Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Telomerase, the reverse transcriptase that maintains telomere DNA, is usually undetectable in adult human tissues, but is positive in embryonic tissues and in cancers. However, in rodents, several organs of normal adult animals express substantial amounts of telomerase activity. To elucidate relevant control mechanisms operating on the tissue-specific expression of telomerase in rodents, we examined the transcriptional regulation of telomerase reverse transcriptase (mTERT) gene in muscle cell differentiation. Reverse transcriptase-polymerase chain reaction analysis showed that the reduction of telomerase activity was caused by the decrease of mTERT mRNA level during myogenesis. Transfections of mTERT promoter showed that the proximal 225-base pair region is the core promoter responsible for basal transcriptional activity and also participates in the reduced transcription after muscle differentiation. Electrophoretic mobility shift assays showed that this region contained the GC-boxes, which bind to Sp1 family proteins, and the E-box, which binds to c-Myc. Furthermore, DNA binding activities of Sp1, Sp3, and c-Myc were down-regulated during myogenesis. These data suggest that Sp1, Sp3, and c-Myc have critical roles of TERT transactivation in mouse, and the lack of these transcription factors cause down-regulation of mTERT gene expression in muscle cells differentiation.
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PMID:Mechanism for the reduction of telomerase expression during muscle cell differentiation. 1127 34

To investigate the regulatory mechanisms of telomerase activity in human melanoma cells, we assessed the enzyme's catalytic activity and the expression of the telomerase subunits, the human telomerase RNA, the human telomerase-associated protein, and the human telomerase reverse transcriptase, in 52 melanoma lesions. Eight normal skin specimens were also studied. Telomerase activity was detected in 84.6% of melanomas, whereas all skin specimens were telomerase negative. Human telomerase-associated protein mRNA and human telomerase RNA were constitutively expressed in all melanoma and skin specimens. Although at a variable level of expression, human telomerase reverse transcriptase mRNA was detected in all but one melanomas, whereas it was never present in skin samples. Reverse transcriptase-polymerase chain reaction experiments were performed using primers within the reverse transcriptase domain of human telomerase reverse transcriptase and revealed the presence of multiple alternatively spliced transcripts in melanoma specimens. Among the 44 telomerase-positive melanomas, one showed the full-length transcript alone whereas in all other specimens a full-length message was present with different combinations of alternatively spliced variants. In these tumors the expression of the full-length transcript was generally equal to or higher than that of the alternatively spliced variants. The ratio full-length transcript to alternatively spliced species ranged from 0.6 to 5.26, with a median value of 1.18. Among the seven telomerase-negative melanomas, one displayed the beta deletion transcript alone, whereas in the remaining six tumors weak expression of the full-length transcript and a more abundant level of alternatively spliced transcripts were found. In these cases human telomerase reverse transcriptase ratio ranged from 0.09 to 1.1, with a median value of 0.40. The results suggest that transcription and alternative splicing of human telomerase reverse transcriptase are regulatory mechanisms controlling telomerase activity in melanoma.
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PMID:Possible regulation of telomerase activity by transcription and alternative splicing of telomerase reverse transcriptase in human melanoma. 1140 73

The objective of this study was to investigate the expression of the catalytic subunit of telomerase, telomerase reverse transcriptase (TRT), and the possible relationship between the TRT expression and poor healing or cancer transformation in radiation-induced chronic human skin ulcer. Rabbit antibody to human TRT and SP immunohistochemical method were used to detect TRT expression in 24 cases of formalin-fixed, paraffin-embedded chronic human skin ulcer tissues induced by radiation, 5 cases of normal skin, 2 of burned skin, and 8 of cancer. The positive rate of TRT expression in chronic radiation ulcers was 58.3% (14/24), of which it was strongly positive in 41.7% cases (10/24) and weakly positive in 16.7% (4/24). TRT expression was 0% in normal (0/5) and burned skin (0/2), and 100% in cancer cases (8/8). The strongly positive expression of TRT was observed almost always in the cytoplasm and nucleus of squamous epithelial cells of the epidermis but it was negative or only weakly positive in the smooth muscle and endothelia of small blood vessels and capillaries, and in fibroblasts. Chronic inflammatory cells, plasmacytes, and lymphocytes were weakly positive for TRT. TRT expression could be involved in the poor healing caused by sclerosis of small blood vessels and lack of granulation tissue and in the cancer transformation of chronic radiation ulcer.
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PMID:Expression of telomerase reverse transcriptase in radiation-induced chronic human skin ulcer. 1193 15

Actively dividing cells show progressive loss of telomeric DNA during successive rounds of replication due to end-replication problem. Telomere shortening has been proposed as a regulatory mechanism that controls the replicative capacity of primary cells before undergoing cellular senescence. In immortal cells including cancer, cellular senescence can be overcome by reactivation of telomerase or by a telomerase-independent mechanism for lengthening telomeres. In this work, we present a novel example of telomere elongation mechanism in a human stomach adenocarcinoma cell line which was selected for resistance to adriamycin. The resistant cell line (MKN/ADR) had long terminal restriction fragments (TRFs) of up to approximately 50 kb, while its parent cell line (MKN-45) had the TRFs, consisting of a smear extending from approximately 4 to approximately 25 kb. The very large TRFs in MKN/ADR cell line were proven to be telomeric by digestion with the exonuclease Bal31. When telomerase activity was examined using the PCR-based telomeric repeat amplification protocol (TRAP) assay, MKN/ADR cell line showed reduced activity to about 10% of that in MKN-45 cell line. The correlation between reduced telomerase activity and mRNA expression of telomerase subunits in MKN/ADR cell line was assessed by the reverse transcriptase-PCR analysis. The level of human telomerase reverse transcriptase (hTERT) mRNA was lower in MKN/ADR cell line than in MKN-45 cell line. This observation correlates with the finding that telomerase activity is reduced about 10-fold in MKN/ADR cell line. Reverse transcriptase-PCR analysis also revealed a close correlation between telomerase-associated protein (TP1) mRNA expression and telomerase activity in MKN/ADR cell line. In contrast, expression levels of human telomerase RNA (hTR) were identical in both MKN/ADR and MKN-45 cell lines. Taken together, these data suggest that telomeres in MKN/ADR cell line may be regulated through a novel mechanism other than telomerase. Although the basis for telomere elongation mechanism in MKN/ADR cell line is not yet understood, the occurrence of alternative mechanism for telomere elongation in drug-resistant cancer cells may have an important implication for use of telomerase inhibitors in human cancer treatment.
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PMID:A novel telomere elongation in an adriamycin-resistant stomach cancer cell line with decreased telomerase activity. 1201 44

Telomerase activity (TA) is increased in human cancers and cell lines and is thought to contribute to their immortality. High TA has been found to correlate with aggressive tumor behavior. The aim of this study was to determine whether increased TA in colorectal carcinoma (CRC) correlates with survival. Formalin-fixed and paraffin-embedded tissue sections from 82 CRC and 6 cases of benign colon with diverticulosis were immunohistochemically stained for telomerase reverse transcriptase (TRT) using the immunoperoxidase method. The percentage of positive nuclei was determined for each case. Survival analysis was performed using the Kaplan-Meier method. TRT immunoreactivity was always nuclear. In normal colonic mucosa, TRT immunoreactivity was detected in the bottom of crypts. However, in normal colon adjacent to CRC, telomerase immunoreactivity was detected throughout the length of the crypts, including the upper third, and frequently in the surface epithelium. Telomerase immunoreactivity in more than 25% of the cancer cell nuclei was associated with significantly poorer patient survival (P = 0.0081). We conclude that increased TA in CRC, as demonstrated by TRT immunostaining, is associated with poorer survival, and that TA is present in normal colonic mucosa and is increased in colonic mucosa near CRC. Additional studies with larger patient samples and multivariate analysis are needed to determine whether TRT expression is an independent prognostic factor in CRC.
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PMID:Immunohistochemical detection of telomerase reverse transcriptase in colorectal adenocarcinoma and benign colonic mucosa. 1219 19

Telomerase reverse transcriptase (TRT) is a tumor-associated antigen expressed in the vast majority of human tumors and is presently one of the most promising target candidates for a therapeutic cancer vaccine. TRT is also expressed at low level in selected tissues and should be considered a self antigen. In the present study we sought to develop cytotoxic T lymphocytes (CTL) responses directed against human (h)TRT peptides with low relative affinity for which the available repertoire is to be preferentially spared from tolerance. This was accomplished by using analogue peptides of hTRT whose relative affinity for the MHC was increased by a targeted (-->Tyr) substitution in position one. By immunizing HLA A2.1 transgenic mice with these analogue peptides, we identified one such low relative affinity peptide (p572) that is endogenously processed and presented by HLA A2.1 in tumor cells, and is recognized by specific CTL. We used the highly immunogenic analogue peptide to successfully induce TRT-specific CTL in cancer patients and normal donors. CTL against p572-lysed human and mouse tumor cells but not activated autologous B cells. This peptide represents, therefore, an important candidate component of a cancer vaccine based on a TRT substrate and validates the strategy of targeting peptides with low affinity for the MHC for cancer immunotherapy.
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PMID:Identification of a human telomerase reverse transcriptase peptide of low affinity for HLA A2.1 that induces cytotoxic T lymphocytes and mediates lysis of tumor cells. 1221 71

To construct the tumor-specific expression vector driven by human telomerase reserve transcriptase gene promoter, we amplified a fragment of the enhanced green fluorescent protein (EGFP) gene from the pEGFP-N1 plasmid and cloned it into the multiple cloning site of the pLNCX vector, then named the recombinant as pLNCX-EGFP. The fragment of human telomerase reverse transcriptase gene promoter (hTERT) was amplified from the human genome with the use of human telomerase reserve transcriptase gene-specific primers and cloned into the pLNCX-EGFP vector, from which the cytomegalovirus promoter had previously been removed through the use of restriction enzymes, in sense orientation relative to the green fluorescent protein coding sequence. Then the expression vector pLNT-EGFP--under the control of the human telomerase reserve transcriptase gene promoter, which contains green fluorescent protein reporter gene-was successfully constructed. To detect the transcriptional activity of the human telomerase reserve transcriptase gene promoter, we conducted transient transfection of this specific expression vector into human lung fibroblast (HLF) cell lines with high telomerase activity and normal human fetal lung fibroblast (WI38) cell lines without telomerase activity. The results of transient transfection showed that the pLNT-EGFP vector strongly expressed the green fluorescent protein reporter gene in telomerase-positive cells but not in telomerase-negative cells.
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PMID:Tumor-specific expression detected with the use of an expression vector driven by human telomerase reverse transcriptase gene promoter. 1561 52

Early detection of disseminated tumor cells in the peripheral blood of patients with early stage gastric cancer could help to improve the outcome after tumor resection. The aim of this study is to evaluate the prognostic significance of tumor-related mRNA for the detection of circulating tumor cells in gastric cancer patients by a reverse-transcriptase polymerase chain reaction (RT-PCR) method. We simultaneously analyzed human telomerase reverse transcriptase (hTERT), cytokeratin-19 (CK-19), cytokeratin-20 (CK-20) and carcinoembryonic antigen (CEA) mRNA (messenger RNA) expression in the peripheral blood of 42 gastric cancer patients and 30 healthy individuals. Additionally, analyses were carried out for the correlation of these four molecular markers with patients' clinicopathologic features, as well as the occurrence of postoperative recurrence/metastasis. Among 42 gastric cancer patients, the prevalence of mRNA for hTERT, CK-19, CK-20, and CEA was 61.9% (26/42), 69% (29/42), 61.9% (26/42), and 78.6% (33/42), respectively. All 30 healthy individuals were negative for hTERT and CEA mRNA, while two were positive for either CK-19 mRNA or CK-20 mRNA. Positive CEA mRNA was significantly correlated with tumor size p=0.008), vessel invasion (p=0.001), depth of tumor invasion (p=0.007), lymph node metastasis (p< 0.001), and TNM stage (p<0.001). In addition, the multivariate logistic regression demonstrated that CEA mRNA expression was an independent and significant predictor for postoperative recurrence/metastasis (p=0.032). Our findings suggest that CEA mRNA may be a more reliable marker than hTERT, CK-19 and CK-20 for the detection of circulating cancer cells in gastric cancer patients' peripheral blood. Patients with positive CEA mRNA expression in peripheral blood have a significantly higher risk of postoperative recurrence/metastasis.
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PMID:Molecular detection of disseminated tumor cells in the peripheral blood of patients with gastric cancer: evaluation of their prognostic significance. 1678 43

Telomerase reverse transcriptase (TRT) is the first bona fide common tumor antigen. While several 9mer peptides of the human TRT have been identified for HLA-A2, little information exists on peptides for the remaining HLA types. Here, we used a multi-step approach to select and characterize a panel of HLA-B7 9mer peptides as candidate immunogens. In sequence, we used algorithm-based predictions, in vivo immunization of HLA-B7 transgenic (Tg) mice, in vitro immunization of human blood lymphocytes from two normal donors and two cancer patients, in vivo processing in HLA-B7 Tg mice and HLA-B7 supertype binding. We found a correlation between the in vivo immunogenicity and the actual HLA-B7 binding avidity of the seven predicted peptides. Furthermore, endogenous processing correlated with in vitro immunogenicity in human PBMC and HLA-B7 supertype binding. Peptide (1123)LPSDFKTIL(1131) (p1123) with the wider spectrum of supertype binding displayed the highest immunogenicity overall and was endogenously processed in several human lymphoblastoid cells. Since no single step of the screening/selection process could substitute for the whole approach, we conclude that the identification of MHC class I-restricted peptides for potential vaccination of cancer patients remains, by and large, an empirical process.
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PMID:Immunogenic HLA-B7-restricted peptides of hTRT. 1707 79

Almost all human malignant tumours exhibit strong telomerase activity, but normal adult tissues, with a few exceptions, do not. hTERT (human telomerase reverse transcriptase) is an essential component of telomerase, and hence it can serve as a parallel sign in the diagnosis and prognosis of cancers. In the present study, we selected a sequence of hTERT containing two antigenic epitopes that have high affinity for HLA-A2 (human leucocyte antigen-A2) as a TAA (tumour-associated antigen) based on a peptide-motif scoring system. The sequence was obtained by reverse-transcriptase PCR and cloned into the Escherichia coli expression vector pGEX-4T-1. The expression product appeared in the form of inclusion bodies. Denatured inclusion-body extract was subjected to SDS/PAGE, and the gel band corresponding to the putative 38 kDa fusion protein (GST-hTERT major tumour-associated antigen) was excised, ground with PBS, mixed with Freund's adjuvant and used to inoculate mice, generating anti-TERT polyclonal antibodies. Western blotting using the leukaemia cell line THP-1 demonstrated that the antibodies were able to detect hTERT expression, implying the potential applicability of the antigenic peptides derived from hTERT as a universal marker in the diagnosis and prognosis of tumours.
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PMID:Characterization of a human telomerase reverse transcriptase sequence containing two antigenic epitopes with high affinity for human leucocyte antigen. 1786 23


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