Inhibition of human T-cell proliferation by mammalian target of rapamycin (mTOR) antagonists requires noncoding RNA growth-arrest-specific transcript 5 (GAS5) - PubMed (original) (raw)

Inhibition of human T-cell proliferation by mammalian target of rapamycin (mTOR) antagonists requires noncoding RNA growth-arrest-specific transcript 5 (GAS5)

Mirna Mourtada-Maarabouni et al. Mol Pharmacol. 2010 Jul.

Abstract

The central importance of the serine/threonine protein kinase mTOR (mammalian Target of Rapamycin) in the control of cell growth and proliferation is well established. However, our knowledge both of the upstream pathways controlling mTOR activity and of the downstream events mediating these effects is still seriously incomplete. We report a previously unsuspected role for the nonprotein-coding RNA GAS5 in the inhibition of T-cell proliferation produced by mTOR antagonists such as rapamycin. GAS5 transcripts are up-regulated during growth arrest and after rapamycin treatment, and GAS5 has recently been shown to be necessary and sufficient for normal T-cell growth arrest. Down-regulation of GAS5 using RNA interference protects both leukemic and primary human T cells from the inhibition of proliferation produced by mTOR antagonists. The GAS5 transcript is a member of the 5' terminal oligopyrimidine class of RNAs, which is specifically controlled at the level of translation by the mTOR pathway, and the effects of GAS5 on the cell cycle provide a novel and important link to the control of proliferation. These observations point to a significant advance in our understanding of the mechanism of action of mTOR inhibitors, which is likely to lead to improvements in immunosuppressive and cancer therapy.

PubMed Disclaimer

Figures

Fig. 1.

Fig. 1.

_GAS5_-specific siRNAs protect against the inhibition of cell proliferation induced by 2.5 μM rapamycin in MOLT-4 and CEM-C7 human T cell lines. MOLT-4 and CEM-C7 T cells were transfected with specific GAS5 siRNAs or negative control siRNA [(−)siRNA] and cultured at 37°C. After 48 h, cells were exposed to rapamycin. MOLT-4 (A) and CEM-C7 (B) viable cell numbers were determined after 48 h by vital dye staining and the LIVE/DEAD assay (Materials and Methods). Results are calculated as the percentage of viable cell numbers relative to controls incubated in the absence of rapamycin (mean ± S.E.M. from five independent experiments). MOLT-4 (C) and CEM-C7 (D) cell proliferation was measured using the BrdU colorimetric ELISA assay. Results are represented as the percentage inhibition of cell proliferation compared with control in the absence of rapamycin. (mean ± S.E.M. from five independent experiments). *, P < 0.01 compared with (−)siRNA. The original data are given as Supplemental Fig. S1.

Fig. 2.

Fig. 2.

siRNAs targeting GAS5 protect the proliferation of MOLT-4 and CEM-C7 T cells from the inhibitory effects of mTOR inhibitors. Forty-eight hours after transfection, siRNA-transfected cells were incubated with temsirolimus (5 nM) or everolimus (10 nM) for 48 h. Viable cell numbers for MOLT-4 (A) and CEM-C7 (B) were determined by vital-dye staining and LIVE/DEAD assay. Results are calculated as the percentage of viable cell numbers relative to controls incubated in the absence of inhibitors (mean ± S.E.M. from five independent experiments). MOLT-4 (C) and CEM-C7 (D) cell proliferation was assessed using the BrdU colorimetric ELISA assay. Results are represented as the percentage inhibition of cell proliferation compared with controls in the absence of rapamycin. (mean ± S.E.M. from five independent experiments). E and F, percentage of cells stained by anti-Ki67 antibody. Data are represented as percentage inhibition of cell proliferation and represent means ± S.E.M. from five independent experiments. *, P < 0.01 compared with (−)siRNA. Original data are provided in Supplemental Fig. S2.

Fig. 3.

Fig. 3.

_GAS5_-specific siRNAs protect MOLT-4 and CEM-C7 T cells from cell cycle perturbations caused by rapamycin. A, MOLT-4; B, CEM-C7. B, T cells were transfected with specific GAS5 siRNAs or negative control siRNA [(−)siRNA] and cultured at 37°C. After 48 h, cells were exposed to 2.5 μM rapamycin. Cell-cycle analysis was carried out after a further 24 h. DNA content was quantified by propidium iodide staining of fixed cells and fluorescence flow cytometry. Results are represented as the means ± S.E.M. (n = 5). *, P < 0.01 compared with control in the absence of rapamycin. Representative histograms are also shown, and full results for all three GAS3 siRNAS are given in Supplemental Tables T6 and T7.

Fig. 3.

Fig. 3.

_GAS5_-specific siRNAs protect MOLT-4 and CEM-C7 T cells from cell cycle perturbations caused by rapamycin. A, MOLT-4; B, CEM-C7. B, T cells were transfected with specific GAS5 siRNAs or negative control siRNA [(−)siRNA] and cultured at 37°C. After 48 h, cells were exposed to 2.5 μM rapamycin. Cell-cycle analysis was carried out after a further 24 h. DNA content was quantified by propidium iodide staining of fixed cells and fluorescence flow cytometry. Results are represented as the means ± S.E.M. (n = 5). *, P < 0.01 compared with control in the absence of rapamycin. Representative histograms are also shown, and full results for all three GAS3 siRNAS are given in Supplemental Tables T6 and T7.

Fig. 4.

Fig. 4.

Changes in GAS5 expression modulate the response of human peripheral blood lymphocytes to mTOR inhibitors. Peripheral blood lymphocytes from a single healthy volunteer were stimulated with 2.5 μg/ml PHA and transfected with siRNAs targeting GAS5 or with negative control (−)siRNA. Forty-eight hours after transfection, siRNA-transfected cells were incubated with either rapamycin 2.5 μM (A–C) or 5 nM temsirolimus or 10 nM everolimus (D–F) for 48 h. A and D, viable cell numbers were determined by vital-dye staining and LIVE/DEAD assay. Results are represented as the percentage viable cell number relative to controls incubated in the absence of mTOR inhibitors (mean ± S.E.M. from six independent experiments), Changes in GAS5 expression modulate the response of human peripheral blood lymphocytes to mTOR inhibitors. Peripheral blood, lymphocytes from a single healthy volunteer were stimulated with 2.5 μg/ml PHA and transfected with siRNAs targeting GAS5 or with negative control (−)siRNA. Forty-eight hours after transfection, siRNA-transfected cells were incubated with either rapamycin 2.5 μM (A–C) or 5 nM temsirolimus or 10 nM everolimus (D–F) for 48 h. A and D, viable cell numbers were determined by vital-dye staining and LIVE/DEAD assay. Results are represented as the percentage viable cell number relative to controls incubated in the absence of mTOR inhibitors (mean ± S.E.M. from six independent experiments), C, DNA content of transfected lymphocytes 24 h after exposure to rapamycin was monitored by PI staining and flow cytometry. The results obtained from one of the GAS5 siRNAs are presented (full results for all three GAS5 siRNAs are given in Supplemental Table T8). Data represent the mean ± S.E.M. from four independent experiments, each carried out with cells from a different donor). B and E, cell proliferation was measured using the BrdU colorimetric ELISA assay. F, percentage inhibition of proliferation as monitored by staining by anti-Ki67 antibody. Results are represented as inhibition of cell proliferation compared with controls in the absence of mTOR inhibitors (mean ± S.E.M. from six independent experiments). *, P < 0.01 compared with controls.

Fig. 4.

Fig. 4.

Changes in GAS5 expression modulate the response of human peripheral blood lymphocytes to mTOR inhibitors. Peripheral blood lymphocytes from a single healthy volunteer were stimulated with 2.5 μg/ml PHA and transfected with siRNAs targeting GAS5 or with negative control (−)siRNA. Forty-eight hours after transfection, siRNA-transfected cells were incubated with either rapamycin 2.5 μM (A–C) or 5 nM temsirolimus or 10 nM everolimus (D–F) for 48 h. A and D, viable cell numbers were determined by vital-dye staining and LIVE/DEAD assay. Results are represented as the percentage viable cell number relative to controls incubated in the absence of mTOR inhibitors (mean ± S.E.M. from six independent experiments), Changes in GAS5 expression modulate the response of human peripheral blood lymphocytes to mTOR inhibitors. Peripheral blood, lymphocytes from a single healthy volunteer were stimulated with 2.5 μg/ml PHA and transfected with siRNAs targeting GAS5 or with negative control (−)siRNA. Forty-eight hours after transfection, siRNA-transfected cells were incubated with either rapamycin 2.5 μM (A–C) or 5 nM temsirolimus or 10 nM everolimus (D–F) for 48 h. A and D, viable cell numbers were determined by vital-dye staining and LIVE/DEAD assay. Results are represented as the percentage viable cell number relative to controls incubated in the absence of mTOR inhibitors (mean ± S.E.M. from six independent experiments), C, DNA content of transfected lymphocytes 24 h after exposure to rapamycin was monitored by PI staining and flow cytometry. The results obtained from one of the GAS5 siRNAs are presented (full results for all three GAS5 siRNAs are given in Supplemental Table T8). Data represent the mean ± S.E.M. from four independent experiments, each carried out with cells from a different donor). B and E, cell proliferation was measured using the BrdU colorimetric ELISA assay. F, percentage inhibition of proliferation as monitored by staining by anti-Ki67 antibody. Results are represented as inhibition of cell proliferation compared with controls in the absence of mTOR inhibitors (mean ± S.E.M. from six independent experiments). *, P < 0.01 compared with controls.

References

    1. Abraham RT, Wiederrecht GJ. (1996) Immunopharmacology of rapamycin. Annu Rev Immunol 14:483–510 -PubMed
    1. Ashwell JD, Lu FW, Vacchio MS. (2000) Glucocorticoids in T cell development and function. Annu Rev Immunol 18:309–345 -PubMed
    1. Bai X, Ma D, Liu A, Shen X, Wang QJ, Liu Y, Jiang Y. (2007) Rheb activates mTOR by antagonizing its endogenous inhibitor, FKBP38. Science 318:977–980 -PubMed
    1. Abdel-Karim IA, Giles FJ. (2008) Mammalian target of rapamycin as a target in hematological malignancies. Curr Probl Cancer 32:161–177 -PubMed
    1. Brunn GJ, Hudson CC, Sekulić A, Williams JM, Hosoi H, Houghton PJ, Lawrence JC, Jr, Abraham RT. (1997) Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin. Science 277:99–101 -PubMed

MeSH terms

Substances

LinkOut - more resources