Synergistic activities of CDK2 and CDK4 in causing extensive proliferation of fully differentiated cells.
(A) The indicated MEL cell CDK transfectants were cultured in the presence of 5 mmol/L HMBA for 48 hours and then plated in plasma clots either in the absence (−) or presence (+) of Dox. The plasma clots were analyzed as described in the legend of Figure 4 after 3 days (■) and 7 days (□) of incubation at 37°C. (B) Plasma clots of a doubly transfected cell line were photographed (original magnification × 10) after staining with benzidine and hematoxylin as described in “Materials and methods.” (C) Logarithmically growing CDK(2 + 4R24C).32 and CDK(2 + 4R24C).40 were treated at 1 × 105 cells per mL with 5 mmol/L HMBA in the presence (shaded symbols) or absence (open symbols) of Dox. The cell densities were maintained at less than 1 × 106 cells per mL by subculturing to 2 × 105 cells per mL with the indicated growth medium every 24 hours as required. The cell densities were measured daily with a Coulter counter (Coulter Electronics, Miami, FL), and the cumulative cell densities were calculated. (D) Total cellular extracts were prepared at the indicated times from cells treated as described in panel C. The extracts were analyzed by nondenaturing PAGE and immunoblotting with an antibody specific for mouse hemoglobin. The lower section shows hemoglobin levels in the rtTA MEL cell parental line after 5 days of treatment with HMBA, with and without Dox.
