The differential staurosporine-mediated G₁ arrest in normal versus tumor cells is dependent on the retinoblastoma protein

Previously, we reported that breast cancer cells with retinoblastoma (pRb) pathway-defective checkpoints can be specifically targeted with chemotherapeutic agents, following staurosporine-mediated reversible growth inhibition in normal cells. Here we set out to determine if the kinetics of staurosporine-mediated growth inhibition is specifically targeted to the G₁ phase of cells, and if such G₁ arrest requires the activity of wild-type pRb. Normal human mammary epithelial and immortalized cells with intact pRb treated with low concentrations of staurosporine arrested in the G₁ phase of the cell cycle, whereas pRb-defective cells showed no response. The duration of G ₁ and transition from G₁ to S phase entry were modulated by staurosporine in Rb-intact cells. In pRb⁺ cells, but not in Rb cells, low concentrations of staurosporine also resulted in a significant decrease in cyclin-dependent kinase 4 (CDK4) expression and activity. To directly assess the role of pRb in staurosporine-mediated G₁ arrest, we subjected wild-type (Rb^+/+) and pRb^-/- mouse embryo fibroblasts (MEFs) to staurosporine treatments. Our results show that whereas Rb^+/+ MEFs were particularly sensitive to G₁ arrest mediated by staurosporine, pRb^-/- cells were refractory to such treatment. Additionally, CDK4 expression was also inhibited in response to staurosporine only in Rb^+/+ MEFs. These results were recapitulated in breast cancer cells treated with siRNA to pRb to down-regulate the pRb expression. Collectively, our data suggest that treatment of cells with nanomolar concentrations of staurosporine resulted in down-regulation of CDK4, which ultimately leads to G₁ arrest in normal human mammary epithelial and immortalized cells with an intact pRb pathway, but not in pRb-null/defective cells.