Subunit Human Telomerase Reverse Transcriptase Research Articles

Human arteries engineered in vitro.

There is a pressing need to develop methods to engineer small-calibre arteries for bypass surgery. We hypothesized that the rate-limiting step that has thwarted previous attempts to engineer such vessels from non-neonatal tissues is the limited proliferative capacity of smooth muscle cells (SMCs), which are the main cellular component of these vessels. Ectopic expression of the human telomerase reverse transcriptase subunit (hTERT) has been shown recently to extend the lifespan of certain human cells. We therefore introduced hTERT into human SMCs and found that the resulting cells proliferated far beyond their normal lifespan but retained characteristics of normal control SMCs. Importantly, using these non-neonatal SMCs, we were able to engineer mechanically robust human vessels, a crucial step towards creating arteries of clinical value for bypass surgery.

Elevated telomerase activity, c-MYC-, and hTERT mRNA expression: association with tumour progression in malignant lipomatous tumours.

The role of telomerase activity in tumour progression of liposarcomas is not well understood. Therefore, we investigated 72 liposarcomas of different histological subtypes for an association between telomeric lengths and telomerase activity, and assessed the association between the catalytic subunit human telomerase reverse transcriptase (hTERT) and its activator c-MYC. Telomeric repeat fragment lengths were determined using radioactive DNA-fingerprint analysis with the telomere-specific probe (TTAGGG)(3), whereas telomerase activity was ascertained using the non-radioactive TRAP-assay. To evaluate the expression of hTERT and c-MYC, we applied real-time RT-PCR using a LightCycler. Eight tumours were investigated by microdissection. The MIB1-proliferation index and hTERT and c-MYC protein expression were determined immunohistochemically. Genetic alterations showed a high degree of tumour specificity. Highly malignant myxoid/round cell liposarcomas showed the longest telomeres, the strongest telomerase activity, and the highest hTERT and c-MYC expression levels compared with the pure myxoid variants (p < 0.001), which are of low malignancy. Pleomorphic liposarcoma was characterized by zero or low hTERT and c-MYC expression and telomerase activity, but long telomeres, underlining their different pathogenetic pathway. Elevated gene expression was accompanied by protein immunopositivity. MIB1-proliferation index did not correlate with other molecular markers. We conclude that hTERT and c-MYC expression are associated with telomerase activity in liposarcomas. Elevated hTERT and c-MYC expression as well as high telomerase activity play a role in the tumour progression of this sarcoma type. Nevertheless, each histological subtype of liposarcomas is defined by a specific molecular pattern. Telomerase activation is the most common pathway in liposarcomas maintaining telomeric length.

Opposing regulatory roles of E2F in human telomerase reverse transcriptase (hTERT) gene expression in human tumor and normal somatic cells.

Telomerase activity is closely correlated with cellular proliferative activity in human tissues. Human cells with high proliferative potential, such as tumor cells or stem cells, exhibit telomerase activity, whereas most normal human somatic cells do not. Telomerase activity is tightly regulated by the expression of its catalytic subunit human telomerase reverse transcriptase (hTERT). Through an expression cloning approach, we identified E2F-1 as a repressor of the hTERT gene in human tumor cells. Ectopic expression of E2F-1 repressed hTERT promoter activity by inhibiting Sp1 activation of the hTERT promoter. In contrast to the repressor function of E2F-1 in human tumor cells, we demonstrated that E2F-1 is an activator of the hTERT gene in normal human somatic cells. Ectopically expressed E2F-1 activated the hTERT promoter through a noncanonical DNA binding site. E2F-1, E2F-2, and E2F-3 (but not E2F-4 and E2F-5) repressed hTERT promoter activity in human tumor cells, whereas they activated it in normal somatic cells. These contrasting effects of E2F transcription factors on the hTERT promoter could underlie the paradoxical biological activities of E2F, which can both promote and inhibit cellular proliferation and tumorigenesis.

Regulation of Telomerase Activity by Alternate Splicing of Human Telomerase Reverse Transcriptase mRNA in a Subset of Neuroblastomas

It has been proposed that the regulation of telomerase takes place at the transcriptional level, the expression of the catalytic subunit human telomerase reverse transcriptase (hTERT) being crucial for telomerase activity (TA). Recently, differential splicing of hTERT mRNA has been demonstrated in various tissues during embryonal development, and it has been suggested that only full-length transcripts translate into functionally active telomerase. With this in view, we analyzed the different hTERT transcripts by reverse transcriptase-polymerase chain reaction in neuroblastic tumors and compared the results with the TA, the tumor growth fraction, and the MYCN status. In a series of 38 neuroblastic tumors, high TA and full-length hTERT transcripts were found in nine samples, whereas nine samples showed absence of both enzymatic activity and hTERT transcripts. Interestingly, in another eight samples, low or absent TA coincided with a lack of full-length hTERT transcripts. Eleven samples contained hTERT transcripts with low or undetectable TA and one sample had low TA but no hTERT transcripts. TA correlated with MYCN amplification and was weakly associated with the proliferative activity. Moreover, a significant correlation with tumor progression was observed. Our findings point at a posttranscriptional regulation of TA in a subset of neuroblastic tumors. Because high TA was detected only in tumors with full-length hTERT transcripts, reverse transcriptase-polymerase chain reaction analysis of archival neuroblastic tumor samples might help to appraise the malignant potential in individual cases.

Regulation of telomerase activity by recombinant human tumor necrosis factor in vitro

OBJECTIVE: To investigate the effect of recombinant human tumor necrosis factor (rhTNF) on telomerase activity and promoter expression of catalytic protein subunit–human telomerase reverse transcriptase (hTERT) in hepatoma cell lines HepG2 cells and HepG1‐6. METHODS: The tartrate‐resistant acid phosphatase– enzyme‐linked immunosorbent assay method (TRAP‐ELISA) was used to determine telomerase activity in HepG2 and HepG1‐6 cells that had been treated with different concentrations of rhTNF. The plasmid TERTluc(800), which contained 800 bp of the hTERT promoter, was transiently transfected into HepG2 cells by using Lipofect, and different concentrations of rhTNF were added into the culture media 2 h later. The activity of the hTERT promoter was tested 48 h after transfection. RESULTS: The telomerase activity of hepatoma cells was suppressed by rhTNF in a dose‐dependent manner. Furthermore, the transient transfection of HepG2 cells with the TERTluc(800) plasmid revealed that hTERT promoter activity is not affected by 10 IU/mL rhTNF, whereas the expression of the hTERT promoter is inhibited by rhTNF at concentrations ranging from 10 to 1000 IU/mL; the extent of inhibition is linearly related to the rhTNF concentration. The activity of the hTERT promoter in cells treated with 1000 IU/mL of rhTNF decreased to 60.3% of that of the control. No extra suppressive effect was observed when a concentration of 10 000 IU/mL was used. CONCLUSIONS: These results suggest that the antitumor activity of rhTNF may be attributed to its inhibitory effect on hTERT promoter expression. Together with other studies, it may indicate that telomerase is not only involved in carcinogenesis, but is also one of the main targets of anticancer cytokines. Further analysis of the molecular mechanism underlying the regulation of telomerase by TNF and isolation of the related transcription factor may provide new means for cancer gene therapy.

Evidence for telomerase involvement in the angiogenesis of astrocytic tumors: expression of human telomerase reverse transcriptase messenger RNA by vascular endothelial cells.

Evidence from recent in vitro studies indicates that reactivation of telomerase, the enzyme that synthesizes the telomere ends of chromosomes, is a crucial event in the unlimited clonal expansion of endothelial cells that precedes the neoplastic conversion of these cells. It is known that high-grade gliomas express telomerase and that, in these neoplasms, proliferating endothelial cells may undergo transformational changes with development of sarcomatous components within the primitive tumor. To assess whether telomerase is involved in the endothelial cell proliferation that characterizes brain tumor angiogenesis, the authors investigated at the single-cell level the expression of messenger (m)RNA for the human telomerase catalytic subunit human telomerase reverse transcriptase (hTERT) by vascular cells of astrocytic tumors. The in situ hybridization (ISH) method was performed by processing histological sections with specific riboprobes for hTERT and for c-myc, an oncogene that is known to upregulate hTERT. Results of the ISH studies were compared with proliferative activity, as estimated by Ki-67 immunostaining. The expression of hTERT mRNA by vascular endothelial cells was related to the histological grade of the tumor because it was detected in five (29%) of 17 low-grade astrocytomas, nine (56%) of 16 anaplastic astrocytomas, and 19 (100%) of 19 glioblastomas multiforme (GBMs). Expression of c-myc mRNA was strictly correlated with that of hTERT mRNA. In low-grade astrocytomas and anaplastic astrocytomas, a dissociation was noted between hTERT mRNA expression and the proliferation rate of endothelial cells. Conversely, GBMs displayed a significant correlation between the level of hTERT mRNA expression and endothelial cell proliferation. Data from an in vitro assay in which human umbilical vein endothelial cells were stimulated to proliferate by adding vascular endothelial growth factor and an ISH study of newly formed vessels surrounding brain infarcts confirmed that expression of hTERT mRNA does not merely reflect the proliferative status of endothelial cells but represents a specific feature of brain tumor neovascularization. The results of this study are consistent with a role of telomerase in the angiogenesis of astrocytic tumors. Expression of hTERT mRNA by tumor vascular cells is an early event during the progression of astrocytic tumors, which precedes endothelial cell proliferation and may represent a first sign of dedifferentiation. Other than elucidating the mechanisms of tumor angiogenesis, these results encourage research on antitelomerase drugs for the treatment of malignant gliomas.

Analysis of human telomerase reverse transcriptase mRNA (hTERT) expression in myxoid liposarcomas using LightCycler real-time quantitative reverse transcriptase-polymerase chain reaction.

We describe a convenient, nonradioactive reverse transcription--polymerase chain reaktion (RT-PCR) method for the rapid and accurate quantitative detection of the human telomerase catalytic subunit human telomerase reverse transcriptase (hTERT) mRNA. The LightCycler TeloTAGGG hTERT Quantification Kit (Roche Molecular Biochemicals) was designed to be used for the highly sensitive and quantitative detection of hTERT mRNA relative to the house-keeping gene porphobilinogen deaminase (PBGD). As a tumor progression model, we investigated 26 myxoid liposarcomas (11 pure myxoid grade I, 15 myxoid/round cell grade II/III) for the hTERT expression level and compared the results of the new method with former measurements performed in silver-stained polyacrylamide gels. Both methods revealed similar results, with real-time RT-PCR being the more accurate quantification technique, which also saves time and material. Elevated hTERT expression (cut-off ratio x 100 at 1.3) was an indicator of round cell components and hence for tumor progression in myxoid liposarcoma. The new method is capable of differentiating between pure myxoid and myxoid/round cell liposarcomas for hTERT-expression more accurately.

Dedifferentiation of human hepatocellular carcinoma up-regulates telomerase and Ki-67 expression.

Dedifferentiation of human hepatocellular carcinoma (HCC) may influence telomerase activation and Ki-67 expression. Laboratory study using human HCC specimens. University hospital. Twelve patients with HCC with specific morphologic patterns (nodule-in-nodule [n = 4] or confluent multinodular [n = 8] type) and histological heterogeneity and who had undergone curative hepatectomy were studied. Of these, 8 patients had 2 different histological grades of HCC cells distributed at various nodules but within the same tumor; 3 patients, 3 different histological grades; and 1 patient, all 4 different histological grades. Tissue samples were retrieved from each nodule of the tumor and not mixed with one another. A total of 42 cancerous tissues from different distinctive nodules of the 12 patients were taken for telomerase and Ki-67 study, and corresponding noncancerous counterparts (n = 12) served as healthy control samples. Telomerase activity was assayed by the telomerase repeat amplification protocol. Expression of messenger RNA (mRNA) by the human telomerase catalytic subunit human telomerase reverse transcriptase (hTERT) was determined using reverse transcription polymerase chain reaction. The relative telomerase activity and hTERT mRNA in each tissue sample was quantified using densitometry and expressed as a percentage of the standardized HeLa cell line. Immunostaining with anti-Ki-67 antibody was used to detect Ki-67 and was expressed as Ki-67 labeling index. Telomerase activity, hTERT mRNA, and Ki-67 labeling index stratified by different histological gradings in each patient was analyzed. The correlations between telomerase activity and hTERT mRNA and between telomerase activity and expression of Ki-67 were plotted. Telomerase activity increased from more to less differentiated foci of HCC cells in each case (generalized linear model, P<.001). Mean +/- SD expression of hTERT mRNA in 43 cancerous tissue samples, even those 4 with negative telomerase activity, was distinguishable from that of the noncancerous controls (0.84 + 0.23 vs 0.41 + 0.11; t test, P =.008). Telomerase activity was correlated to hTERT mRNA expression (Pearson correlation, r(2) = 0.56; P<.001). The Ki-67 labeling index increased from more to less differentiated foci of HCC in each case (generalized linear model, P<.001). Expression of Ki-67 correlated with telomerase activity within differently graded areas within individual tumors (Pearson correlation, r(2) = 0.38; P<. 001). Using the model of human HCC with histological heterogeneity, we determined that dedifferentiation of human HCC induces telomerase activation and Ki-67 expression.

Telomerase activity and expression of the telomerase catalytic subunit, hTERT, in meningioma progression.

In recent reports, 6 to 19% of meningiomas have been classified as atypical or anaplastic/malignant. Some atypical and anaplastic meningiomas appear to arise from benign tumors by progression. Telomerase activation has recently been associated with malignant progression of human tumors. The authors have investigated a series of benign, atypical, and anaplastic/malignant meningiomas for telomerase activity and expression of the telomerase catalytic subunit human telomerase reverse transcriptase (hTERT). A quantitative telomeric repeat amplification protocol was used to detect telomerase enzyme activity in seven (21%) of 34 benign, but in nine (75%) of 12 atypical and in seven (100%) of seven anaplastic/malignant meningiomas. Very high levels of telomerase activity were observed only in highly aggressive tumors. Messenger (m)RNA expression of the catalytic subunit hTERT was found in 11 (33%) of 33 benign, 12 (92%) of 13 atypical, and all seven anaplastic/malignant tumors. All telomerase-positive lesions were also positive for hTERT mRNA, whereas no telomerase activity was detected in six (21%) of 29 hTERT-positive tumors. This indicates that upregulation of hTERT is the rate-limiting step for telomerase activation in the majority of meningiomas. Expression of telomerase and hTERT was seen in all four tumors with gross brain invasion. All recurrent tumors or meningiomas recurring during follow up expressed hTERT. The results are consistent with a role for telomerase activation during the development of malignancy in meningiomas. Hence, expression of telomerase activity and hTERT might prove to be potentially useful markers for the evaluation of these tumors.

Identification of Two RNA-binding Proteins Associated with Human Telomerase RNA

Telomerase plays a crucial role in telomere maintenance in vivo. To understand telomerase regulation, we have been characterizing components of the enzyme. To date several components of the mammalian telomerase holoenzyme have been identified: the essential RNA component (human telomerase RNA [hTR]), the catalytic subunit human telomerase reverse transcriptase (hTERT), and telomerase-associated protein 1. Here we describe the identification of two new proteins that interact with hTR: hStau and L22. Antisera against both proteins immunoprecipitated hTR, hTERT, and telomerase activity from cell extracts, suggesting that the proteins are associated with telomerase. Both proteins localized to the nucleolus and cytoplasm. Although these proteins are associated with telomerase, we found no evidence of their association with each other or with telomerase-associated protein 1. Both hStau and L22 are more abundant than TERT. This, together with their localization, suggests that they may be associated with other ribonucleoprotein complexes in cells. We propose that these two hTR-associated proteins may play a role in hTR processing, telomerase assembly, or localization in vivo.

Genomic organization and promoter characterization of the gene encoding the human telomerase reverse transcriptase (hTERT).

The enzyme telomerase plays a crucial role in cellular proliferation and tumorigenesis. By adding hexameric repeats to chromosome ends, it prevents telomeric loss and, thus, entry into senescence. Recent data suggest that expression of the human telomerase reverse transcriptase subunit (hTERT) represents the limiting factor for telomerase activity. To gain an insight into the mechanisms regulating hTERT expression, we have determined the complete genomic organization of the hTERT gene and isolated the 5'- and 3'- flanking region. The hTERT gene encompasses more than 37kb and consists of 16 exons. We show that all hTERT insertion and deletion variants described so far most likely result from the usage of alternative splice consensus sequences in intron or exon regions. Furthermore, we identified a new hTERT splice variant. Analysis of the DNA sequence surrounding the putative transcriptional start region revealed a TATA-less promoter located in a CpG island. A promoter fragment spanning the first 1100bp upstream of the initiating ATG start codon exhibited high-level activity in HEK-293 cells. Several consensus binding sites for the transcription factor Sp1 as well as a c-Myc binding site were identified in this promoter region. Altogether, these results provide the basis for more detailed studies on the regulation of telomerase activity in normal and cancer cells, and may lead to the development of new cancer therapies.