Cell cycle proteins and hTERT telomerase mRNA expression are independent targets of rapamycin in ovarian cancer cell lines Free

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Introduction: Rapamycin and its derivatives are being investigated in phase I and II clinical trials with some success and limited toxicity. Rapamycin exerts its anti-tumor effects by inhibition of the serine/threonine kinase termed mTOR, resulting in G1 cell cycle arrest. Although mTOR is found in almost all mammalian cells, both normal and cancer cells exhibit varying sensitivities to rapamycin. Thus, our goal was to examine the effects of rapamycin on downstream targets of the mTOR pathway involved in cell cycle control and on telomerase activity in rapamycin-sensitive and resistant ovarian cancer cell lines.

Methods: Rapamycin-sensitive (OVCAR8) and rapamycin-resistant (HEY) ovarian cancer cell lines were used. All experiments were performed in the presence and absence of rapamycin. Cell cycle progression was assessed by flow cytometry. Western blot analysis was performed to evaluate expression of cell cycle proteins. hTERT mRNA levels were quantified by real-time RT-PCR.

Results: Rapamycin induced G1 cell cycle arrest in the rapamycin-sensitive OVCAR8 cells, both in the presence and absence of serum. This effect was not seen in the rapamycin-resistant HEY cells. Exposure to rapamycin resulted in inhibition of CDK4, CDK6, cyclin D1 and p21 in the OVCAR8 cells by 24 hr, but not in the HEY cells. No changes were found in p15 or cyclin D3 expression in either of these cell lines. Treament with rapamycin resulted in decreased phosphorylation of the retinoblastoma (RB) protein in the OVCAR8 cells but not in the HEY cells. A small concordant decrease in total RB was also seen. In both of these cell lines, rapamycin reduced hTERT mRNA expression within 24 hr and this persisted for up to 72 hr.

Conclusions: Rapamycin induced G1 cell cycle arrest and inhibited expression of CDK4, CDK6, cyclin D1 and p21, resulting in decreased RB phosphorylation in rapamycin-sensitive ovarian cancer cells. Rapamycin decreased hTERT mRNA levels in ovarian cells that were both sensitive and resistant to its anti-proliferative effects. This suggests that rapamycin may regulate telomerase activity through an alternative pathway, unrelated to cell cycle control. Further work focused on the interplay between the mTOR signaling pathway and telomerase activity should be helpful in delineating those women for whom rapamycin derivatives may be the most beneficial.