Conocimiento, intención y propósito como elementos de un delito

Previous studies suggest that many neoplastic tissues exhibit a decrease in gap junctional intercellular communication (GJIC). Many hydrocarbons and organochlorine compounds are environmental pollutants known to be carcinogenic. The effect of an organochlorine compound, TCDD, on GJIC in human breast cell lines has not been established. In the present study, we showed that TCDD causes an inhibition in the gap junctional activity in MCF-7 (breast cancer cells). In MCF-7 cells, an increase in the phosphorylated form of gap junctional protein, connexin 43 (Cx43), and PKC α was seen in the presence of TCDD. Gap junctional plaque formation was significantly decreased in MCF-7 cells in the presence of TCDD. Immunoprecipitation studies of PKC α showed that TCDD caused a significant 40% increase in the phosphorylated Cx43 in MCF-7 cells. TCDD also modulated the translocation of PKC α from the cytosol to the membrane and caused a 2-fold increase in the PKC α activity at 50 nM TCDD in MCF-7 cells. Calphostin C, an inhibitor of PKC α, showed a significant inhibition of PKC α activity in the presence of TCDD. Furthermore, TCDD also caused a decrease in the gap junctional activity and Cx43 protein in human mammary epithelial cells (HMEC). However, we observed a shift in the Cx43 plaques towards the perinuclear membrane in the presence of TCDD by confocal microscopy and Western blot. Overall, these results conclude that TCDD decreases GJIC by phosphorylating Cx43 via PKC α signaling pathway in MCF- 7 cells; however, TCDD decreases the GJIC by affecting the localization of Cx43 in HMEC. These new findings elucidate the differential mode of effect of TCDD in the downregulation of GJIC in HMEC and MCF-7 cells.

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3.2 Introduction

Polycyclic aromatic hydrocarbons are ubiquitous environmental and food contaminants formed mainly during the incomplete combustion of organic materials (Blaha et al., 2002). Among these, 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), is a highly persistent environmental pollutant and a known human carcinogen (Safe, 1990, 1995). More than 2-fold increase in the incidence of breast cancer was found in female workers working in TCDD- exposed chemical plant (Manz et al., 1991). TCDD was shown to alter mammary gland differentiation and increased susceptibility for mammary cancer in a carcinogen induced rat mammary cancer model (Jenkins et al., 2006). Many studies indicate the potential role of TCDD in regulating the activity of GJIC (Baker et al., 1995; Chipman et al., 2003; Sharovskaja et al. 2004). But the effect of TCDD on the GJIC in human breast cells has not been studied.

Gap junctional intercellular communication (GJIC) is formed by gap junctions (GJ), dynamic intercellular plasma membrane channels allowing the passage of small molecules (<1 kD) between adjacent cells (Mesnil, 2002; Carystinos et al., 2002; Vine and Bertram, 2002). Gap junctions are formed by the interaction of two hemichannels (connexon) in the adjacent cells, which, in turn are made of six subunits of connexin (Cx) proteins (Carystinos et al., 2002). The structure of connexins includes four hydrophobic membrane-spanning domains, two extracellular loops, one cytoplasmic loop, and an amino and carboxyl terminus in the cytoplasm (Goodenough et al., 1988; Hertzberg et al., 1988; Yeager and Gilula, 1992; Knudsen and Wheelock, 1992). In a cell, GJIC is mainly regulated by the phosphorylation of the C-terminus of connexin proteins (Carystinos et al., 2002). The ability of TPA to inhibit GJIC has been associated with the activation of PKC (Lampe, 1994; Rivedal and Opsahl, 2001; Zampighi et al., 2005). Evidence demonstrated that PKC may inhibit gap junction channels by inhibiting gap junction assembly and channel gating (Tomasetto et al., 1993). PKC α overexpression has been shown to cause tumor growth, and tumor resistance to cytotoxic chemotherapy. In a study conducted on breast cancer patients, PKC activity was found significantly higher in breast tumor tissue compared to normal tissue from the same patient (O'Brian et al., 1989). Studies have shown that many neoplastic cells show fewer or dysfunctional gap junctions and have reduced GJIC (Trosko and Ruch, 1998). Many studies also suggest that the alteration in connexin expression is important, as their forced expression, following cDNA transfection, can promote cell density inhibition and reverse the tumor phenotype (Mesnil, 2002). Tumor growth of communication-deficient human

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tumor cells, SKHep1, was significantly higher than cells transfected with cDNA encoding Cx32 in athymic nude mice (Eghbali et al., 1991). Re-expression of hCx26 and hCx43 exhibited a classical tumor-suppressive behavior restoring growth-regulatory properties to metastatic mammary carcinoma cells and inducing differentiation (Hirschi et al., 1996). Consistently, these studies strongly suggest that the altered expression of connexins leads to loss of growth control and restoration of GJIC causes inhibition of cell proliferation thereby exhibiting tumor suppressive effect. Given the importance of GJIC in cancer, we used breast cancer as the model to test the effect of TCDD on GJIC. Cx43 is the most predominant form in breast epithelium and detected mostly in myoepithelial cells. Therefore, in our present study we observed the effect of TCDD on GJIC by measuring Cx43 expression. The strategy for the prevention and treatment of cancers is based on understanding the underlying mechanisms of carcinogenesis. Environmental pollutants have been shown to affect the GJIC but the mechanism still remains elusive. The aim of our study was to investigate how TCDD can affect the activity of GJIC in human breast cells. We directly examined the effect of TCDD on human breast cancer cells, MCF-7, and primary mammary epithelial cells, HMEC. Our study provides evidence showing that TCDD inhibits the GJIC through traditional and non-traditional pathways in cancerous and non-cancerous breast cells respectively.