Calcium buffer incorporation reversibly inhibits DNA synthesis, nuclear envelope breakdown, and cell division in transformed keratinocytes

Loss of regulation of cell cycle events mediated by changes in cytosolic Ca\sp{2+} ion activity has been implicated in the progression of normal cells to neoplasia. In this study, the Ca\sp{2+} buffer 5,5\sp\prime-difluoro 1,2-bis(2-aminophenoxy) ethane-N,N,N\sp\prime N\sp\prime-tetra-acetic acid (5,5\sp\prime-dfBAPTA, abbreviated dfB ) has been used to modulate cell division in transformed and primary mouse keratinocytes. Exogenous application, via the tetra(acetoxymethyl) ester ( AM ), of 18-20 $\mu$M dfB/AM to the growth media of transformed cells inhibits cell division and DNA synthesis, without compromising the cells\u27 viability, as shown by \sp3H-thymidine incorporation and flow cytometry. Bulk fluorimetry shows that cells treated with dfB/AM are able to buffer Ca\sp{2+} in proportion to the concentration of dfB/AM applied. Primary cultured cells treated with 18-20 $\mu$M dfB/AM die within 3 hours of treatment. Viable dfB/AM treated cultures of transformed cells have a higher proportion of cells in the G\sb2 phase of the cell cycle than do controls, as shown by flow cytometry. This result, in combination with that showing reduced \sp3H-thymidine incorporation, suggests that 18-20 $\mu$M dfB/AM inhibits a pre- or mid-mitotic step. Light, electron, and confocal microscopies show 18-20 $\mu$M dfB/AM-treated cells to have prominent, thickened nuclear envelopes along with actin cytoskeletons that are distinguishable from controls. Upon return to medium that does not contain dfB/AM, treated transformed cells gradually resume their pre-treatment growth and division patterns