CC31

Correlated Cyclin B1 Expression And BrdUrd Incorporation In Irradiated Cell Populations Treated With The G2 Delay Attenuating Agents Staurosporine And Caffeine.

E.A. Walthers, R.A. Albrecht, H.A. Crissman*, Los Alamos National Laboratory, Los Alamos New Mexico, USA.

Comparative effects of staurosporine (Stsp) and caffEine on the characteristic G2 delay in normal human diploid fibroblasts (HSF) and transformed HeLa cells following 6.0 Gy ; irradiation were examined by flow cytometry. Bernhard et al. (Radiat. Res., 140: 393, 1994) have shown a low concentration of Stsp (4.4 nM) attenuates the G2 delay of synchronized HeLa cells exposed to irradiation. Levels of cyclin B1 mRNA were shown by immunoblotting to increase in populations which displayed a reduced G2 delay following irradiation. The effects of caffeine on the G2 delay and cyclin B mRNA expression parallel those of Stsp. Continuous BrdUrd labeling following irradiation of asynchronously growing cells enabled us to distinguish cycling and noncycling G2 cells as well as postmitotic G1 cells, and to observe fluctuations in these populations in the presence of Stsp or caffeine. In combination with BrdUrd labeling studies we were able to simultaneously analyze the cyclin B levels in cycling and non-cycling populations using immunofluorescence and multiparameter flow cytometry. Optimal results for these experiments were obtained using a formalin/methanol fixation which resulted in higher FITC immunofluorescent intensity than EtOH fixation, in addition to eliminating aberrant FITC labeling seen in EtOH fixed cells. Supported by NIH Grant R01 RR06758, the Los Alamos National Flow Resource (NIH Grant P41-RR01315) and the US Department of Energy.