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1.
J Cell Biochem ; 99(1): 241-55, 2006 Sep 01.
Article in English | MEDLINE | ID: mdl-16598789

ABSTRACT

The microbial toxin okadaic acid (OA) specifically inhibits PPP-type ser/thr protein phosphatases. OA is an established tumor promoter with numerous cellular effects that include p53-mediated cell cycle arrest. In T51B rat liver epithelial cells, a model useful for tumor promotion studies, p53 activation is induced by tumor-promoting (low nanomolar) concentrations of OA. Two phosphatases sensitive to these concentrations of OA, PP2A and protein phosphatase 5 (PP5), have been implicated as negative regulators of p53. In this study we examined the respective roles of these phosphatases in p53 activation in non-neoplastic T51B cells. Increases in p53 activity were deduced from levels of p21 (cip1) and/or the rat orthologue of mdm2, two p53-regulated gene products whose induction was blocked by siRNA-mediated knockdown of p53. As observed with 10 nM OA, both phospho-ser15-p53 levels and p53 activity were increased by 10 microM fostriecin or SV40 small t-antigen. Both of these treatments selectively inhibit PP2A but not PP5. siRNA-mediated knockdown of PP2A, but not PP5, also increased p53 activity. Finally, adenoviral-mediated over-expression of an OA-resistant form of PP5 did not prevent increased phospho-ser15-p53, p53 protein, or p53 activity caused by 10 nM OA. Together these results indicate that PP5 blockade is not responsible for OA-induced p53 activation and G1 arrest in T51B cells. In contrast, specific blockade of PP2A mimics p53-related responses to OA in T51B cells, suggesting that PP2A is the target for this response to OA.


Subject(s)
Liver/drug effects , Nuclear Proteins/antagonists & inhibitors , Okadaic Acid/pharmacology , Phosphoprotein Phosphatases/antagonists & inhibitors , Tumor Suppressor Protein p53/metabolism , Alkenes/pharmacology , Amino Acid Sequence , Animals , Antigens, Viral, Tumor/genetics , Antigens, Viral, Tumor/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , G1 Phase/drug effects , Liver/cytology , Liver/metabolism , Molecular Sequence Data , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphoprotein Phosphatases/genetics , Phosphoprotein Phosphatases/metabolism , Polyenes , Protein Phosphatase 2 , Pyrones/pharmacology , RNA, Small Interfering , Rats , Tumor Suppressor Protein p53/drug effects
2.
Prostate ; 61(1): 12-25, 2004 Sep 15.
Article in English | MEDLINE | ID: mdl-15287090

ABSTRACT

BACKGROUND: Designer T cells are T lymphocytes engineered toward specific antibody-type membrane antigens through chimeric immunoglobulin-T-cell receptor (IgTCR) genes that have been used for adoptive cellular immunotherapy. We have extended this approach to prostate specific membrane antigen (PSMA) as a means to attack prostate cancer. METHODS: A chimeric anti-PSMA IgTCR gene was constructed based on an anti-PSMA monoclonal antibody, 3D8. Both T-cell lines and primary cultured human T lymphocytes were transduced with the chimeric anti-PSMA IgTCR construct and were analyzed for IgTCR expression, specific activation by PSMA, cytotoxicity against PSMA-expressing tumor cells in vitro, and retardation of tumor growth in an animal model. RESULTS: The IgTCR was incorporated into the TCR-CD3 complex and formed a functional chimeric complex. The IgTCR-modified T cells were specifically activated through the chimeric receptor with PSMA as measured by IL-2 production and increased CD25 expression and specifically lysed the PSMA-expressing prostate cancer cells in vitro as well as retarded tumor growth in an animal model. CONCLUSIONS: The anti-PSMA designer T cells exhibit an antibody-type specificity that can recognize PSMA expressing tumor cells in a MHC-independent fashion, resulting in T-cell activation, target cell lysis in vitro and inhibition of tumor growth in vivo.


Subject(s)
Antigens, Surface/immunology , Glutamate Carboxypeptidase II/immunology , Immunoglobulin Heavy Chains/immunology , Immunotherapy, Adoptive/methods , Prostatic Neoplasms/immunology , Prostatic Neoplasms/therapy , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/immunology , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Cell Line, Tumor , Cloning, Molecular , Cytotoxicity Tests, Immunologic , Flow Cytometry , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/immunology , Lymphocyte Activation/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Precipitin Tests , Receptors, Antigen, T-Cell/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology
3.
Cancer Res ; 62(10): 2806-12, 2002 May 15.
Article in English | MEDLINE | ID: mdl-12019157

ABSTRACT

Previously, we demonstrated that connexin 43 (cx43) suppressed the growth of human glioblastoma cells. To investigate the molecular mechanisms involved in tumor suppression by cx43, we developed a human cytokine array system, which simultaneously detects the expression of 43 cytokines. By using this new technology, we analyzed the cx43-regulated genes in cx43-transfected cells. The cytokine arrays showed that expression of monocyte chemotactic protein-1 (MCP-1) was profoundly reduced in cx43-transfected cells. RT-PCR, immuno-Western blot, and cDNA microarrays further confirmed this observation. Addition of conditioned medium from control-transfected cells and recombinant MCP-1 to cx43-transfected cells significantly enhanced cx43-transfected cell proliferation and colony formation in soft agar. In contrast, addition of neutralization antibody against MCP-1 significantly inhibited cell proliferation in control-transfected cells. Our results suggested that MCP-1 is involved in the suppression of human glioblastoma cell growth by cx43.


Subject(s)
Chemokine CCL2/biosynthesis , Connexin 43/physiology , Glioblastoma/metabolism , Glioblastoma/pathology , Cell Division/physiology , Chemokine CCL2/genetics , Connexin 43/genetics , Cytokines/biosynthesis , Cytokines/genetics , Down-Regulation , Gene Expression Profiling , Glioblastoma/genetics , Humans , Oligonucleotide Array Sequence Analysis , Transfection
4.
Biochem Biophys Res Commun ; 290(2): 830-8, 2002 Jan 18.
Article in English | MEDLINE | ID: mdl-11785977

ABSTRACT

Identifying immunogenic tumor antigens plays a critical role in developing efficient diagnostic and therapeutic strategies for treatment of cancer. Using a recently developed technology, serological identification of antigens by recombinant expression cloning (SEREX), we identified a total of 8 genes whose expression elicited antibody responses in prostate cancer patients. Of the 8 genes, 5 represented known genes in the GenBank database, 2 were previously uncharacterized genes, and 1 showed sequence homology to a mouse gene. The sequence feature and the expression of one of the novel genes, prostate antigen recognized and identified by SEREX (PARIS-1), are determined in this study. The PARIS-1 cDNA is 3257 bp in length and contains a complete open reading frame of 2751 bp encoding for a primary translation product of 917 amino acids. Using Northern blot hybridization assay, we detected a single species of approximately 3.3 kb PARIS-1 mRNA that is differentially expressed in prostate normal and cancer cells. Western blot analysis confirmed the expression of the PARIS-1 protein in these cells. Structure analysis revealed that PARIS-1 protein contains a TBC domain that is conserved in the family of cell cycle-regulatory and Rab GTPase-activating proteins (Rab-GAP). Thus, the PARIS-1 protein may play a role in regulation of cell differentiation and growth or represent a new member of the Rab-GAP family.


Subject(s)
Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Neoplasm Proteins/genetics , Neoplasm Proteins/immunology , Prostatic Neoplasms/immunology , Amino Acid Sequence , Antibodies/blood , Antibody Specificity/immunology , Antigens, Neoplasm/biosynthesis , Antigens, Neoplasm/isolation & purification , Base Sequence , Blotting, Northern , Blotting, Western , Cell Cycle Proteins/genetics , Cell Line , Clone Cells/cytology , Clone Cells/immunology , Clone Cells/metabolism , Cloning, Molecular/methods , Conserved Sequence/genetics , Gene Expression Profiling , Humans , Male , Middle Aged , Molecular Sequence Data , Neoplasm Proteins/biosynthesis , Prostate/cytology , Prostate/immunology , Prostate/metabolism , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Protein Structure, Tertiary/genetics , RNA, Messenger/metabolism , Sequence Analysis, DNA , Serologic Tests/methods , rab GTP-Binding Proteins/genetics
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