The interdependence of cell communication and rate of cell growth presents unique challenges to our understanding of the differentiation of highly specialized tissues. We have been investigating and comparing signal transduction, rate of cell proliferation, and cell communication in normal and cancerous tissues. Initial studies relied on morphological techniques including electron microscopy, computer assisted morphometry with freeze fracture techniques and enzyme histochemistry. As techniques to examine critical questions regarding development, proliferation, and regeneration following injury, became available our approach has been updated.
We have correlated proliferation rate, cAMP-dependent protein kinase activation and gap junction expression in normal and neoplastic adrenal tissue. Connexin 43 (Cx43) gap junction protein and mRNA were demonstrated in adrenal cell cultures and in the intact gland by immunocytochemistry, western blot, and northern blot techniques. An inverse relationship was detected between gap junction expression and cell proliferation rate in the cortical zones and in cells maintained in cell culture.
In addition to the well characterized surface gap junctions which are expressed at contact sites between cells, annular gap junction profiles were localized within neoplastic cell cytoplasm. These annular gap junctions are thought to have originated at the cell surface and as a part of a degradation process been transported to the cytoplasm. DbcAMP treatment resulted in a decrease in the number of annular gap junction profiles and an increase in the number and size of surface gap junctions. Corresponding with the redistribution and increase in SW-13 surface gap junctions was the decrease in cell proliferation rate after DbcAMP treatment.
To study the effect of suppression of gap junction expression, Cx43 gap junction cDNA antisense has been transfected into adrenal cell populations. Decreased average gap junction number and size and increased cell proliferation rates were measured. These results are consistent with the hypothesis that gap junctions contribute to the control of cell proliferation possibly by allowing passage of molecules between cells.
Current projects ongoing in Dr. Murray's laboratory include integrated approaches that assess the role of gap junctions and cell-to-cell communication in endocrine cell proliferation, migration, differentiation, and hormone production. Four different techniques (time-lapse video microscopy, immunocytochemistry, transmission electron microscopy, and western blot analysis) are being used to examine gap junction protein (connexin) transport, assembly, location and degradation. The effect of over expression and inhibition of gap junctions on adrenal cell function, are being studied with vectors containing cDNA antisense, dominant-negative constructs, siRNA approaches, mimetic peptides, and antibody directed against gap junction genes products. Together these studies are designed to elucidate the role connexin gap junction channels and cell-cell communication in regulating cell population response to physiological stimuli.