Telomerase-associated Protein Research Articles

Nucleolar protein PinX1p regulates telomerase by sequestering its protein catalytic subunit in an inactive complex lacking telomerase RNA.

Human TRF1-binding protein PinX1 inhibits telomerase activity. Here we report that overexpression of yeast PinX1p (yPinX1p) results in shortened telomeres and decreased in vitro telomerase activity. yPinX1p coimmunoprecipitated with yeast telomerase protein Est2p even in cells lacking the telomerase RNA TLC1, or the telomerase-associated proteins Est1p and Est3p. Est2p regions required for binding to yPinX1p or TLC1 were similar. Furthermore, we found two distinct Est2p complexes exist, containing either yPinX1p or TLC1. Levels of Est2p-yPinX1p complex increased when TLC1 was deleted and decreased when TLC1 was overexpressed. Hence, we propose that yPinX1p regulates telomerase by sequestering its protein catalytic subunit in an inactive complex lacking telomerase RNA.

Analysis of MVP and VPARP promoters indicates a role for chromatin remodeling in the regulation of MVP

Multi-drug-resistant cancer cells frequently express elevated levels of ribonucleoprotein complexes termed vaults. The increased expression of vault proteins and their mRNAs has led to the suggestion that vaults may play a direct role in preventing drug toxicity. To further understand vault component up-regulation, the three proteins that comprise the vault, the major vault protein (MVP), vault poly(ADP-ribose) polymerase (VPARP), and telomerase-associated protein-1 (TEP1), were examined with respect to gene amplification and drug-induced chromatin remodeling. Gene amplification was not responsible for increased vault component levels in multi-drug-resistant cancer cell lines. The TATA-less murine MVP and human VPARP promoters were identified and functionally characterized. There was no significant activation of either the MVP or VPARP promoters in drug-resistant cell lines in comparison to their parental, drug-sensitive counterparts. Treatment of various cell lines with sodium butyrate, an inhibitor of histone deacetylase (HDAC), led to an increase in vault component protein levels. Furthermore, treatment with trichostatin A (TSA), a more specific inhibitor of HDAC, caused an increase in MVP protein, mRNA, and promoter activity. These results suggest that up-regulation of MVP in multi-drug resistance (MDR) may involve chromatin remodeling.

Alternative Splicing of Human Telomerase Reverse Transcriptase May Not Be Involved in Telomerase Regulation During all-trans-Retinoic Acid-Induced HL-60 Cell Differentiation

Alternative splicing of the human telomerase reverse transcriptase subunit (hTERT) suppresses telomerase activity during the development of human fetal kidney cells into mature cells. Tumor cell differentiation is the process of turning abnormal tumor cells into ‘normal’ cells accompanied by down-regulation of telomerase activity. However, the precise mechanism of the regulation of telomerase activity in differentiated cells is not fully understood. In this study, we observed the role of alternative splicing of hTERT in the regulation of telomerase activity in all-trans-retinoic acid (ATRA)-induced, differentiated HL-60 cells. ATRA-induced down-regulation of telomerase activity in differentiated HL-60 cells was associated with a decrease in hTERT and an increase in human telomerase-associated protein-1 (hTP1) transcription. Expression of full length variant hTERT α+β+ mRNA decreased in a dose- and time-dependent manner. The drop of hTERT β- mRNA was time-dependent. hTERT α- and hTERT α-b- mRNA were reduced dramatically after ATRA treatment. In the dose-effect study, hTERT α+β+ and hTERT β- maintained a relatively stable ratio when telomerase activity decreased largely from treatment with 1 to 5 μM ATRA. Although the splicing pattern of hTERT mRNA was altered in time-effect research, the change was not related to the ATRA-treated decline of telomerase activity. The expression of alternative splicing variants of hTERT also decreased at the protein level. All these results suggested that alternative splicing of hTERT mRNA may not contribute to the suppression of telomerase activity during ATRA-induced HL-60 leukemia cell differentiation.

Vaults: a ribonucleoprotein particle involved in drug resistance?

Vaults are ribonucleoprotein particles found in the cytoplasm of eucaryotic cells. The 13 MDa particles are composed of multiple copies of three proteins: an M(r) 100 000 major vault protein (MVP) and two minor vault proteins of M(r) 193 000 (vault poly-(ADP-ribose) polymerase) and M(r) 240 000 (telomerase-associated protein 1), as well as small untranslated RNA molecules of approximately 100 bases. Although the existence of vaults was first reported in the mid-1980s no function has yet been attributed to this organelle. The notion that vaults might play a role in drug resistance was suggested by the molecular identification of the lung resistance-related (LRP) protein as the human MVP. MVP/LRP was found to be overexpressed in many chemoresistant cancer cell lines and primary tumor samples of different histogenetic origin. Several, but not all, clinico-pathological studies showed that MVP expression at diagnosis was an independent adverse prognostic factor for response to chemotherapy. The hollow barrel-shaped structure of the vault complex and its subcellular localization indicate a function in intracellular transport. It was therefore postulated that vaults contributed to drug resistance by transporting drugs away from their intracellular targets and/or the sequestration of drugs. Here, we review the current knowledge on the vault complex and critically discuss the evidence that links vaults to drug resistance.

Enhanced expression of p16 in seborrhoeic keratosis; a lesion of accumulated senescent epidermal cells in G1 arrest.

Seborrhoeic keratosis (SK) is a common skin disease associated with skin ageing and photoageing, but only limited studies have been performed on SK and the senescence of keratinocytes. We sought to clarify the genetic basis of SK and the senescence of keratinocytes. Expression of p16, cyclins A, D and E, p21, p53, retinoblastoma (Rb) gene product and telomerase-associated protein 1 (TP1) in SK was examined by immunohistochemistry. DNA fragmentation in SK was detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling method. We cultured keratinocytes from SK lesions and non-lesional epidermis and examined expression of p16, observed morphology of the cultured cells by light and electron microscopy and measured survival time. p16, a cyclin-dependent kinase inhibitor, was expressed in all cells from SK lesions, whereas normal keratinocytes expressed p16 only in the granular cells. Other factors such as cyclins A, D and E, p21, p53, Rb gene product, and TP1, were not expressed in SK cells. These results suggest that p16 expression is a marker of SK and that p16 has a role in the pathogenesis of SK. DNA fragmentation was not detected in four of five SK tissue samples; one of the SK tissue samples showed DNA fragmentation only in the superficial upper layer of an SK lesion, suggesting that apoptosis was inhibited in SK cells. In contrast, normal epidermis showed DNA fragmentation in the granular and squamous layers. Immunohistochemical examination of cultured SK cells also revealed the presence of p16. A greater number of SK cells survived after 3 weeks of culture in comparison with normal keratinocytes. Features of senescence, such as a balloon-like appearance after lengthy culture and increased amounts of tonofilaments in cytoplasm, were observed in SK cells in culture. These results suggest that SK is a benign neoplasm where keratinocytes in a senescent condition and G1 arrest are accumulated.

Expression profile of the telomeric complex discriminates between benign and malignant pheochromocytoma.

Telomerase, a ribonucleoprotein complex that includes the telomerase RNA component, the telomerase-associated protein (TP1), the telomerase catalytic subunit (hTERT), and the heat shock protein 90 (HSP90), is closely related to the malignant potential of human tumors. In pheochromocytomas (PC) it is very difficult to predict malignant potential by conventional histology or immunohistochemical and molecular markers. To test whether the expression of telomerase subunits is reflected in the malignant transition of PCs, we determined their mRNA and/or protein expression in 28 benign and nine malignant PCs and compared the results with telomerase activity. RT-PCR analysis revealed that TP1 was ubiquitously expressed. The telomerase RNA component was found in all malignant (100%) and in 13 of 28 (46%) benign PCs. In contrast, hTERT was clearly associated with aggressive biological behavior. All of the malignant (100%), but only two of 28 benign (7%) PCs expressed hTERT. HSP90 was increased in malignant PCs, but was also expressed at a lower level in benign tumors. High telomerase activity was measurable in hTERT-positive tissues only. Our data indicate that hTERT, HSP90, and telomerase activity are up-regulated in malignant cells of the adrenal medulla. The common expression of hTERT and telomerase activity thus represents an additional prognostic marker that may identify more aggressive tumors.

Evaluation of MDR1, LRP, MRP, and topoisomerase IIα gene mRNA transcripts before and after interferon-α, and correlation with the mRNA expression level of the telomerase subunits hTERT and TEP1 in five unselected human melanoma cell lines

Intrinsic and acquired multidrug-resistance (MDR) and the activity of the enzyme telomerase have been demonstrated in human melanoma. A direct regulation of the MDR pathways and of telomerase by interpheron-alpha (IFN-alpha), which is currently used in the therapy of advanced cutaneous melanoma, has also been hypothesized. In this study, we used five melanoma cell lines not selected in vitro for drug resistance (Me665/2/21, Me665/2/60, HT-144, SK-MEL-28, and SK-MEL-5), which in a previous study, had shown different responses to IFN-alpha in terms of proliferation, apoptosis, telomerase activity and expression of mRNA for the human telomerase reverse transcriptase (hTERT). We investigated the expression of the multidrug resistance (MDR1) gene, multidrug resistance protein (MRP), lung resistance protein (LRP), topoisomerase IIalpha (Topo IIalpha), hTERT, and telomerase-associated protein (TEP1), which is shared by telomerase and vault MDR proteins at the mRNA expression level, using the reverse transcription-PCR (RT-PCR). All cell lines showed an intrinsic expression of hTERT, TEP1, and MDR gene transcripts (only MDR1 mRNA was under the detection level in SK-MEL-28 cells). After IFN-alpha exposure, we observed either no effect, a trend towards a decrease of hTERT, MRP, and Topo IIalpha, or an increase of TEP1, MDR1, and LRP mRNA expression in some cell lines. Effects were usually temporary and not always significant. No correlation was found between hTERT and TEP1 mRNA expression, whereas significant positive correlations were found between TEP1 and MDR1 mRNA, and between TEP1 and LRP mRNA. IFN-alpha modulates differently MDR gene transcripts in human melanoma cell lines. Positive correlation between TEP1 and LRP also seems to identify them as common targets of IFN-alpha effects.

Sudden telomere lengthening triggers a Rad53-dependent checkpoint in Saccharomyces cerevisiae.

Telomeres are specialized functional complexes that ensure chromosome stability by protecting chromosome ends from fusions and degradation and avoiding chromosomal termini from being sensed as DNA breaks. Budding yeast Tel1 is required both for telomere metabolism and for a Rad53-dependent checkpoint responding to unprocessed double-strand breaks. We show that overexpression of a GAL1-TEL1 fusion causes transient telomere lengthening and activation of a Rad53-dependent G2/M checkpoint in cells whose telomeres are short due to the lack of either Tel1 or Yku70. Sudden telomere elongation and checkpoint-mediated cell cycle arrest are also triggered in wild-type cells by overproducing a protein fusion between the telomeric binding protein Cdc13 and the telomerase-associated protein Est1. Checkpoint activation by GAL1-TEL1 requires ongoing telomere elongation. In fact, it is turned off concomitantly with telomeres reaching a new stable length and is partially suppressed by deletion of the telomerase EST2 gene. Moreover, both telomere length rebalancing and checkpoint inactivation under galactose-induced conditions are accelerated by high levels of either the Sae2 protein, involved in double-strand breaks processing, or the negative telomere length regulator Rif2. These data suggest that sudden telomere lengthening elicits a checkpoint response that inhibits the G2/M transition.

The telomerase-associated protein p43 is involved in anchoring telomerase in the nucleus

Telomere replication of eukaryotic chromosomes is achieved by a specialized enzyme, the telomerase. Although the biochemistry of end-replication is well understood, little is known about the organization of the end-replication machinery, its regulation throughout the cell cycle or the biological function of the telomerase-associated proteins. Here we investigate the function of the telomerase-associated protein p43 within the macronucleus of the ciliated protozoa Euplotes. It has been shown that p43 binds in vitro to the RNA subunit of telomerase and shares homology with the La autoantigen family. It therefore has been suggested that it is involved in the assembly and/or nuclear retention of telomerase. We show that the p43-telomerase complex is bound to a subnuclear structure in vivo and is resistant to electroelution. Upon inhibition of p43 or telomerase expression by RNAi, which in this study was used for the first time in spirotrichs, this complex is no longer retained in the nucleus. Further analysis revealed that the p43-telomerase complex is bound to the nuclear matrix in vivo and that after inhibition of p43 expression, telomerase is released from this structure, strongly suggesting that p43 is involved in anchoring of telomerase in the nucleus. This is the first in vivo demonstration of the biological function of this telomerase-associated component involved in telomere replication and allows us to propose a model for the organization of the end-replication machinery in the eukaryotic cell.

Biochemical aspects of telomerase function

Arthur Kornberg “never met a dull enzyme” (For the Love of Enzymes: The Odyssey of a Biochemist, Harvard University Press, 1989) and telomerase is no exception. Telomerase is a remarkable polymerase that uses an internal RNA template to reverse-transcribe telomere DNA, one nucleotide at a time, onto telomeric, G-rich single-stranded DNA. In the 17 years since its discovery, the characterization of telomerase enzyme components has uncovered a highly conserved family of telomerase reverse transcriptases that, together with the telomerase RNA, appear to comprise the enzymatic core of telomerase. While not as comprehensively understood as yet, some telomerase-associated proteins also serve crucial roles in telomerase function in vivo, such as telomerase ribonudeoprotein (RNP) assembly, recruitment to the telomere, and the coordination of DNA replication at the telomere. A selected overview of the biochemical properties of this unique enzyme, in vitro and in vivo, will be presented.

Markers for the development of early prostate cancer.

Biochemical and genetic changes precede histologically identifiable changes accompanying cell transformation often by months or years. De-expression of the extracellular matrix adhesive glycoprotein tenascin and the cell-to-cell adherent protein E-cadherin have been suggested as markers of early neoplastic change in prostate epithelial cells. Previous studies have been inconclusive, probably due to epitope masking. This study examined 2,378 biopsy cores from 289 prostates using a heat antigen retrieval protocol at low pH to improve the accuracy of detection. Tenascin and E-cadherin de-expression was correlated with purinergic receptor and telomerase-associated protein labelling, as well as prostate-specific antigen (PSA) levels and Gleason scores. E-cadherin was a poor marker, as it was expressed in all lesions except carcinomas of the highest Gleason score. Tenascin was maximally expressed in the extracellular matrix and acinar basement membrane in normal and prostatic intraepithelial neoplasia tissue. In prostate cancer tissue, tenascin expression did not correlate with Gleason score but was significantly de-expressed as purinergic receptor and telomerase-associated protein expression increased. Marked changes in tenascin, telomerase-associated protein, and purinergic receptor expression were apparent before any histological abnormalities were visible by haematoxylin and eosin (H&E) stain, making these potential markers for early and developing prostate cancer. Moreover, the potential increased accuracy of diagnosis of underlying prostate cancer using purinergic receptor translocation (PRT) assessment suggests that PSA levels may be more accurate than has generally been supposed when apparent false negatives arising from H&E-based diagnoses are correctly categorized.

The formation of vault-tubes: a dynamic interaction between vaults and vault PARP

Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles that are composed of a major vault protein (MVP), two minor vault proteins [telomerase-associated protein 1 (TEP1), vault poly(ADP-ribose) polymerase (VPARP)] and small untranslated RNA molecules. Not all expressed TEP1 and VPARP in cells is bound to vaults. TEP1 is known to associate with the telomerase complex, whereas VPARP is also present in the nuclear matrix and in cytoplasmic clusters (VPARP-rods). We examined the subcellular localization and the dynamics of the vault complex in a non-small cell lung cancer cell line expressing MVP tagged with green fluorescent protein. Using quantitative fluorescence recovery after photobleaching (FRAP) it was shown that vaults move temperature independently by diffusion. However, incubation at room temperature (21 degrees C) resulted in the formation of distinct tube-like structures in the cytoplasm. Raising the temperature could reverse this process. When the vault-tubes were formed, there were fewer or no VPARP-rods present in the cytoplasm, suggesting an incorporation of the VPARP into the vault-tubes. MVP molecules have to interact with each other via their coiled-coil domain in order to form vault-tubes. Furthermore, the stability of microtubules influenced the efficiency of vault-tube formation at 21 degrees C. The dynamics and structure of the tubes were examined using confocal microscopy. Our data indicate a direct and dynamic relationship between vaults and VPARP, providing further clues to unravel the function of vaults.

Function of HSP90 and p23 in the telomerase complex of thyroid tumors.

Recently, studies on endocrine tumors revealed a potential role of telomerase in the dedifferentiation and/or malignant transition of these tumors. Telomerase is a ribonucleoprotein complex that includes the telomerase RNA component (hTR), the telomerase-associated protein (TP1), and the telomerase catalytic subunit (hTERT). Previously, the chaperones p23 and HSP90 have been described as additional telomerase regulators. To test whether the interactions of these genes are reflected in the dedifferentiation of thyroid tumors, we determined their mRNA and/or protein expression in 30 normal (tumor-free) thyroid tissues (NT), 35 follicular adenomas (FAD), 42 papillary carcinomas (PTC), 38 follicular carcinomas (FTC), 25 poorly differentiated carcinomas (PDTC), and 34 undifferentiated carcinomas (UTC). We then compared the results with telomerase activity. RT-PCR analysis revealed that TP1 was ubiquitously expressed. hTR was found in 50-94% of malignant tumors, in contrast to 7% of NT and 26% of FAD. hTERT was clearly associated with aggressive biological behavior. Ninety-two to 100% of the malignant tumors were positive for hTERT protein, whereas NT and FAD were negative in 100% and 94%, respectively. HSP90 mRNA and protein showed a close relationship to hTERT. p23 protein was negative in NT and positive in 3% of FAD, 39% of FTC, 40% of PTC, 44% of PDTC and 47% of UTC. High telomerase activity was measurable in hTERT and HSP90-positive tissues only. Our data show that the common expression of hTERT and HSP90 regulates telomerase activity in thyroid carcinomas. Chaperone p23 is involved in the telomeric complex to a lesser extent, but its expression is stronger in carcinomas than in non-malignant thyroid tissues. The expression profile of telomerase components represents an additional prognostic marker that may identify more aggressive thyroid tumors.

HSP90 is a key for telomerase activation and malignant transition in pheochromocytoma.

Recent studies on a limited number of pheochromocytomas (PCs) revealed a potential role of telomerase in the malignant transition of these tumors. Telomerase is a ribonucleoprotein complex that includes the telomerase RNA component (hTR), the telomerase-associated protein (TP1), the telomerase catalytic subunit (hTERT), and the heat-shock protein 90 (HSP90). The interactions between these subunits and the activation machinery of telomerase are still unclear. To test whether the expression and regulation of telomerase subunits are reflected in the malignant transition of PCs, we determined their mRNA and/or protein expression in 28 benign and 9 malignant PCs and compared the results with telomerase activity. Reverse transcriptase polymerase chain reaction analysis revealed that TP1 was ubiquitously expressed. hTR was found in all malignant (100%) and in 13/28 (46%) benign PCs. By contrast, hTERT was clearly associated with aggressive biologic behavior. All the malignant (100%) but only 2/28 benign (7%) PCs expressed hTERT. HSP90 was increased in malignant PCs but was also expressed at a lower level in benign tumors. High telomerase activity was measurable in only hTERT-positive tissues. Our data indicate that hTERT, HSP90, and telomerase activity are upregulated in malignant cells of the adrenal medulla. Overexpression of HSP90 is an important factor in the activation of telomerase via hTERT. The common expression of hTERT and telomerase activity thus represents an additional prognostic marker that may identify more aggressive tumors.

The use of RNAi to analyze gene function in spirotrichous ciliates

The use of small double-stranded RNA to inhibit expression of genes has proven to be a powerful tool to analyze the biological function of genes in various eukaryotic organisms. We have established the RNAi technology in three genera of spirotrichous ciliates, Euplotes, Oxytricha and Stylonychia . Here we describe this method to silence gene expression in these three genera. Expression of the following genes was inhibited: the catalytic subunit of telomerase (TERT) and the telomerase-associated protein p43 in Euplotes and the a-subunit of the telomere-binding protein (αTeBP) of Stylonychia and Oxytricha . In addition, we report how the biological function of a developmentally regulated gene can be elucidated by this technique in Stylonychia.

Radiation-induced changes of telomerase activity in a human Ewing xenograft tumor.

The effect of ionizing irradiation on telomerase activity and further associated biological factors was evaluated in a human Ewing tumor xenograft model on nude mice. The human Ewing tumor cell line STA-ET-1 was established in a nude mouse model. Initially, the dose-response relationship for the tumor model was established. For the radiation experiments two dose levels were chosen: 5 Gy and 30 Gy. After 5 Gy, there was no significant growth delay whereas after 30 Gy there was a marked growth delay without the induction of a complete remission. Tumors were examined 6, 12, 24, 48, 72, and 96 hours post irradiation. After irradiation with 30 Gy further time points were 6, 9, 12 and 15 days. For each dose and time group, three tumors were evaluated. There was a reduction of telomerase activity after 5 Gy to 50% (not statistically significant) after 3 days; however, after 30 Gy there was a reduction of telomerase activity to 23% of the initial value after 6 days (p = 0.001). Telomerase activity correlated with the expression of human telomerase reverse transcriptase (hTERT), but not with the expression of telomerase-associated protein (TP1) and human telomerase RNA (hTR). The maximal amounts of necrosis or apoptosis after 30 Gy were 19% and 6.9%, respectively. Ionizing radiation reduces telomerase activity and the expression of hTERT which cannot be explained by the induction of necrosis or apoptosis alone. The reduction of telomerase activity may contribute to delayed cell death after radiotherapy. The combined use of radiation and specific telomerase inhibitors may be a potentially synergistic treatment strategy.

The Est1 Subunit of Saccharomyces cerevisiae Telomerase Makes Multiple Contributions to Telomere Length Maintenance

The telomerase-associated Est1 protein of Saccharomyces cerevisiae mediates enzyme access by bridging the interaction between the catalytic core of telomerase and the telomere-binding protein Cdc13. In addition to recruiting telomerase, Est1 may act as a positive regulator of telomerase once the enzyme has been brought to the telomere, as previously suggested by the inability of a Cdc13-Est2 fusion protein to promote extensive telomere elongation in an est1-Delta strain. We report here three classes of mutant Est1 proteins that retain association with the telomerase enzyme but confer different in vivo consequences. Class 1 mutants display a telomere replication defect but are capable of promoting extensive telomere elongation in the presence of a Cdc13-Est2 fusion protein, consistent with a defect in telomerase recruitment. Class 2 mutants fail to elongate telomeres even in the presence of the Cdc13-Est2 fusion, which is the phenotype predicted for a defect in the proposed second regulatory function of EST1. A third class of mutants impairs an activity of Est1 that is potentially required for the Ku-mediated pathway of telomere length maintenance. The isolation of mutations that perturb separate functions of Est1 demonstrates that a telomerase holoenzyme subunit can contribute multiple regulatory roles to telomere length maintenance.

Down-regulation of telomerase activity via protein phosphatase 2A activation in salvicine-induced human leukemia HL-60 cell apoptosis

Salvicine is a novel topoisomerase II inhibitor possessing significant antitumor activity, both in vitro and in vivo. The antitumor effect of salvicine is associated with its ability to induce tumor cell apoptosis. Telomerase plays an important role in the apoptotic pathway. However, little is known about the mechanisms of telomerase regulation during apoptosis induced by anticancer drugs. This study investigated the regulation of telomerase activity in salvicine-induced human leukemia HL-60 cell apoptosis. Salvicine treatment resulted in HL-60 cell apoptosis and down-regulation of telomerase activity in a time- and concentration-dependent manner. Repression of telomerase activity preceded a decrease in expression of the telomerase catalytic subunit (hTERT) and telomerase-associated protein (TP1) at the mRNA level, suggesting that the salvicine-induced decrease in telomerase activity may be additionally regulated by mechanisms other than telomerase subunit transcription. We observed that okadaic acid (OA), a protein phosphatase inhibitor, prevented the induction of apoptosis and the down-regulation of telomerase activity by salvicine. The significant increase in protein phosphatase 2A (PP2A) activity induced by salvicine treatment was blocked completely by OA. Moreover, although salvicine induced HL-60 cell apoptosis in a caspase-3-dependent manner, a specific caspase-3 inhibitor, Z-DEVD-FMK, did not prevent a decrease in telomerase activity or an increase in PP2A activity in apoptotic HL-60 cells, ruling out a role for caspase-3 in PP2A activation by salvicine. The results collectively suggest that the salvicine-induced decline in telomerase activity is not a consequence of HL-60 cell apoptosis and that it may be caused principally by the dephosphorylation of telomerase components mediated by PP2A activation.

Telomere length and telomerase activity in the process of human fibroblast immortalization.

To examine the telomere maintenance mechanism in the process of human fibroblast immortalization, we studied telomere length, telomerase activity, chromosome instability, and minisatellite alterations in human fibroblasts following the introduction of the human papillomavirus type 16 E6 gene or E7 gene, or both. One cell line immortalized by E6 alone maintained short telomeres, and its telomerase activity was positive. Fairly long and heterogeneous telomeres were maintained in all four E7-immortalized cell lines lacking telomerase activity. Of the three clones immortalized by both E6 and E7, two cell lines with telomerase activity maintained short telomeres, and the other cell line, which lacked telomerase activity, maintained long and heterogeneous telomeres. Although all immortal cell lines expressed mRNAs for human telomerase RNA component and telomerase-associated protein, expression of mRNA for human telomerase reverse transcriptase was detected only in the telomerase-positive cell lines. All immortal cell lines showed both chromosomal abnormalities, including structural and numerical changes, and minisatellite alterations detected by DNA fingerprinting. These findings indicate the existence of different telomere maintenance mechanisms in telomerase-positive and -negative fibroblast cell lines immortalized by E6, E7, or E6/ E7, and the possible involvement of chromosome instability and minisatellite alterations in the activation of the telomere maintenance mechanisms.

The Product of the Survival of Motor Neuron(SMN) Gene is a Human Telomerase-associated Protein

Telomerase is a ribonucleoprotein (RNP) complex that is minimally composed of a protein catalytic subunit, the telomerase reverse transcriptase (TERT), and an RNA component, the telomerase RNA. The survival of motor neuron (SMN) gene codes for a protein involved in the biogenesis of certain RNPs. Here, we report that SMN is a telomerase-associated protein. Using in vitro binding assays and immunoprecipitation experiments, we demonstrate an association between SMN and the telomerase RNP in vitro and in human cells. The specific immunopurification of SMN from human 293 cells copurified telomerase activity, suggesting that SMN associates with a subset of the functional telomerase holoenzyme. Our results also indicate that the human telomerase RNA and the human (h) TERT are not associated with Sm proteins, in contrast to Saccharomyces cerevisiae telomerase. Immunofluorescence analysis showed that hTERT does not specifically colocalize with wild-type SMN in gems or Cajal bodies. However, a dominant-negative mutant of SMN (SMNDeltaN27) previously characterized to elicit the cellular reorganization of small nuclear RNPs caused the accumulation of hTERT in specific SMNDeltaN27-induced cellular bodies. Furthermore, coexpression of SMNDeltaN27 and hTERT in rabbit reticulocyte lysates decreased the efficiency of human telomerase reconstitution in vitro. Our results establish SMN as a novel telomerase-associated protein that is likely to function in human telomerase biogenesis.