CONNEXIN EXPRESSION AND INTERCELLULAR COMMUNICATION IN 2-DIMENSIONAL AND 3-DIMENSIONAL IN-VITRO CULTURES OF HUMAN BLADDER-CARCINOMA

The identification of gap-junctional proteins (connexins) and the prep aration of related antibodies provides new tools to study patterns of intercellular communication in tumors. Focusing on the biology of huma n bladder carcinoma, we compared the expression of gap-junctional prot eins (connexins Cx26, Cx32, and Cx43) with a dye-coupling assay for ga p-junctional intercellular communication in three cell lines with diff erent urothelial differentiation. The cell lines HCV-29, RT4, and J82 were initially grown as monolayers of different ages, Connexin express ion was found mostly positive over the time of culture and found const antly negative only in J82 cells for Cx26 and HCV-29 cells for Cx32. I n HCV-29 cells, Cx26 increased in Positivity over the time of culture, Western blotting with the antibodies confirmed the findings. Comparis ons of dye transfer using Lucifer Yellow showed an increase of couplin g in the normal urothelial cell line HCV-29 in contrast to a decrease of coupling in the tumor cell lines, Data were extended by multicellul ar spheroid (MCS) co-cultures with the stromal fibroblast line NI, In three-dimensional cultures as MCSs, Cx26 was increased in proximity of RT4 tumor cells to fibroblasts, and positivity was maintained in J82 cells, E-cadherin expression in cell lines showed no change in depende nce of growth state, The data suggest that Cx26 plays a role in negati ve growth control or differentiation of urothelial cells, Preliminary comparative data on normal and neoplastic urothelium show all three co nnexins in normal urothelium, in contrast to varying amounts of Cx43 a nd low amounts of Cx32 in tumors and evident loss of Cx26 in low-grade tumors, Discrepancies between monolayer and MCS cultures are most lik ely due to higher differentiation in MCSs, and the continuation of sys tematic work with heterologous MCSs Is indicated for more information on the role of gap-junctional proteins in human tumors.