Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT.

The maintenance of chromosome termini, or telomeres, requires the action of the enzyme telomerase, as conventional DNA polymerases cannot fully replicate the ends of linear molecules. Telomerase is expressed and telomere length is maintained in human germ cells and the great majority of primary human tumours. However, telomerase is not detectable in most normal somatic cells; this corresponds to the gradual telomere loss observed with each cell division. It has been proposed that telomere erosion eventually signals entry into senescence or cell crisis and that activation of telomerase is usually required for immortal cell proliferation. In addition to the human telomerase RNA component (hTR; ref. 11), TR1/TLP1 (refs 12, 13), a protein that is homologous to the p80 protein associated with the Tetrahymena enzyme, has been identified in humans. More recently, the human telomerase reverse transcriptase (hTRT; refs 15, 16), which is homologous to the reverse transcriptase (RT)-like proteins associated with the Euplotes aediculatus (Ea_p123), Saccharomyces cerevisiae (Est2p) and Schizosaccharomyces pombe (5pTrt1) telomerases, has been reported to be a telomerase protein subunit. A catalytic function has been demonstrated for Est2p in the RT-like class but not for p80 or its homologues. We now report that in vitro transcription and translation of hTRT when co-synthesized or mixed with hTR reconstitutes telomerase activity that exhibits enzymatic properties like those of the native enzyme. Single amino-acid changes in conserved telomerase-specific and RT motifs reduce or abolish activity, providing direct evidence that hTRT is the catalytic protein component of telomerase. Normal human diploid cells transiently expressing hTRT possessed telomerase activity, demonstrating that hTRT is the limiting component necessary for restoration of telomerase activity in these cells. The ability to reconstitute telomerase permits further analysis of its biochemical and biological roles in cell aging and carcinogenesis.

Specific association of human telomerase activity with immortal cells and cancer.

Synthesis of DNA at chromosome ends by telomerase may be necessary for indefinite proliferation of human cells. A highly sensitive assay for measuring telomerase activity was developed. In cultured cells representing 18 different human tissues, 98 of 100 immortal and none of 22 mortal populations were positive for telomerase. Similarly, 90 of 101 biopsies representing 12 human tumor types and none of 50 normal somatic tissues were positive. Normal ovaries and testes were positive, but benign tumors such as fibroids were negative. Thus, telomerase appears to be stringently repressed in normal human somatic tissues but reactivated in cancer, where immortal cells are likely required to maintain tumor growth.

Telomerase activity and survival of patients with node-positive breast cancer.

BACKGROUND: Shortening of telomeres (specialized structures at the ends of chromosomes) beyond a certain length may signal a cell to stop dividing and to enter senescence. A ribonucleoprotein enzyme, telomerase, is a key component in maintaining telomere length. Because the majority of cancers express telomerase but most normal somatic tissues do not, we measured the level of telomerase expression in primary breast cancer specimens for correlation with traditional prognostic indicators and disease outcome. METHODS: Telomerase activity was measured in frozen human breast cancer specimens by use of the Telomeric Repeat Amplification Protocol (TRAP) assay. The level of telomerase activity was expressed as total product generated (TPG) and was corrected for specimen cellularity by expressing it as a ratio of TPG to the sample's 28S ribosomal RNA content. RESULTS: A preliminary study of 150 breast cancer specimens demonstrated that telomerase activity correlated with the fraction of cells in S phase of the cell cycle (r(sp) = .23). In a larger prognostic study of 398 tumors from patients with lymph node-positive breast cancer, telomerase expression correlated with S-phase fraction, progesterone receptor level, DNA ploidy, and lymph node status. After correcting for sample cellularity, increasing TPG levels were associated with decreased disease-free survival (P = .041) and overall survival (P = .009) of the patients. The telomerase activity level remained strongly predictive of death (P = .027) and marginally predictive of disease recurrence (P = .08) after adjustment for other prognostic factors. All P values are two-sided. CONCLUSIONS: Telomerase activity in human breast cancers is associated with a more aggressive tumor phenotype in patients.

Telomerase activity and prognosis in primary breast cancers.

PURPOSE: Recent studies associate telomerase activity with prognostic factors and survival. We compared quantitative telomerase activity in primary tumors with traditional prognostic factors and outcome in a group of invasive but nonmetastatic breast cancers. PATIENTS AND METHODS: Telomerase activity was measured in 203 invasive breast cancers by the quantitative telomeric repeat amplification protocol method. Telomerase expression was compared with 28S rRNA level, tumor content, and clinical variables, including outcome. For clinical correlations, telomerase activity was standardized by two methods: (1) a correction for cellularity using 28S rRNA levels, and (2) a correction for the histologically determined invasive proportion of the specimen. RESULTS: Telomerase activity was found in 82% of breast cancers with measurable 28S rRNA levels. Telomerase activity was associated with the proliferative index (P <.01) of the tumor but not with any other prognostic variable. Neither uncorrected nor corrected telomerase activity was associated with relapse-free or overall survival in this study. CONCLUSION: Telomerase activity level was associated with the proliferative index of invasive breast cancers, but its measurement in samples from this group of nonmetastatic breast cancer patients did not predict survival.

Complete sequences and organization of the rrnA operon from campylobacter jejuni TGH9011 (ATCC43431).

The rrnA ribosomal RNA (rRNA) operon of Campylobacter jejuni (Cj) TGH9011 (ATCC43431) was cloned and sequenced to completion. rRNAs were then characterized by primer extension and S1 nuclease mapping analysis. The secondary structure models of Cj 16S and 23S rRNAs were constructed, and the models were compared to the corresponding models from other eubacterial rRNA. The analysis presented a typical 5'-promoter-16S-tRNAs-23S-5S-terminator-3' prokaryotic rRNA operon structure. However, an unusual organization of the intercistronic tRNAs was observed where the two tRNAs, tRNA(Ala) and tRNA(Ile), were present in the order 5'-16S-tRNA(Ala)-tRNA(Ile)-23S-3', which is opposite of the typical 5'-16S-tRNA(Ile)-tRNA(Ala)-23S-3' structure observed in other bacteria.

Clinical implications of telomerase in cancer.

Cellular immortality is believed to be a critical step in tumorigenesis. As an important component of the telomere maintenance mechanism, the activation of the enzyme telomerase is tightly associated with cellular immortality and cancer. Telomerase expression is detected in a majority of tumours, but is absent in most somatic tissues and correlates to clinical outcome in a number of cancer types. Telomerase expression is associated with the stage of differentiation but not necessarily with the rate of cell proliferation. Data also indicate that inhibition or absence of telomerase may result in cell crisis in cancer cells and tumour regression in cancer patients. These results suggest that cancer therapy based on telomerase inhibition could be a more effective and safer treatment for cancer, as well as provide a more accurate means for diagnosing and predicting clinical outcome in cancer. Complete understanding of the role of telomerase in tumorigenesis through well-designed clinical studies will have a significant clinical impact on the treatment and diagnosis of cancer.

Telomerase expression in primary neuroblastomas.

Maintenance of chromosomal telomeres is necessary for continued cell growth, and this is carried out in germline tissues by telomerase. In contrast to most somatic tissues, many tumours have telomerase activity. The RNA component of human telomerase (hTR) was measured by Northern analyses of 150 primary untreated neuroblastomas and compared with clinical stage at diagnosis. hTR expression > 33 (relative to cell line control = 100) was seen in 41% of all tumours and the frequency of hTR > 33 increased with stage of disease. Expression of hTR may be involved in progression of neuroblastoma.

Localization of telomerase hTERT protein and survivin in placenta: relation to placental development and hydatidiform mole.

OBJECTIVE: To find if a difference in telomerase or survivin expression exists between non-neoplastic tissues and hydatidiform moles, and explore expression of those proteins in normal placental development, post-term gestation, and preeclampsia. METHODS: Formalin-fixed placental tissues were selected from collections of the Department of Pathology at the University of Colorado. Five specimens of each trimester, five each of preeclamptic and post-term placentas, and 23 molar pregnancies were selected. The telomerase catalytic protein hTERT was localized in placental tissues using the catalyzed signal amplification system, and survivin was localized by conventional immunoperoxidase method. Staining was graded on a scale of zero to 4. RESULTS: hTERT staining was detected in sections of 42 of 48 specimens (23 of 23 hydatidiform moles, 19 of 25 non-neoplastic placental tissues). The intensity of staining for hTERT was higher in hydatidiform moles (mean 3.3, median 3) compared with levels in non-neoplastic placental tissues (mean 0.92, median 1) (P <.001). Survivin was detected in 39 of 48 specimens (22 of 23 hydatidiform moles, 17 of 25 non-neoplastic placental tissues). Compared with non-neoplastic tissues (mean 0.88, median 1), survivin levels were elevated in hydatidiform moles (mean 1.35, median 1) (P =.031). CONCLUSION: Survivin and telomerase were increased in hydatidiform moles, suggesting that regulation of apoptosis and stabilization of telomere length might be involved in neoplastic transformation of the placenta. The patterns of expression observed for survivin and telomerase in non-neoplastic placental tissues suggest that the control of apoptosis and stabilization of telomeric DNA might also be involved in normal gestational development.

Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP).

The telomeric repeat amplification protocol (TRAP) assay has been used to test telomerase activity in numerous cancer specimens. We describe primers, controls and quantification methods for the TRAP assay to accurately measure the level of telomerase activity in clinical samples. The assay is reliable and reproducible in routine analyses and can be used to estimate the processivity of telomerase activity.

Telomerase activity modulation in the prevention of prostate cancer.

Telomerase is a ribonucleoprotein that stabilizes chromosomes by maintaining their telomeric ends. Although telomerase is normally expressed in reproductive tissues, it is virtually absent in most normal somatic tissues. During carcinogenesis, cells activate telomerase to protect chromosomal ends from the telomere erosion that occurs with replication. Prevention of telomere loss by activation of telomerase allows for the cellular immortalization that is a characteristic of cancer cells. Recent studies have shown that genetic instability arising from critical telomere shortening is a mechanism through which cancer cells attain multiple genetic aberrations that characterize a malignant clone. Thus, the timing of telomerase activation during carcinogenesis is likely to play an important role in modulating the genetic instability that determines the malignant phenotype. Earlier activation of telomerase should minimize genetic aberrations in neoplastic cells and lead to less aggressive tumors, or may prevent carcinogenesis. In this article, we discuss recent data on telomerase expression in prostate cancer, propose a model that relates the dynamics of telomerase activation to the evolution of different prostatic malignancies, and discuss the potential application of telomerase activation as a strategy for the prevention of prostate cancer.

Telomerase and cancer.

The data reviewed here suggest that telomere dynamics and telomerase expression are fundamentally involved in cellular aging and cancer. Of particular importance is the stabilization of telomeres by activation of telomerase and the association of this process with cell immortality and human malignancies. Thus, we believe that cell immortalization is required for long-term growth of the vast majority of malignant or metastatic tumors and that advances in telomere biology and telomerase inhibition will improve the way cancers are diagnosed and treated. We look forward to the clinical evaluation of these bold predictions.

Mechanism of telomerase induction during T cell activation.

The progressive shortening of the ends of chromosomes (telomeres) during cell division may serve as a mitotic clock for replicative senescence. Telomerase, a ribonucleoprotein which synthesizes telomeric DNA and maintains telomere length, is absent from most normal somatic cells but is expressed in immortal cells. Low levels of telomerase activity have been detected in peripheral blood mononuclear cells (PBMC) and hematopoietic cells and an increase in telomerase activity during T cell activation has recently been reported. In this study, we show that the increase in telomerase activity during T cell activation was transient and did not prevent the loss of telomeres in long-term T cell cultures. Analysis of the mechanism of telomerase induction showed that the increase in telomerase activity was accompanied by an increase in the levels of hTR, the RNA component of human telomerase. Moreover, telomerase induction occurred in the presence of aphidicolin, indicating that DNA synthesis was not required. Increased telomerase expression was observed when PBMC were activated with phorbol myristate acetate (PMA) and ionomycin, indicating that it was independent of early transmembrane signals. It was, however, linked to the T cell signal transduction pathway, as inhibiting protein kinase C with bisindolylmaleimide prevented the increase in telomerase activity.

Fine mapping of the three rRNA operons on the updated genomic map of Campylobacter jejuni TGH9011 (ATCC 43431).

The three rRNA gene loci of Campylobacter jejuni TGH9011 (ATCC 43431) were cloned. All three rRNA operons were shown to possess a contiguous 16S-23S structure and contain intercistronic tRNA(Ala) and tRNA(Ile). The three RNA operons and additional 14 genetic markers were mapped in the updated genomic map of C. jejuni TGH9011, which now has a total of 24 genetic markers.

Physical map of Campylobacter jejuni TGH9011 and localization of 10 genetic markers by use of pulsed-field gel electrophoresis.

The physical map of Campylobacter jejuni TGH9011 (ATCC 43430) was constructed by mapping the three restriction enzyme sites SacII (CCGCGG), SalI (GTCGAC), and SmaI (CCCGGG) on the genome of C. jejuni by using pulsed-field gel electrophoresis and Southern hybridization. A total of 25 restriction enzyme sites were mapped onto the C. jejuni chromosome. The size of the genome was reevaluated and was shown to be 1,812.5 kb. Ten C. jejuni genetic markers that have been isolated in our laboratory were mapped to specific restriction enzyme fragments. Furthermore, we have accurately mapped one of the three rRNA operons (rrnA) and have demonstrated a separation of the 16S and 23S rRNA-encoding sequences in one of the rRNA operons.

A secreted DNA-binding protein that is translated through an internal ribosome entry site (IRES) and distributed in a discrete pattern in the central nervous system.

Internal initiation of translation, a mechanism infrequently used by cellular messages, avoids the requirement of a methyl cap structure for translation of messenger RNAs. The mRNA transcript encoding the DNA-binding protein MYT2 represents one of the exceptional cellular messages that contains an internal ribosome entry site (IRES). The RNA pseudoknot structure located in the 5' untranslated region of MYT2 functions to promote translation in vivo. MYT2 was cloned by its specific binding to a TTCCA motif in the promoter region of a glial-specific gene, myelin proteolipid protein. MYT2 also recognizes single-stranded nucleic acids. In the central nervous system, MYT2 protein is found in oligodendrocyte progenitor cells, subsets of neurons, and cells of the choroid plexus together with ciliated ependymal cells. MYT2 protein can also be secreted from cells, an atypical event for a DNA-binding protein. The presence of an internal ribosome entry site in MYT2, together with the unusual localization of MYT2, suggests that this nucleic acid-binding protein may be in the class of proteins involved in cellular growth control and survival in the nervous system.

Differential expression of telomerase activity in hematopoietic progenitors from adult human bone marrow.

The loss of telomeric DNA may serve as a mitotic clock which signals cell senescence and exit from cell cycle. Telomerase, and enzyme which synthesizes telomeric repeats de novo, is required to maintain telomere lengths. In humans, significant telomerase activity has been found in cells with essentially unlimited replicative potential such as reproductive cells in ovaries and testes, immortal cell lines and cancer tissues, but not in most normal somatic cells or tissues. We have now examined telomerase expression in subpopulations of hematopoietic cells from adult human bone marrow using a sensitive polymerase chain reaction-based telomeric repeat amplification protocol. Telomerase activity was found at low levels in the highly enriched primitive hematopoietic cells (CD34+CD71loCD45RAlo) and was increased transiently when these cells were cultured in the presence of a mixture of cytokines. In contrast, the early progenitors (CD34+CD71+) expressed telomerase activity at a higher level which was subsequently downregulated in response to cytokines. Telomerase activity remained low in the more mature CD34-cells upon exposure to cytokines. Taken together, our results suggest that telomerase is expressed at a basal level in all hematopoietic cell populations examined, is induced in a primitive subset of hematopoietic progenitor cells and is downregulated upon further proliferation and differentiation of these cells. We have previously observed telomere shortening in cytokine-stimulated primitive hematopoietic cells. The low and transient activation of telomerase activity described here thus appears insufficient to maintain telomere lengths in cultured hematopoietic cells.

Telomerase activity by TRAP assay and telomerase RNA (hTR) expression are predictive of outcome in neuroblastoma.

BACKGROUND: Recent studies have associated telomerase with prognostic factors and survival in neuroblastoma. PROCEDURE: We examined telomerase activity by telomere repeat amplification protocol (TRAP) and expression of the RNA component of telomerase (hTR) by Northern blotting in 106 primary neuroblastoma tumors and 22 established cell lines. RESULTS: Overall survival at 5 years for all 106 tumors was significantly better for patients with undetectable TRAP (75% vs. 59%; P = 0.03) or low hTR expression (84% vs. 43%; P < 0.0001), and especially for patients whose tumors had both low hTR expression and undetectable TRAP (all patients, 91% vs. 54%, P = 0.0002; for 17 stage IV-S tumors, 100% vs. 72%, P = 0.04). Strong expression of hTR was seen in 22 cell lines from aggressive tumors, and all maintained telomere length, but 3/22 were TRAP negative. CONCLUSIONS: These data suggest that both hTR expression and telomerase activity via the TRAP assay should be performed concurrently to predict survival in neuroblastoma patients, particularly in stage 4-S.

Phylogenetic and molecular characterization of a 23S rRNA gene positions the genus Campylobacter in the epsilon subdivision of the Proteobacteria and shows that the presence of transcribed spacers is common in Campylobacter spp.

The nucleotide sequence of a 23S rRNA gene of Campylobacter coli VC167 was determined. The primary sequence of the C. coli 23S rRNA was deduced, and a secondary-structure model was constructed. Comparison with Escherichia coli 23S rRNA showed a major difference in the C. coli rRNA at approximately position 1170 (E. coli numbering) in the form of an extra sequence block approximately 147 bp long. PCR analysis of 31 other strains of C. coli and C. jejuni showed that 69% carried a transcribed spacer of either ca. 147 or ca. 37 bp. Comparison of all sequenced Campylobacter transcribed spacers showed that the Campylobacter inserts were related in sequence and percent G+C content. All Campylobacter strains carrying transcribed spacers in their 23S rRNA genes produced fragmented 23S rRNAs. Other strains which produced unfragmented 23S rRNAs did not appear to carry transcribed spacers at this position in their 23S rRNA genes. At the 1850 region (E. coli numbering), Campylobacter 23S rRNA displayed a base pairing signature most like that of the beta and gamma subdivisions of the class Proteobacteria, but in the 270 region, Campylobacter 23S rRNA displayed a helix signature which distinguished it from the alpha, beta, and gamma subdivisions. Phylogenetic analysis comparing C. coli VC167 23S rRNA and a C. jejuni TGH9011 (ATCC 43431) 23S rRNA with 53 other completely sequenced (eu)bacterial 23S rRNAs showed that the two campylobacters form a sister group to the alpha, beta, and gamma proteobacterial 23S rRNAs, a positioning consistent with the idea that the genus Campylobacter belongs to the epsilon subdivision of the class Proteobacteria.