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Epithelial cell adhesion molecule (EpCAM) (CD326) is a surface glycoprotein expressed by invasive carcinomas and some epithelia. Herein, we report that EpCAM regulates the composition and function of tight junctions (TJ). EpCAM accumulated on the lateral interfaces of human colon carcinoma and normal intestinal epithelial cells but did not co-localize with TJ. Knockdown of EpCAM in T84 and Caco-2 cells using shRNAs led to changes in morphology and adhesiveness. TJ formed readily after EpCAM knockdown; the acquisition of trans-epithelial electroresistance was enhanced, and TJ showed increased resistance to disruption by calcium chelation. Preparative immunoprecipitation demonstrated that EpCAM bound tightly to claudin-7. Co-immunoprecipitation documented associations of EpCAM with claudin-7 and claudin-1 but not claudin-2 or claudin-4. Claudin-1 associated with claudin-7 in co-transfection experiments, and claudin-7 was required for association of claudin-1 with EpCAM. EpCAM knockdown resulted in decreases in claudin-7 and claudin-1 proteins that were reversed with lysosome inhibitors. Immunofluorescence microscopy revealed that claudin-7 and claudin-1 continually trafficked into lysosomes. Although EpCAM knockdown decreased claudin-1 and claudin-7 protein levels overall, accumulations of claudin-1 and claudin-7 in TJ increased. Physical interactions between EpCAM and claudins were required for claudin stabilization. These findings suggest that EpCAM modulates adhesion and TJ function by regulating intracellular localization and degradation of selected claudins. [ABSTRACT FROM AUTHOR]

C1 domains, the recognition motif of the second messenger diacylglycerol and of the phorbol esters, are classified as typical (ligand-responsive) or atypical (not ligand-responsive). The C1 domain of Vav1, a guanine nucleotide exchange factor, plays a critical role in regulation of Vav activity through stabilization of the Dbl homology domain, which is responsible for exchange activity of Vav. Although the C1 domain of Vav1 is classified as atypical, it retains a binding pocket geometry homologous to that of the typical C1 domains of PKCs. This study clarifies the basis for its failure to bind ligands. Substituting Vav1-specific residues into the C1b domain of PKCδ, we identified five crucial residues (Glu9, Glu10, Thr11, Thr24, and Tyr26) along the rim of the binding cleft that weaken binding potency in a cumulative fashion. Reciprocally, replacing these incompatible residues in the Vav1 C1 domain with the corresponding residues from PKCδ C1b (δC1b) conferred high potency for phorbol ester binding. Computer modeling predicts that these unique residues in Vav1 increase the hydrophilicity of the rim of the binding pocket, impairing membrane association and thereby preventing formation of the ternary C1-ligand-membrane binding complex. The initial design of diacylglycerol-lactones to exploit these Vav1 unique residues showed enhanced selectivity for C1 domains incorporating these residues, suggesting a strategy for the development of ligands targeting Vav1. [ABSTRACT FROM AUTHOR]

Abstract: Bryostatin 1 has attracted considerable attention both as a cancer chemotherapeutic agent and for its unique activity. Although it functions, like phorbol esters, as a potent protein kinase C (PKC) activator, it paradoxically antagonizes many phorbol ester responses in cells. Because of its complex structure, little is known of its structure-function relations. Merle 23 is a synthetic derivative, differing from bryostatin 1 at only four positions. However, in U-937 human leukemia cells, Merle 23 behaves like a phorbol ester and not like bryostatin 1. Here, we characterize the behavior of Merle 23 in the human prostate cancer cell line LNCaP. In this system, bryostatin 1 and phorbol ester have contrasting activities, with the phorbol ester but not bryostatin 1 blocking cell proliferation or tumor necrosis factor alpha secretion, among other responses. We show that Merle 23 displays a highly complex pattern of activity in this system. Depending on the specific biological response or mechanistic change, it was bryostatin-like, phorbol ester-like, intermediate in its behavior, or more effective than either. The pattern of response, moreover, varied depending on the conditions. We conclude that the newly emerging bryostatin derivatives such as Merle 23 provide powerful tools to dissect subsets of bryostatin mechanism and response. [Copyright &y& Elsevier]

Background: Oncolytic adenoviruses are under study as a potential anticancer treatment. Their preclinical antitumor activity; however, has not been effectively translated to the clinic. A contributing factor may be the clinical relevance of immunodeficient mouse tumor xenograft models used to study these vectors. While these demonstrate viral replication in the xenograft, they are limited by the lack of natural host immune responses to the virus, associated toxicity, dissemination and systemic viral replication. Cotton rats (Sigmodon hispidus) are semi-permissive for the replication of human adenovirus and have been used to study these vectors. Until recently this model lacked a transplantable tumor to assess oncolytic activity. We characterized three syngeneic transplantable cotton rat tumor cells lines that support adenovirus replication and oncolysis in this immunocompetent rodent model.Methods: Three tumor cell lines: CCRT, LCRT and VCRT were derived from a spontaneously arising osteosarcoma, and fibrosarcomas of the breast and jaw in animals from an inbred cotton rat colony. The ability of the cells to be infected with adenovirus was examined using an adenovirus expressing green fluorescent protein (Ad.GFP). Viral burst assays were used to determine titers of infective adenovirus produced by each cell line at 4 and 72 hours following infection with wild type adenovirus type-5 (Ad5). Transmission electron microscopy (TEM) was used to visualize the relative number of virions at each time point. We also evaluated replication and oncolysis of the cell lines in vivo.Results: Using Ad.GFP, the cell lines were readily infected and demonstrated GFP expression comparable to human A549 cells. All cell lines showed significant higher (P<0.05) intracellular titers of adenovirus and in supernatants 72 hours after infection compared to 4 hours after infection with Ad5 indicating viral replication and increased number of functional virions. In addition, all the cell lines exhibited greater expression of adenoviral hexon protein at 72 hours than at 4 hours after infection, indicating late transcriptional unit gene activation. The cotton rat cell lines formed tumors in cotton rats when injected subcutaneously. On days 5 and 8, PCR demonstrated 2 to 3-log greater number of Ad5 genome copies in tumors injected with Ad5 compared to those injected with a non- replicating adenovirus (Ad.null). This was consistent for CCRT, LCRT and VCRT. Ad5 slowed tumor growth in cotton rats subcutaneously injected with CCRT and VCRT cells. Despite in vitro oncolysis, no anti-tumor effect was seen in the LCRT tumors with the virus rapidly cleared from the tumor.Conclusions: CCRT, LCRT and VCRT, three novel transplantable cotton rat tumor-derived cell lines demonstrate replication of Ad5 both in vitro and in vivo. In addition, CCRT and VCRT demonstrated in vivo oncolysis. The varied antitumor effect in this model mirrors the results of human clinical trials highlighting the potential relevance of these cotton rat tumor models for assessing replication competent adenovirus vectors.Molecular Therapy (2006) 13, S123–S123; doi: 10.1016/j.ymthe.2006.08.379 [ABSTRACT FROM AUTHOR]

Background: Interleukin-15 (IL-15) is a critical cytokine involved in the activation, proliferation and survival of natural killer (NK) and CD8+ T cells. The high-affinity IL-15 receptor (IL-15Rα) expressed on monocytes and dendritric cells supplies IL-15 in trans to NK and T cells. Tumor cells engineered to express IL-15Rα may enhance the immune response to tumor. We studied the antitumor activity of adenoviral-mediated transfer of the murine IL-15Rα gene in a mouse breast cancer model.Methods: An E1–, E3– adenovirus expressing the murine IL-15Rα (Ad.mIL-15Rα) under the control of the CMV promoter and a control vector expressing no transgene (Ad.null) were generated. The expression of mIL-15Rα was examined in murine TS/A mammary carcinoma cells after infection with Ad.mIL-15Rα. The ability of Ad.mIL-15Rα to enhance NK cytolytic killing of transfected TS/A cells in the presence and absence of IL-15 was examined. TS/A cells infected ex vivo (MOI 30) with Ad.mIL-15Rα or Ad.null were subcutaneously (s.c.) injected (5 x 105) into groups of BALB/c mice. The mice were monitored for tumor growth. To study the effect of Ad.mIL-15Rα on pre-established tumors, mice with 150 mm3 TS/A tumors were intratumorally injected with 3 x 109 pfu of Ad.mIL-15Rα or Ad.null, and the tumor growth monitored. To investigate the mechanism of action, depletion of specific immune cell populations was carried out using anti-asialo- GM1, anti-CD4 and anti-CD8 antibodies.Results: Infection of TS/A cells with Ad.mIL-15Rα at an MOI 30 resulted in 60% of the cells expressing IL-15Rα as detected by flow cytometry. The addition of IL-15 enhanced NK cell killing by 2-fold at low NK cell:target cell ratios (1:1). No enhancement by IL-15 was seen at high ratios (10:1). On day 24 mean tumor volumes of mice injected with TS/A cells infected with Ad.mIL-15Rα were 256.2 ±37.1 mm3 compared to 1054.1 ±154.3 mm3 for mice injected with TS/A cells transduced with Ad.null (p <0.05) and 1225.8 ±123.2 mm3 for the untreated tumors (p <0.05). In animals with established tumors, Ad.mIL-15Rα reduced tumor size as measured on day 21 (265.3 ±37.1 mm3) compared to tumors injected with Ad.null (746.1±157.2 mm3) or PBS (799.2 ±121.0 mm3) (p <0.05). Depletion of NK cells abolished the effectiveness of Ad.mIL-15Rα; depletion of CD8+ T cells reduced the treatment effectiveness, whereas depletion of CD4+ T cells had no effect. Immunohistochemical staining of tumors injected with Ad.mIL-15Rα showed extensive infiltration of NK and CD8+ T cells compared to tumors treated with Ad.null, or untreated TS/A tumors.Conclusion: Adenoviral-mediated transfer of the mIL-15Rα gene into tumor demonstrated antitumor activity in the TS/A syngeneic breast cancer model. The effect appears to be mediated by the action of NK cells and CD8+ T cells. IL-15Rα gene transfer represents a cytokine receptor-mediated immunotherapy approach to activate the innate and adaptive immune systems against cancer.Molecular Therapy (2006) 13, S165–S165; doi: 10.1016/j.ymthe.2006.08.494 [ABSTRACT FROM AUTHOR]