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TERT suppresses apoptotis at a premitochondrial step by a mechanism requiring reverse transcriptase activity and 14–3-3 protein-binding ability¹

PEISU ZHANG^*, SIC L. CHAN^*, WEIMING FU^*, MARTY MENDOZA^* and MARK P. MATTSON^*,,2

^* Laboratory of Neurosciences, National Institute on Aging Gerontology Research Center, Baltimore, Maryland, USA; and
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

²Correspondence: Laboratory of Neurosciences, National Institute on Aging GRC 4F01, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA. E-mail: mattsonm{at}grc.nia.nih.gov

SPECIFIC AIMS

We tested the hypothesis that reverse transcriptase (RT) activity and nuclear localization are essential for the antiapoptotic function of the catalytic subunit of telomerase (TERT). Another major objective was to determine whether telomerase prevents apoptosis at an early, premitochondrial step in the cell death cascade by suppressing a signal emanating from the nucleus.

PRINCIPAL FINDINGS

1. Mutations of the RT domain and 14–3-3 protein-binding region of TERT abolish telomerase activity without affecting nuclear localization
We determined the relationships between the subcellular localization of TERT, its enzyme activity, and its ability to prevent apoptosis by producing TERT mutants with an enzymatically inactive RT domain (RTmt) or a disrupted 14–3-3 binding region (14–3-3mt). Wild-type (WT) and mutant forms of TERT and 14–3-3 were then overexpressed (WT, RTmt, and 14–3-3mt TERTs) in three human cell lines known to differ in their expression of telomerase components: HeLa cells express TERT and the telomerase RNA component, GM847 cells lack TERT but contain the RNA component, and WI38 cells express neither TERT nor the RNA component. Levels of telomerase activity were increased in HeLa cells overexpressing WT TERT but not in HeLa cells overexpressing RTmt or 14–3-3mt TERT. No telomerase activity was detected in control-untransfected or vector-transfected GM847 and WI38 cells. Stable overexpression of WT TERT resulted in the appearance of telomerase activity in GM847/alternative lengthening of telomere (ALT) cells, whereas overexpression of RTmt or 14–3-3mt did not; similar results were obtained in cells transiently transfected with WT, RTmt, and 14–3-3mt forms of TERT (data not shown). No telomerase activity was detected in WI38/ALT cells overexpressing WT or mutant forms of TERT, consistent with the lack of the telomerase RNA component in these cells.

To determine the subcellular localization of WT and mutant forms of TERT, we overexpressed green fluorescent protein-tagged WT and 14–3-3mt TERT proteins in HeLa cells and GM847 cells. WT and 14–3-3mt TERT were localized to the nucleus in both cell types. To confirm the subcellular localization of TERT, we overexpressed WT, RTmt, and 14–3-3mt forms of hTERT in GM847 cells and then immunostained the cells with an hTERT antibody. All three forms of TERT were localized in the nucleus. Thus, the 14–3-3 binding domain of TERT does not appear to play an essential role in the nuclear localization of TERT.

2. RT activity and 14–3-3 protein-binding ability are required for the antiapoptotic action of TERT
To establish the requirements of RT activity and 14–3-3 binding in the antiapoptotic function of TERT, we determined the sensitivity of HeLa cells overexpressing WT or mutant forms of TERT and 14–3-3 to apoptosis induced by the bacterial alkaloid staurosporine (STS). WT TERT protected the cells against apoptosis, whereas RTmt and 14–3-3mt forms of TERT did not (Fig. 1 ). As DNA damage is a trigger of apoptosis in a variety of physiological and pathophysiological settings, we determined the functional requirements of TERT in suppressing DNA damage-induced apoptosis. Vector-transfected HeLa cells and cells overexpressing WT, RTmt, or 14–3-3mt TERT were exposed for 24 h to the DNA-damaging agent etoposide, and the percentage of cells exhibiting apoptotic nuclei was quantified. Cells overexpressing WT TERT exhibited increased resistance to etoposide-induced apoptosis compared with vector-transfected cells (Fig. 1) . In contrast, cells overexpressing RTmt or 14–3-3mt TERT did not exhibit increased resistance to etoposide-induced apoptosis. Cells undergoing apoptosis exhibit nuclear DNA cleavage into oligonucleosome-sized fragments, which can be detected as a "ladder" when the DNA is subjected to electrophoresis. There was a clear decrease in the extent of DNA laddering in cells overexpressing WT TERT but not in cells overexpressing RTmt or 14–3-3mt TERT, indicating that RT activity and 14–3-3 binding are each required for suppression of internucleosomal DNA cleavage.

View larger version (50K): [in this window] [in a new window]   Figure 1. Requirement of RT activity, 14–3-3 binding, and the telomerase RNA component for the antiapoptotic function of TERT. The indicated cell lines were exposed to the indicated concentrations of STS (4 h and 8 h for GM847 cells or 8 h for WI38 cells) or etoposide (8 h for GM847 cells and 18 h for WI38 cells), and the percentage of apoptotic cells in each culture was quantified. Values are the mean and SD of determinations made in at least 3 separate experiments and 3 replicates for each experiment. *, P < 0.01; **, P < 001 compared with corresponding values for vector, RTmt, and 14–3-3mt cells (ANOVA with Scheffe post-hoc tests).

To more clearly establish the requirements for the antiapoptotic action of TERT, we determined the vulnerablity of cells that lack TERT (GM847) or TERT and the telomerase RNA component (WI38) to apoptosis induced by STS and etoposide. The vulnerabilty of GM847 cells to apoptosis induced by STS and etoposide was significantly decreased by overexpression of WT but not by overexpression of RTmt or 14–3-3 mutant forms of TERT (Fig. 1) . In contrast, WT did not protect WI38 cells against apoptosis induced by STS or etoposide. Thus, telomerase activity, 14–3-3 protein-binding ability, and the telomerase RNA component are each required for prevention of apoptosis.

3. Telomerase prevents release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria in cells exposed to an apoptotic insult
As mitochondrial alterations play pivotal roles in apoptosis, we determined whether TERT exerts its antiapoptotic action at a premitochondrial step in the cell death pathway. We first assessed the localization of cytochrome c in GM847 cells overexpressing WT, RTmt, or 14–3-3mt forms of TERT under basal conditions and after exposure to STS. As expected, cytochrome c immunoreactivity was colocalized with mitochondria (labeled with Mitotracker Red) in cells not exposed to STS. After exposure to STS, many vector-transfected cells exhibited a delocalization of cytochrome c immunoreactivity, consistent with release of cytochrome c from mitochondria. WT TERT reduced the number of cells releasing cytochrome c, whereas RTmt and 14–3-3mt forms of TERT did not.

In many cells undergoing apoptosis, AIF is released from mitochondria and translocates to the nucleus where it plays a key role in DNA degradation. In untreated, control cells, AIF immunoreactivity was located in punctate extranuclear compartments consistent with a mitochondrial localization (Fig. 2 ). In vector-transfected cells exposed to STS, many of the cells exhibited nuclear AIF immunoreactivity. Overexpression of WT TERT but not RTmt or 14–3-3mt forms of TERT prevented nuclear translocation of AIF in cells exposed to STS (Fig. 2) . Immunoblot analyses of mitochondrial proteins isolated from GM847 cells overexpressing WT and mutant forms of TERT revealed that considerably more AIF was retained in the mitochondria of cells overexpressing WT TERT as compared with vector-transfected cells and to cells overexpressing RTmt and 14–3-3mt forms of TERT (Fig. 2) . In an additional experiment, we found that overexpression of WT TERT but not RTmt or 14–3-3mt forms of TERT significantly decreased the level of activated caspase-3 in STS-treated GM847 cells (data not shown). Collectively, these findings suggest that TERT prevents apoptosis by inhibiting an early step in the cell death cascade prior to mitochondrial alterations and caspase activation.

View larger version (31K): [in this window] [in a new window]   Figure 2. TERT prevents AIF release from mitochondria and nuclear translocation by a mechanism requiring RT activity and 14–3-3 protein-binding ability. a) GM847 cells expressing the indicated forms of TERT were exposed to 500 nM STS for 4 h; additional vector-transfected cells were left untreated (Control). Cells were then double-stained with propidum iodide (PI) to label nuclei and an antibody against AIF. The images at the bottom show the merged images, and the yellow color represents sites of colocalization of PI and AIF. b) Mitochondria were isolated, and proteins were separated by electrophoresis and subjected to immunoblot analysis using antibodies against AIF and heat shock protein (HSP)-60. Note that much more AIF was retained in the mitochondria of STS-treated cells overexpressing WT TERT than in STS-treated cells overexpressing RTmt (RT) or 14–3-3mt TERT. GM, GM847.

CONCLUSIONS AND SIGNIFICANCE

The present findings demonstrate that RT activity and the ability to bind to 14–3-3 proteins are essential for the antiapoptotic action of TERT. Although the molecular basis of the antiapoptotic function remains to be determined, the requirements for RT activity and nuclear localization narrow the possibilities. Nuclear DNA damage is a prominent trigger of apoptosis and can set in motion a cell death cascade involving the tumor suppressor protein p53, the proapoptotic Bcl-2 family member Bax, mitochondrial alterations, and caspase activation. Apoptosis induced by etoposide and STS involves cytochrome c release from mitochondria and activation of caspase-3. In the case of etoposide, the trigger for mitochondrial alterations leading to cytochrome c release involves a DNA damage response in which p53 is activated, and the proapoptotic protein Bax translocates to the mitochondria. STS also induces p53, Bax translocation, cytochrome c release, and activation of caspase-3. STS and related inhibitors of protein kinase C can induce DNA damage characterized by single-strand breaks and may facilitate DNA damage by impairing DNA protective mechanisms. Recent findings suggest that telomeric DNA sequences may be preferentially damaged by toposiomerase II inhibitors. Our data therefore suggest that telomerase may prevent or repair damage to telomeric DNA and thereby suppress the DNA damage response that triggers apoptosis (Fig. 3 ). Suppression of a nuclear DNA damage-related signal is also consistent with our evidence that TERT suppresses apoptosis at an early stage prior to mitochondrial alterations and caspase activation.

View larger version (19K): [in this window] [in a new window]   Figure 3. Working model for the antiapoptotic function of telomerase. Apoptotic signals induce alterations in the nucleus that may include DNA damage. The nuclear alterations trigger an apoptotic cascade involving release of cytochrome c (Cyt c) and AIF from mitochondria and caspase activation. TERT acts in the nucleus to suppress DNA damage and/or apoptotic signals triggered by DNA damage; this action of TERT requires binding to 14–3-3 proteins as well as the telomerase RNA component (TR). DESP, Death effector substrate proteins; Apaf-1, apoptotic protease activating factor-1.

Previous studies have shown that 14–3-3 can prevent apoptosis, but the specific binding partners of 14–3-3 that mediate this antiapoptotic effect have not been established. 14–3-3 may bind and inactivate proapoptotic Bcl-2 family members such as Bax, Bad, and p53. Previous studies suggested that binding of 14–3-3 to TERT plays an important role in the retention of TERT in the nucleus. However, we did not find that TERT with a mutant 14–3-3 binding domain was excluded from the nucleus. Nevertheless, 14–3-3mt TERT was completely ineffective in protecting cells against apoptosis. These findings suggest that interaction of TERT with 14–3-3 proteins in the nucleus is critical for the antiapoptotic function of TERT.

Our findings suggest that agents that inhibit the RT activity of telomerase or its interaction with 14–3-3 proteins may promote apoptosis of tumor cells expressing telomerase.

Beyond their implications for mechanisms of carcinogenesis and cancer therapy, the present findings suggest roles for telomerase in embryogenesis and cell responses to injury. Telomerase activity and TERT expression are high throughout the body during early embryogenesis and then decrease rapidly as progenitor cells cease dividing and differentiate. Apoptosis plays important roles in the development of many different tissues, as is evident in the abnormal phenotypes caused by increased or decreased apoptosis as a result of disruption of anti- and proapoptotic genes, respectively. Evidence that telomerase activity is important for normal development comes from analyses of later generation mice lacking the telomerase RNA component and studies of cultured embryonic brain neurons in which TERT expression was reduced using antisense technology. Future studies will likely reveal important roles for TERT and 14–3-3 proteins in the regulation of cell fate during embryogenesis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0603fje; to cite this article, use FASEB J. (February 19, 2003) 10.1096/fj.02-0603fje

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