Anti-Proliferative Effect of Rapamycin on Human T Cell Leukemia Cell Line Jurkat by Cell Cycle Arrest and Telomerase Inhibition.

Rapamycin exerts its biological activity by inhibiting the kinase mammalian target of rapamycin (mTOR), which is a key regulator of cell growth and survival in many cell types. Its constitutive activation has been involved in pathogenesis of various cancers; the critical functions of mTOR have led to the development of mTOR inhibitors (MTIs) as novel anticancer agents. Recently, the anticancer effects of rapamycin are presently evaluated in various solid tumors; however, the use of rapamycin in acute lymphoblastic leukemia (ALL) is poorly documented. We try to examine the ability of rapamycin to suppress growth and its mechanism in Human T Cell Leukemia cell line Jurkat, and meanwhile to explore its ability to regulate telomerase. Cell proliferation was assessed after exposure to rapamycin by MTT assay. Apoptotic cells were determined by flow cytometric detection of annexin V binding assay. And cell cycle was monitored by flow cytometric detection of DNA content assay. Proteins important for cell cycle progression and Akt/mTOR signaling cascade were assessed by Western blot. Telomerase activity was quantified by TRAP assay. hTERT mRNA levels were determined by semi-quantitative RT-PCR. Rapamycin significantly inhibited proliferation of Jurkat in a dose and time-dependent manner with IC50 values for 24hr, 48hr, and 72hr were 344nM, 92nM and 16nM. To further determine the mechanism of growth inhibition by rapamycin, we found rapamycin did not increase the amount of cells in annexin V+ fraction 72 hr after treatment, confirming that rapamycin did not promote apoptosis in Jurkat cells. However, G1 phase arrest was induced by rapamycin (10nM) since 16hr after treatment, and ratio of G1 continued to grow as treatment was prolonged to 32hr, which indicated rapamycin inhibited cell cycle progress. By Western blot analysis, we found rapamycin could up-regulate the level of cyclin-dependent kinase inhibitors (CDKIs) of p27Kip1 as well as p21waf1, down-regulate CyclinD3, but had no significantly effect on expression of Cdk4, Cdk6 or CyclinD2, which demonstrated that G1 cell cycle arrest induced by rapamycin in Jurkat cells, was mediated by affecting cyclin D3, P27Kip1and p21waf1. Moreover, we characterized the signaling pathways affected by rapamycin, and found phosphorylation of mTOR downstream cascade targets, sucha as p70S6K and S6 were significantly reduced by rapamycin, but phospho- Akt level was not affected, which in agreement with established models that rapamycin functions downstream of Akt in Akt/mTOR signaling. Interestingly, Activation of telomerase is seen in Jurkat cells and thought to be a critical element in leukemia pathogenesis. Treatment with rapamycin decreased telomerase activity dose-dependently, which was accompanied with down-regulation of the catalytic subunit, telomerase reverse transcriptase (TERT). However, the exact transcription factors, which modulate hTERT gene transcription and are targeted by the mTOR pathway, need to be further identified. Conclusion:Rapamycin displayed potent antileukemic effect in Human T Cell Leukemia cell line by inhibition of cell proliferation through G1 cell cycle arrest and also through suppression of telomerase activity, suggesting rapamycin may have potential clinical implications in treatment of some leukemia.