Overexpression of parathyroid hormone-related protein enhances apoptosis in the rat intestinal cell line, IEC-6

We used the rat intestinal cell line, IEC-6, to study potential effects of overexpression of PTH-related protein (PTHrP) on apoptosis. A clonal line o f PTHrP-overexpressing cells was established by stably transfecting parenta l cells with PTHrP complementary DNA in a sense orientation (sense). A simi larly transfected line stably, transfected with empty vector, served as con trol (vector). Immunoreactive PTKrP, measured in culture medium, showed tha t sense cells secreted approximately 30 times as much PTHrP as did vector c ontrol cells. Apoptosis induced by serum withdrawal was evaluated by severa l methods. DNA laddering was demonstrable in sense-transfected cells as ear ly as 12 h after serum withdrawal but not until later time points in vector -transfected control cells. Flow cytometric analysis of propidium iodide-st ained cells showed a greater increase in the sub-G1 (apoptotic) population in sense cells, compared with vector. Fluorescent microscopy with Hoechst 3 3258 dye showed increased nuclear fragmentation and condensation in sense c ells. Studies of apoptotic gene expression by ribonuclease protection assay , and protein by Western blot analysis, showed an enhanced ratio of Bax to Bcl-x(L) in sense cells. Mutation of the PTHrP nuclear localization amino a cid sequence negated the ability of PTHrP to enhance apoptosis.