Calcium-mediated modulation of microtubule assembly in human breast epithelial cells

Normal human breast epithelial cells obtained from a reduction mammoplasty (S130) have been maintained in culture for up to a year in Ham's F12:Dulbecco's medium, with 5% equine serum and a low calcium concentration (0.04 mM). These cells undergo senescence and terminal differentiation if they are switched to high Ca2+ medium (1.05 mM). To clarify the mechanism by which Ca2+ regulates the growth of these cells, we studied the role of tubulin assembly-disassembly and the morphologic changes subsequent to high Ca2+ switch. An early Passage (9) of S130 breast epithelial cells growing in low Ca2+ medium was analyzed. Of a total of 785 counted cells, 720 (92%) were rounded and 65 (8%) were flat, elongated, and fibroblastlike. When the cells were switched to high Ca2+ medium, out of 553 cells, only 111 (20%) were rounded and the remaining 442 (80%) were elongated and fibroblastlike. Immunocytochemical localization of tubulin, using the immunogold silver enhancement technique, showed that the majority of low Ca2(+)-grown cells did not display a network of tubulin fibers, whereas high Ca2(+)-grown cells revealed extensive cytoplasmic network of polymerized tubulin, which seemed to stretch out the cells. Experiments designed to determine the mechanisms of tubulin polymerization in these cells revealed that: a) Cells grown in high Ca2+ medium containing 0.1 mM colchicine had a reduced proportion of elongated cells; b) treatment of the cells with the calcium ionophore A23187 in low calcium medium resulted in an increase in the number of elongated cells which had more polymerized tubulin; and d) treatment of the cells with cyclic-AMP in low Ca2+ medium had no observable effect on cell morphology. These results indicate that high levels of Ca2+ either favor tubulin polymerization or stabilize the polymerized state.