Protection of Normal Proliferating Cells Against Chemotherapy by Staurosporine-Mediated, Selective, and Reversible G₁ Arrest

Abstract

Background: A major limiting factor in human cancer chemotherapy is toxicity in normal tissues. Our goal was to determine whether normal proliferating cells could be protected from chemotherapeutic agents by taking advantage of the differential drug sensitivity of cell cycle G₁ checkpoint in normal and cancer cells. Methods: Normal mammary epithelial cells and mammary cancer cells were initially treated with staurosporine at a cytostatic (i.e., nonlethal) concentration, which preferentially arrests normal cells in the G₀/G₁ phase of the cell cycle without affecting the proliferation of tumor cells. After the selective arrest of normal cells in G₀/G₁, both normal and tumor cells were treated with doxorubicin or camptothecin, two cytotoxic (i.e., lethal) chemotherapeutic agents. Cells were then allowed to recover in drug-free medium for 12 days. Results: After pretreatment of both normal and tumor cells with staurosporine followed by treatment with doxorubicin or camptothecin, tumor cells were selectively killed by chemotherapeutic agents, whereas normal cells resumed proliferation after the drugs were removed. Pretreatment with staurosporine also protected normal circulating lymphocytes that had been induced to proliferate in vitro with phytohemagglutinin from chemotherapeutic agents. Staurosporine-induced arrest of normal cells in G₀/G₁ phase was reversible, and arrested cells tolerated doses of camptothecin that were more than 100-fold higher than necessary to eradicate all tumor cells in culture. Staurosporine-mediated G₀/G₁ arrest targets the retinoblastoma protein (pRb) pathway and was accompanied by a rapid decrease in cyclin-dependent kinase (CDK) 4 protein levels, increased binding of CDK inhibitors p21 and p27 to CDK2, and inhibition of CDK2 activity in normal cells. Conclusions: Breast cancer cells with defective checkpoints regulated by the pRb pathway can be targeted specifically with chemotherapeutic agents, following staurosporine-mediated, selective and reversible G₀/G₁ arrest in normal cells.