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Dual regulation of telomerase activity through c-Myc-dependent inhibition and alternative splicing of hTERT

¹ Deutsches Krebsforschungszentrum, Division of Skin Carcinogenesis, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
² Klinik für Allg. Chirurgie und Thoraxchirurgie, Molecular Oncology, University of Kiel, D-24105 Kiel, Germany
³ University of Marburg, Department of Haematology and Oncology, Baldinger Str. D-35033 Marburg, Germany
⁴ University of Heidelberg, Department of Immunology, Im Neuenheimer Feld 305, D-69120 Heidelberg, Germany

View larger version (59K): [in a new window]   Fig. 1. Effect of TGF-ß1 signaling on proliferation and telomerase activity in either untransfected HaCaT cells (a), hTERT- (HaCaT-TERT) (b) or c-Myc (HaCaT-myc) transfectants (c). Upper panels: Western blot showing phosphorylation of Smad2 and expression of c-Myc after TGF-ß1 treatment. Middle panels: BrdU incorporation assay. Untreated cells are taken as 100%. Lower panel: TGF-ß1 downregulation of telomerase activity in HaCaT and HaCaT-myc cells and telomerase maintenance in HaCaT-TERT cells. Telomerase activity was measured by conventional TRAP assay. From each sample an RNase-inactivated negative control was assayed in parallel. IC, internal control.

View larger version (28K): [in a new window]   Fig. 2. Effect of TGF-ß1 treatment on hTERT expression. (a) hTERT transcription in HaCaT (upper gel) and HaCaT-myc cells (lower gel) after TGF-ß1 treatment. hTERT total mRNA expression was measured by RT-PCR using primers 1784S and 1928A (Ulaner et al., 1998). (b) hTERT splicing after TGF-ß1 treatment. RT-PCR was carried out with primers TERT-HT2026F and TERT-HT2482R (Kilian et al., 1997). fl, full length; , splice; ß, ß splice. (c) hTERT expression in HaCaT-TERT cells: RT-PCR as in (b), but to remain in the exponential phase only 30 cycles of amplification were performed. The endogenous and ß splice variants are not detectable owing to high expression of the exogenous hTERT transcript. Untransfected HaCaT cells (control and treated with TGF-ß1 for 96 hours) were amplified under the same conditions and included for comparison of the expression level of the endogenous with the exogenous gene and as control for the TGF-ß1 activity.

View larger version (40K): [in a new window]   Fig. 3. Splicing modulation by TGF-ß1 is reversible. (a) hTERT alternative splicing in HaCaT-myc cells after 96 hours of TGF-ß1 treatment and after additional growth for 96 hours without TGF-ß1. fl, full length; , splice; ß, ß splice. (b) TRAP assay of parallel cultures. IC, internal control.

View larger version (28K): [in a new window]   Fig. 4. hTERT splicing regulation in dispase-detached HaCaT cells. RT-PCR for hTERT alternative splicing. Increase in the ß splice variant within 8 hours of detachment of HaCaT sheets with dispase is shown. Densitometric analysis of expression of the different splice variants is shown below the gel. fl, full length; , splice; ß, ß splice.

View larger version (70K): [in a new window]   Fig. 5. Regeneration of a epidermis-like epithelium includes changes in hTERT splicing. (a) Organotypic cocultures of HaCaT cells with fibroblasts after one and three weeks. After three weeks the cells have formed an stratified epidermis-like epithelium with all characteristic layers (Bar=50 µm). (b) hTERT splicing pattern of HaCaT, HaCaT-myc and HaCaT-TERT one and three weeks after plating. With regeneration of the epithelium, the full-length hTERT transcript predominates. fl, full length; , splice; ß, ß splice.

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