Kinetics of staurosporine-arrest of human diploid fibroblasts

• Published in: Cytometry (Baltimore); (United States) Vol. 6
• Main Authors: Stevenson, A.P., Bustos, L.D., Dickson, J.A., Habbersett, R.C., Crissman, H.A.
• Format: Conference Proceeding
• Language: English
• Published: United States 01.01.1993
• Subjects: 550200 > Biochemistry; BASIC BIOLOGICAL SCIENCES; CELL CYCLE; CELL TRANSFORMATIONS; CONTROL; CYTOLOGICAL TECHNIQUES; ENZYME INHIBITORS; ENZYMES; FLUORESCENCE; LASERS; LUMINESCENCE; ONCOGENIC TRANSFORMATIONS; ORGANIC COMPOUNDS; PHOSPHORUS-GROUP TRANSFERASES; PHOSPHOTRANSFERASES; PROTEINS; QUANTITY RATIO; SORTING; TRANSFERASES 550300 > Cytology
• ISSN: 0196-4763; 1097-0320

The authors have previously shown that the general protein kinase inhibitor, staurosporine (Stsp), has a differential effect on the progression of normal and transformed cells. Low levels of Stsp (1.0 ng/ml) reversibly arrested normal diploid cells in early G1 phase, whereas concentrations of Stsp as high as 50-75 ng/ml had no effect on G1 progression in transformed cells. High concentrations of Stsp arrested normal cells in G1 and G2 phases but blocked transformed cells only in G2 phase of the cell cycle. To follow the accumulation of cells in G1 and/or G2 phases, asynchronous cultures of human diploid fibroblasts were treated with 0, 1, 5, 10, 20, 30 or 50 ng/ml staurosporine for 18 hours in the presence of bromodeoxyuridine. The kinetics of labeling with BrdU were determined with a two-laser flow cytometric technique that monitored emission of fluorescence from DNA-species stains that differed in the degree of BrdU-induced quenching of their fluorescence signals. Bivariate plots of Hoechst/mithramycin fluorescence signal ratio vs. Hoechst fluorescence identified the arrested cells as cohorts of cells in different positions across cell cycle at the time of Stsp addition.