FIG.2.

CDK2 is the principal kinase that phosphorylates p21 at G₂/M phase. (A) Ten, 20, and 40 μM concentrations of butyrolactone dissolved in dimethyl sulfoxide were added, along with nocodazole (0.8 μg/ml), to H460 cells and incubated at 37°C for 18 h with 5% CO₂. Western analysis shows complete dephosphorylation of p21 even at the lowest concentrations of butyrolactone used. (B) Transient transfection of wild-type CDK2 or DN-CDK2 in U2OS cells showed reduced phosphorylation of p21 at Nocodazole-arrested G₂/M phase. Cell cycle profile of wild-type CDK2 or DN-CDK2-transfected arrested U20S cells in G₂/M phase after treatment with nocodazole for 18 h. (C) Transient transfection of pSuper Retro CDK2 RNAi in U2OS cells showed reduced CDK2 expression in asynchronous or nocodazole-treated cells and also inhibited phosphorylation of p21 in G₂/M-enriched cells. However, no change in CDK2 levels or p21 phosphorylation was observed in control vector-transfected cells. (D) Caffeine, an ATM/ATR kinase inhibitor, inhibits p21 phosphorylation when added at 2 or 4 mM concentrations to the exponentially growing H460 cells at 6 h before or at the same time as nocodazole but not if added after 6 h of nocodazole treatment. (E) Two different concentrations (50 or 100 μM) of a 24-amino-acid p21 peptide including the NLS/PCNA region (see Materials and Methods for sequence) dissolved in water were added to asynchronous H460 cells without FBS for 3 h, followed by 0.8-μg/ml nocodazole with complete medium and incubation for another 18 h. Cells were harvested and dissolved in whole-cell lysis buffer, electrophoresed on SDS-PAGE, and immunoblotted with p21 antibodies. There was almost complete inhibition of p21 phosphorylation with or without UV exposure in nocodazole-treated cells at the higher dose, while less inhibition was observed at the lower dose tested. (F) H460 cells were FBS depleted for 30 h, followed by aphidicolin treatment for another 24 h and release from aphidicolin block for 8 or 10 h. p21 peptide was added after 4 h of aphidicolin release without FBS for 1 h to adsorb the peptide, with the cells subjected to 10% FBS added and incubated at 37°C for another 4 or 6 h before cells were harvested. Western analysis showed dephosphorylation of p21 protein after 8 or 10 h of release from aphidicolin with the 100 μM peptide-treated cells. (G) Immunofluorescence staining for p21 protein expression in H460 cells treated with p21 peptide with or without nocodazole. Nuclear p21 protein translocation was blocked by the p21 peptide with evidence cytoplasmic localization in p21- plus nocodazole-treated H460 cells (d) but not in nocodazole-treated cells in the absence of peptide (c). The asynchronous H460 cells showed cytoplasmic or nuclear localization of the protein (a). p21 protein was detected throughout the cells in p21 peptide-treated cells not exposed to nocodazole (b). Inset shows an enlarged view of cells from panel G-d. (H) Treatment with p21 peptide along with nocodazole in H460 and HCT116 cells resulted in a significant population of annexin V-positive cells in both cell lines (panels H-d) compared to the controls (panels H-a, H-b, and H-c).