Hypoxic stress induces reversible hypophosphorylation of pRB and reduction in cyclin A abundance independent of cell cycle progression.

The effect of hypoxic stress on the phosphorylation state of the product of retinoblastoma susceptibility gene (pRB) and cyclin A abundance was examined in CV-1P monkey kidney cells. Flow cytometric DNA histogram analysis and [3H]-thymidine incorporation assays demonstrated that hypoxia inhibited cell cycle progression and cell division. Within 6-12 h of hypoxia, pRB became hypophosphorylated and cyclin A abundance fell below detection limits. Hypophosphorylation of pRB and loss of cyclin A detection occurred without progression of cells through S-phase. These effects were found to be reversible by reoxygenation of the hypoxic cultures. Cells were shown to resume DNA synthesis within 12-16 h of reoxygenation concomitant with pRB hyperphosphorylation and an increase in cyclin A detection. These data demonstrate that hypoxic stress blocks the progression of these cells through the phases of the cell cycle and suggests that the effect might arise from the down regulation of key cell cycle controlling elements. The data also raise the possibility that maintaining pRB in a hyperphosphorylated state may be crucial for S-phase progression as well as S-phase entry.