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First published online March 19, 2008
doi: 10.1242/10.1242/jcs.024646

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Growth arrest in human T-cells is controlled by the non-coding RNA growth-arrest-specific transcript 5 (GAS5)

¹ Institute for Science and Technology in Medicine, Huxley Building, Keele University, Keele, ST5 5BG, UK
² King's College London, Department of Haematological Medicine, The Rayne Institute, 123 Coldharbour Lane, London, SE5 9NU, UK

View larger version (24K): [in this window] [in a new window]   Fig. 1. GAS5 overexpression inhibits the growth of T-cell lines and promotes spontaneous apoptosis. (A) CEM-C7 cells were transiently transfected with GAS5 (Image clone 3585621) in pCMVSPORT6, or pCMVSPORT6 (without insert). Numbers of viable cells were determined by vital-dye staining (mean ± s.e.m. from five independent experiments). (B,C) Apoptosis was determined 48 hours post transfection using CaspaTag (mean ± s.e.m. from five independent experiments) of which representative photographs are shown in C. (D) DNA contents of CEM-C7/pCMVSPORT6 and CEM-C7/GAS5 were quantified by PI staining and fluorescence flow cytometry (mean ± s.e.m. from five independent experiments). Representative results are shown. (E) Colony-forming assay was performed 24 hours after transfection. Results are expressed as the mean ± s.e.m. and are representative of data obtained from five independent experiments. *P<0.01 compared with vector only.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Growth arrest is induced by the expression of different GAS5 ESTs. (A) Representation of GAS5 ESTs used in this study. Boxes numbered 2 to12 represent exons. U79, U80 U47, U81 and U74 represent the box C/D small nucleolar RNAs (snoRNAs). Horizontal lines represent the intronic sequences. Upward arrows show the position of the stop codon in the putative ORF (ORF not present in GAS5 1B or GAS5 2B). Exons, introns and snoRNAs are not drawn to scale (Gas5 1B, 1249bp, IMAGE clone 6194071; Gas5 2B, 1270 bp, IMAGE clone 2598129; Gas5 01, 1396 bp, IMAGE clone 3585621; Gas5 3A, 1802 bp, IMAGE clone 5739605; Gas5 4A, 1802 bp, IMAGE clone 2761825). (B) Histogram representing the expression level of GAS5 RNA in CEM-C7 transfected with different GAS5 ESTs relative to cells transfected with pCMVSPORT6, as determined by real-time RT-PCR. Results are represented as mean ± s.e.m. from four separate experiments. (C) CEM-C7 cells were transiently transfected with the different GAS5 ESTs in pCMVSPORT6 expression vector. Numbers of viable cells were determined by vital-dye staining. Data represent the mean ± s.e.m. from four independent experiments. *P<0.01 compared with vector only.

View larger version (10K): [in this window] [in a new window]   Fig. 3. Overexpression of GAS5 induces growth arrest in Jurkat T-leukemic cells. (A,B) Jurkat T cells were transiently transfected with GAS5-01 constructs or with the vector only and numbers of colonies (A) and of viable cells (B) were determined. Data represent the mean ± s.e.m. from five independent experiments. (A,C) Long-term survival was assayed by a colony-forming assay (A) 24 hours after transfection, and DNA content from Jurkat/pCMVSPORT6- and Jurkat/GAS5-transfected cells was quantified by PI staining and fluorescence flow cytometry (C). Representative results from five experiments are shown. Data represent the mean ± s.e.m. from five independent experiments. *P<0.01 compared with vector only.

View larger version (23K): [in this window] [in a new window]   Fig. 4. GAS5-specific siRNAs regulate the growth of CEM-C7 cells and delay growth arrest. CEM-C7 T-leukemic cells were transfected with specific siRNAs targeting GAS5 or with negative control siRNA [(–)siRNA] and cultured at 37°C. (A) After 72 hours the expression of endogenous GAS5 in the siRNA transfected CEM-C7 cells was determined by real-time RT-PCR (mean ± s.e.m. from six separate experiments). (B) Viable cell numbers were determined by vital-dye staining (mean ± s.e.m. from six independent experiments). (C) Caspase activation, as a marker of apoptosis, was determined 72 hours after transfection (mean ± s.e.m. from six independent experiments). (D) DNA content of CEM-C7 transfected with negative-control siRNA or specific GAS5 siRNAs was monitored at different time points by PI staining and flow cytometry (see Table 1 for quantification). *P<0.01 compared with (–)siRNA.

View larger version (16K): [in this window] [in a new window]   Fig. 5. siRNAs targeting GAS5 protect the long-term survival of CEM-C7 T-leukemic cells after serum withdrawal or dexamethasone treatment. CEM-C7 cells were treated with GAS5 siRNAs and cultured either without serum or in the presence of dexamethasone (1 µM) for 72 hours. Colony-forming ability was then determined by plating in soft agar. Data are the mean ± s.e.m. from six independent experiments. *P<0.01 compared with (–)siRNA.

View larger version (29K): [in this window] [in a new window]   Fig. 6. Changes in GAS5 expression modulate the survival of human peripheral blood lymphocytes. Peripheral blood lymphocytes were stimulated with 2.5 µg/ml PHA and transfected with siRNAs targeting GAS5 or with negative control (–)siRNA. (A,B) Numbers of viable cells were determined by vital-dye staining, at the indicated time points (mean ± s.e.m. from seven independent experiments), either (A) in the presence of serum or (B) in its absence (mean ± s.e.m. from seven independent experiments). (C) Apoptosis was determined at different time points using CaspaTag (mean ± s.e.m. from five independent experiments). (D) The expression of endogenous GAS5 in lymphocytes transfected with GAS5-specific siRNA relative to cells transfected with negative control siRNA was determined 72 hours post transfection, by real-time RT-PCR (mean ± s.e.m. from seven separate experiments). (E) Peripheral blood lymphocytes were cultured in complete RPMI medium supplemented with PHA for 5 days and transiently transfected with pCMVSPORT only, or with pCMVSPORT containing GAS5 ESTs, using the Nucleofector (Amaxa; program T-23). After 4 hours, the medium was changed to medium that contains 2.5 µg/ml of PHA. The level of GAS5 overexpression was determined by real-time RT-PCR and was found to be four- to fivefold. Numbers of viable cells were determined by vital-dye staining. Data represent the mean ± s.e.m. from seven independent experiments. *P<0.01 compared with vector only. (F) Expression analysis of GAS5 splice variants by RT-PCR. RNA from PHA-stimulated (untransfected) primary lymphocytes was extracted at 24, 48, 72 and 96 hours. RNAs were reverse transcribed, and PCR was performed on the cDNA using GAS5-forward primer located in exon 9 and GAS5-reverse primer in exon 12. Three PCR fragments were observed. As verified by sequence analysis, all products correspond to GAS5 splice variants. (G) GAS5 expression in stimulated primary lymphocytes increases with time in culture. Expression of endogenous GAS5 in primary lymphocytes was determined by real-time PCR, using the comparative CT method, normalised with 18S RNA as an internal control. Results are represented as the mean ± s.e.m. from three separate experiments. *P<0.01 compared with (–)siRNA.