Search

The transition from low grade astrocytoma to glioblastoma multiforme is almost always accompanied by the loss of genetic markers from chromosome 10. Recently two genes, PTEN/MMAC1/TEP1 and DMBT, have been isolated from chromosome 10q. We have analysed these two genes for mutations in 21 primary glioblastomas. An exon by exon screen of the PTEN gene using SSCP failed to identify any mutations in this tumour series. In contrast, 38% of tumours showed intragenic homozygous deletions in the DMBT gene. The fact that the majority of gliomas do not carry mutations in either of these genes suggests that there may still be other genes on chromosome 10 which are important in the development of glioblastoma multiforme.

Telomerase is a specialized type of reverse transcriptase which catalyzes the synthesis and extension of telomeric DNA (for review, see ref.1). This enzyme is highly active in most cancer cells, but is inactive in most somatic cells2. This striking observation led to the suggestion that telomerase might be important for the continued growth3 or progression4 of cancer cells. However, little is known about the molecular mechanism of telomerase activation in cancer cells. Human telomerase reverse transcriptase (hTRT) has recently been identified as a putative human telomerase catalytic subunit5,6. We transfected the gene encoding hTRT into telomerase-negative human normal fibroblast cells and demonstrated that expression of wild-type hTRT induces telomerase activity, whereas hTRT mutants containing mutations in regions conserved among other reverse transcriptases did not. Hepatocellular carcinoma (2O samples) and non-cancerous liver tissues (19 samples) were examined for telomerase activity and expression of hTRT, the human telomerase RNA component (hTR; encoded by TER)7 and the human telomerase-associated protein (HTLP1; encoded by 7EP7)8,9. A significant correlation between hTRT expression and telomerase activity was observed. These results indicate that the hTRT protein is the catalytic subunit of human telomerase, and that it plays a key role in the activation of telomerase in cancer cells.

The chromosomal locations of the human TEP1 (telomerase protein component 1) and mouse Tep1 genes, which were originally named TLP1 (telomerase protein 1) or TP1 (telomerase-associated protein 1), were determined by direct R-banding FISH and a molecular linkage analysis with interspecific backcross mice. The human TEP1 and mouse Tep1 genes were mapped by FISH to human chromosome 14q11.2 and to the C2-D1 band of mouse chromosome 14, respectively. By means of genetic linkage mapping, the mouse gene was further localized as being 2.7 cM distal toD14Mit18andD14Mit134and 2.0 cM proximal toD14Mit5on mouse chromosome 14, where conserved linkage homology with human chromosome 14q11–q12 has been identified.

The maintenance of chromosome termini, or telomeres, requires the action of the enzyme telomerase, as conventional DNA poly-merases cannot fully replicate the ends of linear molecules1–4. Telomerase is expressed and telomere length is maintained in human germ cells and the great majority of primary human tumours5,6. However, telomerase is not detectable in most normal somatic cells; this corresponds to the gradual telomere loss observed with each cell division7–9. 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 proliferation10. In addition to the human telomerase RNA component (hTR; ref. 11), TP1/TLP1 (refs 12,13), a protein that is homologous to the p80 protein associated with the Tetrahymena enzyme14, 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 (SpTrt!) telomerases15,17, 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 homologues17,18. 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.

Catalytic protein subunits of telomerase from the ciliate Euplotes aediculatus and the yeast Saccharomyces cerevisiae contain reverse transcriptase motifs. Here the homologous genes from the fission yeast Schizosaccharomyces pombe and human are identified. Disruption of the S. pombe gene resulted in telomere shortening and senescence, and expression of mRNA from the human gene correlated with telomerase activity in cell lines. Sequence comparisons placed the telomerase proteins in the reverse transcriptase family but revealed hallmarks that distinguish them from retroviral and retrotransposon relatives. Thus, the proposed telomerase catalytic subunits are phylogenetically conserved and represent a deep branch in the evolution of reverse transcriptases.

Research on the association of the ribonucleoprotein enzyme, telomerase, with human cancer has expanded rapidly in recent years. Essentially all major types of cancer have been screened and the presence of telomerase activity has been detected in the vast majority of cases. In this article we provide a summary, in table form, of the current data.

The telomerase ribonucleoprotein catalyzes the addition of new telomeres onto chromosome ends. A gene encoding a mammalian telomerase homolog called TP1 (telomerase-associated protein 1) was identified and cloned. TP1 exhibited extensive amino acid similarity to theTetrahymenatelomerase protein p80 and was shown to interact specifically with mammalian telomerase RNA. Antiserum to TP1 immunoprecipitated telomerase activity from cell extracts, suggesting that TP1 is associated with telomerase in vivo. The identification of TP1 suggests that telomerase-associated proteins are conserved from ciliates to humans.

Eukaryotic chromosomes are capped with repetitive telomere sequences that protect the ends from damage and rearrangements. Telomere repeats are synthesized by telomerase, a ribonucleic acid (RNA)-protein complex. Here, the cloning of the RNA component of human telomerase, termed hTR, is described. The template region of hTR encompasses 11 nucleotides (5'-CUAACCCUAAC) complementary to the human telomere sequence (TTAGGG)n. Germline tissues and tumor cell lines expressed more hTR than normal somatic cells and tissues, which have no detectable telomerase activity. Human cell lines that expressed hTR mutated in the template region generated the predicted mutant telomerase activity. HeLa cells transfected with an antisense hTR lost telomeric DNA and began to die after 23 to 26 doublings. Thus, human telomerase is a critical enzyme for the long-term proliferation of immortal tumor cells.

Telomerase is a ribonucleoprotein DNA polymerase that catalyzes the de novo synthesis of telomeric simple sequence repeats. We describe the purification of telomerase and the cloning of cDNAs encoding two protein subunits from the ciliate Tetrahymena. Two proteins of 80 and 95 kDa copurified and coimmuno-precipitated with telomerase activity and the previously identified Tetrahymena telomerase RNA. The p95 subunit specifically cross-linked to a radiolabeled telomeric DNA primer, while the p80 subunit specifically bound to radiolabeled telomerase RNA. At the primary sequence level, the two telomerase proteins share only limited homologies with other polymerases and polymerase accessory factors.

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.

Evolving data demonstrate the pathogenetic significance of chromosomal ends telomeres and telomerase activity in the molecular pathogenesis of many hematological disorders. Furthermore, the presence of eroded telomeres and enhanced telomerase activity in hematopoietic cells has been associated with poor prognosis both in myeloid and lymphoid malignancies. The aim of the present study was to evaluate telomere length and telomerase activity in patients with Ph1-negative Chronic Myeloproliferative Disorders (Ph−-CMPD) either at diagnosis or during the course of the disease and to assess their possible clinical utility. Sixty-six bone marrow and 60 peripheral blood samples were obtained from 80 Ph−-CMPD patients (aged 58.57±16.42 years) and 18 healthy age-matched controls (aged 53.94±15.16 years). Thirty-six patients diagnosed suffering from Polycythemia Vera, 36 from Essential Thombocythemia, 4 from Idiopathic Myelofibrosis and 4 from Unclassified CMPD. Twenty-six samples were studied at diagnosis, whereas 54 during the course of the disease. Telomere length analysis of individual chromosome ends was performed on bone marrow metaphases using Telomere/Centromere Quantitative-Fluorescence In Situ Hybridization (T/C Q-FISH) (Dako A/S, Denmark). Telomerase activity was determined in bone marrow purified CD34(+) and CD20(+) cells as well as in peripheral blood CD3(+) T-lymphocytes and granulocytes with the PCR-based Telomeric Repeat Amplification Protocol (TRAP) assay (Roche, Germany). Gene expression of telomerase-associated proteins (hTERT, hTER, TEP1, TRF-1 and TRF-2) was assayed by Real-Time Multiplex PCR (Maximbio, USA). Ph−-CMPD patients showed significantly more eroded telomeres (P=0.010) and increased telomerase activity in CD34(+) cells (P=0.005) compared to healthy age-matched individuals. However, there was no statistical difference in telomere length (P=0.451) and enzyme activity (P=0.538) among different groups of Ph−-CMPD. Telomerase activity was not detected in the remaining hematopoietic cells both in patients and healthy controls, which was closely correlated with downregulation in hTERT mRNA expression. hTER, TEP1, TRF-1 and TRF-2 showed no apparent differential expression of mRNA in all hematopoietic cell fractions. Chromosomal aberrations (+8, +9, del13q14, del20q12) were found by FISH in 37% Ph−-CMPD patients with reduced telomere lengths (P=0.001) and enhanced telomerase activity (P=0.014), especially during the course of the disease (P=0.028). The patients with shortened telomeres displayed a higher incidence of having thrombotic or hemorrhagic events during follow-up (P=0.011), treatment failure (P=0.024) and disease progression to myelofibrosis, myelodysplastic syndromes, secondary leukemia or death (P=0.137). Nevertheless, telomerase expression was not correlated with the above complications. The event free survival (survival without complications, e.g. myelofibrosis, myelodysplastic syndromes, secondary leukemia and death) was significantly shorter in patients with reduced telomere lengths (Log Rank P=0.033), who demonstrated a 7,71-fold higher probability of having complications within five years from the initial diagnosis (95% CI=2,04–31,49 P

Telomere shortening and increased telomerase activity is associated with disease progression in chronic myeloid leukemia (CML). In order to gain insight into the biology of telomerase and its regulation in CML we have evaluated the expression levels of the components of telomerase, its regulators, and several telomeric associated proteins in the hematopoietic stem cells (HSC)/leukemic blast cells of 21 patients with this disorder, as compared to the HSC population of a group of healthy individuals, using quantitative real time PCR. In this study we have made the surprising observation that hTERT, the catalytic component of telomerase, is down-regulated in the CD34+/blast cells of the majority of CML patients in chronic phase (CP) (7 out of 9, p=0.0339) relative to CD34+ cells from normal controls. Furthermore hTERT was found to be significantly down-regulated in 2 out of 3 patients in accelerated phase (AP) and in 9 out of 9 patients in blast crisis (BC) relative to normal controls (p=0.0003). The expression levels of hTR, the telomerase RNA template, and the telomeric- associated proteins TEP1, TRF1, TRF2 and tankyrase were all up-regulated in the CD34+/blast cells of the majority of the CML patients in CP and AP, relative to normal control CD34+ cells. With the exception of TEP1, the expression levels of all these factors were highest in CP CML and, in the majority of patients, decreased during disease progression. We speculate that the up-regulation of tankyrase (positive regulator of telomere length) and TRF2 (protects critically shortened telomeres) may contribute to telomere maintenance in CML. The expression levels of hTERT and c-myc, a positive regulator of hTERT expression, correlate in all stages of CML (R2=0.623). The expression levels of c-myc decreased with disease progression, falling below the levels observed in normal CD34+ cells in the majority of patients in BC. The expression levels of hTERT were increased in the CD34+ cells of three additional CP CML patients in remission following successful treatment with imatinib, relative to both untreated CP CML patients (p=0.0126) and to normal individuals, reflecting a restoration of haematopoiesis towards normal. In CML the turnover of all types of Ph+ stem and progenitor cells is known to be increased. We suggest that the reduced (and presumably inadequate) expression levels of hTERT in CML CD34+/blast cells, and the consequent telomere shortening with each division, is the direct cause of the shortened telomeres frequently observed in CML patients. These data challenge the widely accepted view that hTERT expression is always increased in tumorigenesis. It is possible that the Bcr-Abl fusion product may play a role in the down-regulation of hTERT observed in the HSC of CML patients in this study.

Background Ribonucleoproteins carry out a variety of important tasks in the cell. In this study we show that a number of these contain a novel module, that we speculate mediates RNA-binding.

The telomerase enzyme of Tetrahymena synthesizes repeats of the telomeric DNA sequence TTGGGG de novo in the absence of added template. The essential RNA component of this ribonucleoprotein enzyme has now been cloned and found to contain the sequence CAACCCCAA, which seems to be the template for the synthesis of TTGGGG repeats.

We have cloned and characterized the rat telomerase protein component 1 gene (TLP1), which is related to the gene for Tetrahymena p80. The cDNA encodes a 2629 amino acid sequence and produces the TLP1 proteins p240 and p230. The anti-TLP1 antibody specifically immunoprecipitated the telomerase activity. Moreover, p240 and p230 were copurified with telomerase activity in a series of extensive purification experiments. These results strongly suggest that the TLP1 proteins are components of, or are closely associated with, the rat telomerase. A pulse-chase experiment showed that p240 is modified to p230 in vivo. p230 was the dominant form in telomerase-positive cells, suggesting that modification of the TLP1 protein may regulate telomerase activity in vivo.