH synthesis. Downregulation of Rac1 by transfection of small interfering RNA (siRNA) duplexes or the use of a specific Rac1 inhibitor decreased Aplidin-induced JNK activation and cytotoxicity. Our results show that Aplidin induces apoptosis by increasing the GSSG/GSH ratio, a necessary step for induction of oxidative stress and sustained JNK activation through Rac1 activation and MKP-1 downregulation.
