To evaluate the stability of cells arrested in metaphase, cell viability, RNA content, and chromatin structure (the latter probed by the DNA in situ sensitivity to acid-induced denaturation) were studied in uniform-age mitotic CHO cell populations maintained either at 37Β°C (in the presence of Colcemid) or at 0-4Β°C for up to 6 h. No significant changes in cell viability and RNA content were seen throughout the experiment for both groups of cells. The sensitivity of DNA in situ to denaturation was significantly increased during the initial 40 min of cell arrest in mitosis. However, no further chromatin changes for
In stathmokinetic experiments, cells remain arrested in metaphase for an extended time. Likewise, in cytogenetic studies, long incubations with mitotic blockers are often required to accumulate adequate numbers of metaphase cells, especially in the case of primary cultures of human tumors. There are conflicting reports in the literature regarding stability of mitotic cells. In practical terms, the maximum allowable duration of stathmokinesis is estimated from the linearity of the slope representing cumulative number of mitotic cells in asynchronous populations (14). Whereas in some studies the linear accumulation (or stability) of metaphase cells was observed for up to 8 (15) or even 12 h (8,161, in other reports degeneration of metaphase cells was described after 2.5 or 3 h (12). The subject of instability of cells arrested in mitosis is reviewed by Wright and Appleton (20).
This study aims to examine changes in cell viability, RNA content, and chromatin sensitivity to denaturation as a function of time of cell arrest in mitosis. Most of the published data have been obtained on asynchronous cultures in the course of stathmokinesis. The cumulative populations of metaphase cells in these studies represented cells of different age. In contrast, the present study is done on synchronized metaphase cells of uniform age. Also, in contrast to the earlier studies, we estimate not only the physical integrity or viability ofcells but their RNA content and chromatin structure as up to 6 h were evident regardless of whether cells were kept at 37Β°C with Colcemid or at 0-4Β°C in its absence. The data indicate that neither significant deterioration of metaphase cells nor progressive chromatin changes are expected during stathmokinesis experiments in vitro or during the metaphase cell arrest in cytogenetic studies lasting up to 6 h. Also, no RNA turnover can be detected in mitotic cells during this time interval. Key terms: Stathmokinesis, DNA denaturation, cytogenetics, RNA turnover, mitosis, flow cytometry, acridine orange well. The studies on RNA content in metaphase cells offer a unique opportunity to evaluate RNA turnover in individual cells in which DNA transcription is suppressed naturally during the cell cycle. The data on chromatin may reveal whether or not there are chromosomal changes during the extended mitotic arrest. This information would be of interest in cytogenetic studies and in analysis of drug effects on metaphase cell chromatin, as will be discussed later.
MATERIALS AND METHODS
Chinese hamster ovary line (CHO) cells obtained from
Los Alamos National Laboratory were maintained as growing monolayer cultures in F10 medium supplemented with 10% fetal bovine serum and antibiotics (17). Populations of synchronized mitotic cells were obtained from 500 ml Falcon culture flasks of exponentially growing cells by shaking the flasks to dislodge loosely attached dividing cells (9,13). After the first shake ("clean-Supported by USPHS Grants CA28704 and CA23296.