The hTERTalpha splice variant is a dominant negative inhibitor of telomerase activity

Abstract

The telomerase catalytic subunit (hTERT) is an essential component of the holoenzyme complex that adds telomeric repeats to the ends of human chromosomes. Maintenance of telomeres by telomerase or another mechanism is required for cell immortalization, and loss of telomeric DNA has been proposed as a trigger for cellular senescence. Available evidence suggests that regulation of telomerase activity primarily depends on transcriptional control of hTERT. However, several human tissues as well as some normal cell strains have been shown to express low levels of hTERT mRNA even though they lack telomerase activity. We have previously identified six splice variants of hTERT, including a "deletion" variant (hTERTalpha) that is missing conserved residues from the catalytic core of the protein. Several of the deletion variants have been detected in normal and developing human tissues. We now show that hTERTalpha inhibits endogenous telomerase activity, which results in telomere shortening and chromosome end-to-end fusions. Telomerase inhibition induced a senescence-like state in HT1080 cells and apoptosis in a jejunal fibroblast cell line. These results suggest a possible role for hTERT splice variants in the regulation of telomerase activity.

Figure 1

Deletion variant hTERTα is expressed in some telomerase-positive immortalized cell lines. (A). The sites where the α and β deletion variants occur are shown relative to the conserved reverse transcriptase domains of hTERT [12], and hTERTα employs an alternative splice site within exon 6 (based on genomic sequence [19,20]). (B) GC-rich RT-PCR was performed on total cellular RNA from five telomerase-positive immortal cell lines (indicated above each lane). Primers were used that spanned the 430-bp region encompassing the α and β deletion sites. (M) indicates DNA size markers. Deletion variants are noted, including β variant and α+β variant previously described [12]. RT-PCR for GAPDH, to verify quality of the RNA, is shown in the bottom panel. (C) Telomerase-negative GM847 cells were transiently transfected with plasmid overexpressing hTERT or the deletion variant hTERTα. Telomerase activity was reconstituted by expression of hTERT but not by hTERTα. Protein lysis buffer (LB) was used as a negative control for PCR, and the internal positive control is indicated (IC).

Figure 2

Overexpression of hTERTα inhibits endogenous telomerase activity in stable clones of JFCF-6T/2H and HT1080. (A–B) The TRAP assay was performed on 2 µg of protein lysate from each of the indicated G418-selected clonal cell lines. Lanes 1 and 14, full length hTERT controls; lanes 2 through 5, JFCF-6T/2H stable clones expressing hTERTα; lanes 6 and 13, empty vector controls; lanes 7 and 8, lysis buffer; lanes 9 through 12, HT1080 stable clones expressing α variant; lane 15, D712A dominant negative control. Results of RT-PCR to check for plasmid transcription are shown below the corresponding clone. IC indicates internal control for the PCR step of the TRAP assay. (C) Example of Southern analysis on stable clones overexpressing dominant negative inhibitors of telomerase. JFCF' indicates JFCF-6T/2H cells. HT1080 or JFCF-6T/2H were transfected with empty vector (neo), pIRES-hTERTα (α) or 3-1 dominant negative control. Telomeric DNA, (T₂AG₃)₃, was used as a probe. Positions of size markers are indicated on the right.

Figure 3

hTERTα expression causes loss of telomeric DNA and chromosome end-to-end fusions. (A) FISH analysis using Cy3-conjugated telomeric PNA probe on metaphases of HT1080 stable clones overexpressing empty vector (upper panel) or pIRES-hTERTα (lower). DNA was counterstained with DAPI and images merged. Undetectable telomeres (green arrows) and fused or circularized chromosomes (yellow arrows) are indicated. (B) FISH analysis using Cy3-conjugated telomeric PNA probe on metaphases of JFCF-6T/2H stable clones overexpressing empty vector (upper panel), or pIRES-hTERTα (lower). DNA was counterstained with DAPI and images merged. Undetectable telomeres (green arrows) and fused or broken chromosomes (yellow arrows) are indicated.

Figure 4

Dominant negative forms of hTERT induce either apoptosis or a senescence-like state. (A) Photomicrographs of HT1080 stable clones are shown, “control,” (upper panel) was transfected with empty vector. Dominant negative (DN) 3-1 control is shown with staining for endogenous β-galactosidase activity (middle). β-Galactosidase staining of an hTERTα transfected stable clone is also shown (lower). (B) JFCF-6T/2H cells are shown: control, empty vector (upper); dominant negative (DN) 3-1 control (middle); α variant transfected stable clone, DAPI stained (lower). (C) Stable clones of HeLa are shown: empty vector (top) and β-galactosidase stained cells expressing DN 3-1 control (bottom).