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First published online 1 February 2005
doi: 10.1242/jcs.01673

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Ectopic mTERT expression in mouse embryonic stem cells does not affect differentiation but confers resistance to differentiation- and stress-induced p53-dependent apoptosis

¹ National Cancer Centre, 11, Hospital Drive, Singapore 169610, Republic of Singapore
² Oncology Research Institute, NUMI, National University of Singapore, Singapore 117597, Republic of Singapore
³ Department of Physiology, National University of Singapore, 8, Medical Drive, Singapore 117597, Republic of Singapore
⁴ Department of Biochemistry, National University of Singapore, 8, Medical Drive, Singapore 117597, Republic of Singapore

View larger version (43K): [in a new window]   Fig. 1. Generation of mTERT-overexpressing ES cells. (A) Expression of telomerase was determined by RT-PCR in vector-transfected and mTERT-expressing ES cells. 3 µg total RNA was reverse-transcribed into cDNA and two sets of primers were used to amplify the genes encoding mTERT and tubulin simultaneously. (B) Protein level of mTERT was determined by western-blot analysis using 100 µg nuclear extract from vector-transfected and mTERT-expressing ES cells. Topoisomerase-I levels were also determined, which represent loading in each lane. (C) Telomerase activity of vector-transfected and mTERT-expressing ES cells was determined by a TRAP assay. Decreasing amounts of protein (50 ng, 10 ng, 2 ng and 0.4 ng) were used in the assay.

View larger version (40K): [in a new window]   Fig. 2. Differentiation properties of mTERT-overexpressing ES cells. (A) Differentiation of ES cells was induced with 0.3 µM retinoic acid for 6 days. Cell morphology of non-induced and induced cells was monitored with phase-contrast microscopy. (B) Expression of mTERT in differentiated ES cells was determined by RT-PCR in vector-transfected and mTERT-expressing ES cells. (C) Protein level of mTERT in differentiated ES cells was determined by western-blot analysis using 100 µg nuclear extract from vector-transfected and mTERT-expressing ES cells. Topoisomerase-I levels were also determined, which represent loading in each lane. (D) Vector-transfected or mTERT-expressing ES cells were induced to differentiate with 0.3 µM retinoic acid for 2 days, 4 days and 6 days. The loss of surface expression of a stem-cell-specific antigen (ECMA-7) was monitored over time by flow cytometry. (E) Expression of stem-cell and differentiation markers was determined by RT-PCR. ES cells were induced to differentiate with retinoic acid for 6 days. Cells were harvested at different days of differentiation and RNA was extracted for RT-PCR analysis.

View larger version (16K): [in a new window]   Fig. 3. Ectopic mTERT expression affects cell growth under physiological conditions. (A) Growth curve of pcDNA-transfected and mTERT-expressing ES cells. Representative experiment is shown. Data represent means±s.d. from one of the experiments in duplicate. (*) represents P<0.05 in a one-tailed unpaired Student's t test. (B) Proliferating cells were identified by labelling with BrdU for 30 minutes and followed by staining with FITC-conjugated anti-BrdU antibody. Representative flow-cytometry results are shown. The gated area represents the BrdU+ proliferating cells. (C) Basal cell death was determined in pcDNA-transfected and mTERT-overexpressing ES cells by PI exclusion. Data represent means±s.d. from one of the experiments in triplicate. (*) represents P<0.05 in a one-tailed unpaired Student's t test. (D) Undifferentiated ES cells were induced to differentiate with 0.3 µM retinoic acid for 3 days and the cumulative cell death over the course of differentiation was determined as described in Fig. 3C. Data represent means±s.d. from one of the three independent experiments in duplicate. (*) indicates P<0.05 in a one-tailed unpaired Student's t test.

View larger version (13K): [in a new window]   Fig. 4. mTERT-overexpressing ES cells are more resistant to stress-induced cell death. Undifferentiated ES cells were treated with various doses of H2O2 for 24 hours (A) and with cisplatin (B) or doxorubicin (C) for 12 hours, and cell death was determined by PI exclusion. Data represent means±s.d. from one of the three independent experiments in duplicate. (*) indicates P<0.05 in a one-tailed unpaired Student's t test.

View larger version (27K): [in a new window]   Fig. 5. mTERT overexpression protects ES cells from stress-induced apoptosis. Vector-transfected (left) and mTERT-overexpressing (right) ES cells were treated with 0.6 µg ml–1 doxorubicin (top) or 1 mM H2O2 (middle) for 12 hours. Untreated controls are shown at the bottom. Apoptosis was determined by staining cells with FITC-conjugated annexin-V and PI, and analysed by flow cytometry. Representative results are shown from one of the experiments in duplicate.

View larger version (31K): [in a new window]   Fig. 6. Production of catalytically inactive mTERT does not protect ES cells from apoptosis. (A) Expression of a gene encoding catalytically inactive mTERT was determined by RT-PCR in vector-transfected and inactive-mTERT-producing ES cells. 3 µg RNA was extracted from undifferentiated ES cells and reverse transcribed into cDNA for PCR. (B) Telomerase activity of vector-transfected and inactive-mTERT-producing ES cells was determined by a TRAP assay. Decreasing amounts of protein (50 ng and 5 ng) were used in the assay. (C) The indicated undifferentiated ES cells were treated with various doses of doxorubicin for 12 hours, and cell death was determined by PI exclusion. Data represent means±s.d. from one of the three independent experiments in duplicate.

View larger version (30K): [in a new window]   Fig. 7. Role of p53 in mTERT-mediated resistance to cell death. (A) p53 protein levels of untreated or cells treated with 5 µM cisplatin, 0.3 µg ml–1 doxorubicin and 0.5 mM H2O2 were determined by western blotting. Actin levels were determined to show loading. (B) Expression of p53 target genes (bax, noxa and puma) in vector-transfected and mTERT-expressing ES cells was determined by RT-PCR. Total RNA was extracted from untreated cells and ES cells treated with 5 µM cisplatin or 0.3 µg ml–1 doxorubicin and reverse transcribed into cDNA for PCR of p53 target genes. (C) Activation of caspase-3 was determined after doxorubicin treatment (0.3 µg ml–1) of pcDNA-transfected and mTERT-overexpressing cells by staining with FITC-conjugated cleaved caspase-3-specific antibody and flow cytometry.

View larger version (23K): [in a new window]   Fig. 8. Effect of p53 inhibition and deficiency on mTERT-mediated resistance to cell death. (A) Pifithrin (PFT) (10 µM) was added to cells 24 hours before cisplatin treatment in order to inhibit p53. Cell death was then determined upon treatment with various doses of cisplatin in the presence of PFT by PI exclusion. Data represent means±s.d. from one of the experiments in duplicate. (B) Expression of p53 in undifferentiated CCE ES cells (p53+/+) and P1.1 ES cells (p53–/–) was determined by RT-PCR. 3 µg RNA was extracted from undifferentiated ES cells and reverse transcribed into cDNA for PCR. (C) Expression of mTERT in the indicated undifferentiated ES cells was determined by RT-PCR as described above. (D) Undifferentiated CCE ES cells (p53+/+) and P1.1 ES cells (p53–/–) were treated with various doses of doxorubicin for 12 hours, and cell death was determined by PI exclusion. Data represent means±s.d. from one of the experiments in duplicate.

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