We have shown that metaphase durations are prolonged in experimentally transformed cells and cells derived from many tumors. A variety of observations suggested that this prolongation might be related to alterations in calcium regulation, and preliminary studies from our own laboratory are consistent with this concept. Using our computer-based quantitative video intensification microscopy system (QVIM) and the fluorescent probes chlorotetracycline (for membrane-associated Ca?2+?) and quin2 (for free Ca?2+?), we have obtained preliminary data that suggest that Ca?2+? regulation may indeed be altered in the neoplastic cells and that these alterations may parallel the changes in metaphase durations.
The aim of our work in the coming year is to solidify and extend these observations. In this work we will: (1) enlarge the number of normal and transformed cell lines in our sample; (2) determine whether alterations in mitochondrial metabolism, as indicated by altered rhodamine 123 fluorescence levels, are associated with altered Ca?2+? pools and metaphase durations; and (3) measure chlorotetracycline and quin2 fluorescence in normal and neoplastic cells during the course of the cell cycle using a combination of time lapse cinemicrography, QVIM, and the Brdu monoclonal antibody technique for labeling S-phase cells. We will thus document changes in membrane-associated and free intracellular Ca?2+? pools as cells progress through interphase, determine whether changes occur at the time of onset of DNA synthesis, and determine whether differences exist in these Ca?2+? pools between normal and neoplastic cells. The data should increase our understanding of the role of Ca?2+? in the regulation of cell division and the cell cycle and indicate whether such regulation is different in normal versus neoplastic cells. (N)
