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Calcium buffer incorporation reversibly inhibits DNA synthesis, nuclear envelope breakdown, and cell division in transformed keratinocytes
Abstract
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' viability, as shown by $\sp3$H-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 $\sp3$H-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.
Subject Area
Cellular biology|Molecular biology
Recommended Citation
Cishek, Dawn M, "Calcium buffer incorporation reversibly inhibits DNA synthesis, nuclear envelope breakdown, and cell division in transformed keratinocytes" (1996). Doctoral Dissertations Available from Proquest. AAI9638947.
https://scholarworks.umass.edu/dissertations/AAI9638947