Connexin 43 suppresses human glioblastoma cell growth by down-regulation of monocyte chemotactic protein 1, as discovered using protein array technology.

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.

Inhibition of PP2A, but not PP5, mediates p53 activation by low levels of okadaic acid in rat liver epithelial cells.

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.

Anti-prostate specific membrane antigen designer T cells for prostate cancer therapy.

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.

Serological cloning of PARIS-1: a new TBC domain-containing, immunogenic tumor antigen from a prostate cancer cell line.

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.