ABSTRACT
The nucleolar protein PinX1 has been proposed to be a putative tumor suppressor due to its binding to and inhibition of the catalytic activity of telomerase, an enzyme that is highly expressed in most human cancers in which it counteracts telomere shortening-induced senescence to confer cancer cell immortalization. However, the role of PinX1 in telomere regulation, as well as in cancer, is still poorly understood. In this study, we showed that the PinX1 protein is constitutively expressed in various human cells regardless of their telomerase activity and malignant status. Most interestingly, we found that silencing PinX1 expression by a potent short hairpin RNA construct led to a robust telomere length shortening and growth inhibition in telomerase-positive but not in telomerase-negative human cancer cells. We further showed that silencing PinX1 significantly reduced the endogenous association of telomerase with the Pot1-containing telomeric protein complex, and therefore, could account for the phenotypic telomere shortening in the affected telomerase-positive cancer cells. Our results thus reveal a novel positive role for PinX1 in telomerase/telomere regulations and suggest that the constitutive expression of PinX1 attributes to telomere maintenance by telomerase and tumorigenicity in cancer cells.
Subject(s)
Cell Transformation, Neoplastic/metabolism , Telomerase/metabolism , Telomere/metabolism , Tumor Suppressor Proteins/deficiency , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cell Cycle Proteins , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , DNA Damage , Etoposide/pharmacology , Gene Silencing , Humans , Mice , Mice, Inbred BALB C , RNA, Small Interfering/genetics , Shelterin Complex , Telomerase/biosynthesis , Telomere/genetics , Telomere-Binding Proteins/metabolism , Transfection , Tumor Suppressor Proteins/biosynthesis , Tumor Suppressor Proteins/geneticsABSTRACT
Telomerase maintains telomere length and has been implicated in both aging and carcinogenesis of human cells. This enzyme is a specialized ribonucleoprotein (RNP) complex, minimally consisting of two essential components: the protein catalytic subunit TERT (telomerase reverse transcriptase) and the integral RNA moiety TR (telomerase RNA, TERC). Both TERT and TR have been found to localize to nucleoli within the nucleus, leading to the suggestion of nucleoli as the site for telomerase RNP biogenesis in human cells. However, whether this statement is true or not has not yet been convincingly demonstrated. Here, we identify that residues 965-981 of the human TERT polypeptide constitute an active nucleolar-targeting signal (NTS) essential for mediating human TERT nucleolar localization. Mutational inactivation of this NTS completely disrupted TERT nucleolar translocation in both normal and malignant human cells. Most interestingly, such a TERT mutant still retained the capacity to activate telomerase activity, maintain telomere length and extend the life-span of cellular proliferation, as does wild-type TERT, in BJ cells (normal fibroblasts). Therefore, our data suggest that TERT nucleolar localization is unrelated to telomerase function in human cells.
Subject(s)
Telomerase/genetics , Telomerase/metabolism , Amino Acid Sequence , Animals , Catalytic Domain , Cell Line , Cell Nucleolus/metabolism , Humans , Mice , Nuclear Localization Signals/genetics , Nuclear Localization Signals/metabolism , RNA/genetics , RNA/metabolism , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , Sequence Homology, Amino Acid , Telomere/genetics , Telomere/metabolismABSTRACT
The factor PinX1 has been shown as a telomerase inhibitor evolutionarily conserved in both the yeast and the human being. yPinX1 inhibits telomerase activity by sequestering yTERT (telomerase reverse transcriptase) from uniting with yTR (telomerase template RNA) in the nucleolus of yeast cells. However, the mechanism underlying the action of hPinX1 on telomerase regulation in human cells is not known. We here demonstrated that hPinX1 actually has an effect on mediating hTERT nucleolar localization and this effect is mediated by a novel domain enclosed within the central section of the polypeptide. Interestingly, we showed that a reported cancerous mutant form of hPinX1, in which residues of Ser254 and Cys265 are, respectively, mutated to Cys and Tyr, lost the activity on mediating hTERT nucleolar localization. Finally, we provided evidence that mediation of hTERT nucleolar localization and telomerase enzymatic inhibition are two separated function of hPinX1 on telomerase regulation in human cells.
Subject(s)
Cell Nucleolus/metabolism , Telomerase/metabolism , Tumor Suppressor Proteins/metabolism , Binding Sites/genetics , Cell Cycle Proteins , Cell Line, Tumor , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Mutation , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Nuclear Localization Signals/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Telomerase/antagonists & inhibitors , Telomerase/genetics , Transfection , Tumor Suppressor Proteins/genetics , Red Fluorescent ProteinABSTRACT
We have constructed a 27-kDa hTERT C-terminal polypeptide (hTERTC27) devoid of domains required for telomerase activity and demonstrated that it is capable of nuclear translocation/telomere-end targeting. Here we showed that expression of a low level of hTERTC27 renders hTERT positive HeLa cells sensitive to H(2)O(2)-induced oxidative stress and subsequent cell senescence. The senescence-associated gene, the cyclin/cdk inhibitor p21(Waf1), was up-regulated. This occurs without changing the expression of endogenous hTERT, causing significant telomere shortening or inhibiting telomerase activity. Results from this study suggest for the first time that in addition to telomerase activity, the C-terminus of hTERT also plays a role in hTERT-mediated cellular resistance to oxidative stress.
Subject(s)
Cellular Senescence , Hydrogen Peroxide/pharmacology , Oxidants/pharmacology , Oxidative Stress , Telomerase/chemistry , Telomerase/metabolism , Clone Cells , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/biosynthesis , DNA-Binding Proteins , HeLa Cells , Humans , Peptides/genetics , Peptides/pharmacology , Recombinant Fusion Proteins/pharmacology , Telomerase/geneticsABSTRACT
The COOH-terminus of telomerase reverse transcriptase (hTERT) has been shown to participatein the nuclear translocation of TERT. Here, we constructed plasmids expressing the COOH-terminal M(r) 27,000 polypeptide of hTERT (hTERTC27) withthe telomerase RNA-binding domains and the reverse transcriptase domains deleted. We showed that ectopic overexpression of this polypeptide caused a defect in telomere maintenance in hTERT-positive HeLa cells, which led to senescence-like growth arrest and apoptosis. The hTERTC27 appears to work by inducing telomere dysfunction, exemplified by significantly increased anaphase chromosome end-to-end fusion events in transfected cells. Significantly, it had no effect on the cellular telomerase enzymatic activity or telomere length. The in vivo effect was further demonstrated as HeLa cells stably expressing hTERTC27 have significantly lower growth rate and reduced tumorigenicity in nude mice xenografts. Results from this study revealed an important function for the COOH terminus of hTERT in maintaining the integrity of telomere structure and chromosome ends, as well as in cell senescence and apoptosis. Furthermore, hTERTC27 provides a new strategy for cancer therapy by inducing telomere dysfunction in cancer cells without affecting the telomerase enzymatic activity.
