Abstract
Gap junctions, composed of connexins, provide a pathway of direct intercellular communication for the diffusion of small molecules between cells. Evidence suggests that connexins act as tumor suppressors. We showed previously that expression of connexin-43 and connexin-32 in an indolent prostate cancer cell line, LNCaP, resulted in gap junction formation and growth inhibition. To elucidate the role of connexins in the progression of prostate cancer from a hormone-dependent to -independent state, we introduced connexin-43 and connexin-32 into an invasive, androgen-independent cell line, PC-3. Expression of these proteins in PC-3 cells resulted in intracellular accumulation. Western blot analysis revealed a lack of Triton-insoluble, plaque-assembled connexins. In contrast to LNCaP cells, connexins could not be cell surface-biotinylated and did not reside in the cell surface derived endocytic vesicles, in PC-3 cells, suggesting impaired trafficking to the cell surface. Intracellular accumulation of connexins was observed in several androgen-independent prostate cancer cell lines. Transient expression of alpha-catenin facilitated the trafficking of both connexins to the cell surface and induced gap junction assembly. Our results suggest that impaired trafficking, and not the inability to form gap junctions, is the major cause of communication deficiency in human prostate cancer cell lines.
Highlights
Cell-cell and cell-matrix adhesion is involved in maintaining the structural integrity of cells in tissues and in governing a wide array of cell behavior [1,2,3]
Because bi-directional signaling between cell adhesion molecules and Cxs may be important in initiating the formation of gap junctions, we investigated if forced expression of Cx43 and Cx32 into an invasive, androgen-independent prostate cancer (PCA) cell line PC-3, with deficient cadherin-mediated adhesion due to the deletion of the ␣-catenin gene [7], would abrogate its malignant phenotype in a manner similar to that of LNCaP cells, which have functional cadherin-mediated adhesion
We had observed that epithelial cells in well differentiated prostate tumors assembled Cxs into gap junctions whereas those in invasive and poorly differentiated prostate tumors did not and, instead, contained Cxs that were localized intracellularly [20]
Summary
Materials—Cell culture media were obtained from Invitrogen. Defined fetal bovine and dialyzed fetal calf sera were from HyClone Laboratories (Logan, UT). The retroviral packaging cell lines PA317 (ATCC CRL 9078) and PG13 (ATCC CRL 10686) were grown in RPMI containing 10% defined fetal bovine serum as described previously [20, 28]. 5 ϫ 104 cells were seeded in 6-well clusters containing glass coverslips and allowed to grow to confluence They were washed 3 times with PBS, fixed for 10 min, and immunostained at room temperature with various antibodies. Cells were rinsed briefly with PBS and incubated with DMEM without dextrans for 30 min at 37 °C, rinsed again three times with PBS before fixing (with 2% paraformaldehyde), and immunostained for connexins as described (see “Antibodies and Immunostaining”). Cells (5 ϫ 104) were seeded on glass coverslips in 6-well culture plates (for immunostaining) or 100-mm dishes (for Western blotting) containing 2 and 12 ml of complete medium, respectively, and incubated at 37 °C. Cell culture medium from freshly confluent 6-cm dishes was removed and replaced with 2.5 ml of medium containing rhodamine-conjugated fluorescent dextrans (10 kDa, 1 mg/ml; fixable) and LY (0.05%)
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