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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The identification of circulating tumor cells in the peripheral blood of patients with malignant melanoma by detection of melanoma associated protein transcripts using the reverse transcriptase polymerase chain reaction (RT-PCR) technique has been introduced as a noninvasive and sensitive technique for early detection of tumor progression and metastatic disease. An alternative approach is the analysis of S-100 protein in the serum of melanoma patients by a luminoimmunometric assay (LIA). In this study, the sensitivities of RT-PCR and LIA were compared. Seventy-seven blood samples of 59 melanoma patients were analyzed for tyrosinase, Melan-A/MART-1, MAGE-3, gp100, and p97 expression by multimarker RT-PCR; 540 serum samples of 352 melanoma patients were analyzed for S-100 protein concentration by LIA. In stage III 23.8% and in stage IV 37.5% of the samples were positive for at least one marker in multimarker RT-PCR, versus 8.1% and 48.1% of elevated S-100 levels analyzed by LIA, respectively. In a direct comparison, 31 identical samples were analyzed by multimarker RT-PCR and by S-100 LIA. In stage III 18.2% and in stage IV 45% of the samples were positive by multimarker RT-PCR versus 45.5% and 80% by S-100 LIA, respectively. S-100 LIA was more sensitive in detection of metastatic disease in melanoma patients than multimarker RT-PCR and should be evaluated in further studies. RT-PCR might be more useful in the analysis of micrometastases in anatomic compartments other than peripheral blood.
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PMID:Tumor markers in peripheral blood of patients with malignant melanoma: multimarker RT-PCR versus a luminoimmunometric assay for S-100. 1054 77

Microphthalmia (MITF) gene product, a transcription factor of the basic-helix-loop-helix type, is thought to play a role in the regulation of genes encoding the enzymes necessary for melanogenesis. These include tyrosinase, TRP-1 and TRP-2. Melanocyte-specific isoform of microphthalmia, MITF-M, is expressed in normal and malignant melanocytes. The presence of two other isoforms of microphthalmia, MITF-A and MITF-H, which differ from MITF-M in the amino-terminus, was demonstrated also in some non-melanocytic lineages. Here we have analyzed the presence of all three known isoforms of MITF mRNA in a panel of 17 human melanoma cell lines by a reverse transcriptase-polymerase chain reaction using isoform-specific primers. While, as expected, the predominant form in melanoma cell lines was MITF-M, low amounts of MITF-A mRNA was found in almost all melanomas, as well as in most of 20 tumor cell lines of the non-melanocyte origin (lung and colon carcinomas, osteosarcomas and neuroblastomas). The expression of MITF-H was not detected, with a few exceptions, in the tested cell lines. Pax3 transcription factor was reported earlier to regulate positively the melanocyte-specific promoter of the MITF gene. We found here that the Pax 3 mRNA was expressed in all melanoma cell lines, even in those that had repressed the MITF-M and were amelanotic. This suggests that additional factors, besides Pax3, are required for the MITF expression. The MSG1 (melanocyte-specific gene 1), a gene originally isolated from melanocytes and containing a strong transcription activation domain, was also found expressed in all melanomas and most non-melanocyte tumor cell lines. Together, these data indicate that the MITF-M isoform is the major type of MITF mRNA present in human melanoma cell lines and show that the expression of the isoform MITF-A and the MSG1 is not restricted to malignant melanocytes and occurs in a wide range of tumor cell lines.
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PMID:Expression of genes for microphthalmia isoforms, Pax3 and MSG1, in human melanomas. 1064 12

The presence of tyrosinase mRNA in the peripheral blood cells of melanoma patients has been recently studied as a possible marker of haematogenous dissemination. However, considerable variations in the rates of detection have been noted. We determined the presence of tyrosinase mRNA-positive circulating cells using reverse transcriptase-polymerase chain reaction (RT-PCR) in 35 patients with stage I melanoma, two patients with stage II melanoma and two patients with stage III melanoma. Among the patients with stage 1, 13 were tested before and after surgery (< 1 h). Twenty healthy subjects served as negative controls. Out of the melanoma patients, the tyrosinase gene was expressed in three of the 52 samples tested. Tyrosinase mRNA was present in the circulating cells of only one patient with stage I melanoma after intra-congenital naevi resection. However, two other stage I patients developed rapidly lethal metastasis within the following 6 months, despite the lack of detectable tyrosinase mRNA. None of stage II patients were positive for the tyrosinase transcripts, while both patients with stage III melanoma showed enzyme expression. Our results confirm those of previous studies, showing that a small proportion of stage I melanoma patients have tyrosinase-positive circulating cells. Moreover, the lack of tyrosinase mRNA detection in the blood does not necessarily exclude metastatic progression. Therefore, this study indicates that the detection of tyrosinase mRNA-positive circulating cells by RT-PCR is not a predictive biomarker of a metastasis risk in patients with stage I melanoma.
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PMID:The detection of tyrosinase mRNA in the peripheral blood of stage I melanoma patients is not of clinical relevance in predicting metastasis risk and survival. 1080 11

The level of expression of melanoma antigens (MA) may modulate the host immunologic response. Thus, the accurate measurement of MA expression may allow proper patient selection for antigen-specific therapies and yield important information for the evaluation of clinical results. In this study, we measured the absolute levels of MA messenger ribonucleic acid (mRNA) in tumor cell lines utilizing real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). mRNA levels of MART-1, gp100, tyrosinase, TRP-1 and TRP-2 melanoma differentiation antigens and MAGE-1, MAGE-3 and ESO-1 cancer testis (CT) antigens were compared in 24 early-passage (<5 passages in culture) and 12 archival melanoma cell lines. MA mRNA expression was extremely variable among cell lines, occasionally reaching levels comparable to ribosomal RNA (rRNA). gp100 and MART-1 mRNA levels correlated with protein expression measurement obtained by FACS analysis. More significantly, a threshold of gp100 mRNA expression required for T-cell stimulation and target-cell killing was identified. This threshold level corresponded to approximately 500 mRNA copies per 10(8) copies of rRNA. Our results suggest that the measurements of MA mRNA levels may yield useful information relevant to the interpretation of clinical outcome during antigen-specific treatments.
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PMID:Threshold levels of gene expression of the melanoma antigen gp100 correlate with tumor cell recognition by cytotoxic T lymphocytes. 1084 96

Several studies have evaluated the use of polymerase chain reaction (PCR) amplification of tyrosinase mRNA to detect melanoma cells in blood. However, contradictory results have been obtained from different groups. We therefore have developed and validated a quantitative PCR method for tyrosinase and tyrosinase-related protein-2 (TRP-2) mRNA. An important methodological finding was that high concentrations of reverse transcriptase or RNA sample inhibited the following PCR. This could be abolished by dilution of the cDNA sample before the PCR. Standard curves with a linear range over at least five logs were obtained with dilutions of melanoma cell cDNA. Controls (RNA and cDNA) consisting of melanoma cells (1000/ml) added to blood were analysed repeatedly over 3 months, resulting in means between 880 and 1074 AU/ml. The RNA controls were stable, whereas the cDNA controls, as well as the calibrators, showed a tendency to change over time. The variation in the RNA controls was 25% for tyrosinase and 22% for TRP-2. Seven stage III-IV melanoma patients were tested for tyrosinase and TRP-2 transcripts in blood drawn from a peripheral vein and from a Port-a-cath. Tyrosinase mRNA was found in three patients (0.8-12.4 AU/ml). For TRP-2, the same amount was found in the patients as in healthy donors. No differences were seen between blood from a peripheral vein and from the Port-a-cath. We here present fast and sensitive methods for the quantification of tyrosinase and TRP-2 mRNA in blood.
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PMID:Quantitative analysis of tyrosinase and tyrosinase-related protein-2 mRNA from melanoma cells in blood by real-time polymerase chain reaction. 1089 Mar 74

The specificity and sensitivity of the nested reverse transcriptase polymerase chain reaction (RT-PCR) on tyrosinase was studied, for the detection of micrometastases of malignant melanoma. The specificity was assessed in the blood of six healthy donors, four patients with non-melanoma cancers of which one patient was treated with granulocyte-colony stimulating factor. Lymph nodes of nine patients without malignant melanoma were tested and four cell lines of various other tumours. Six of the nine non-melanoma lymph nodes were positive in this assay. The sensitivity was tested in a spike experiment in vitro, using a melanoma cell line. The detection limit was ten melanoma cells per 10(7) peripheral blood lymphocytes.
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PMID:Limitations of the nested reverse transcriptase polymerase chain reaction on tyrosinase for the detection of malignant melanoma micrometastases in lymph nodes. 1090 68

The presence of lymph node metastases is the best prognostic factor for predicting relapse or survival in melanoma patients. It has been demonstrated that melanoma metastases spread through the first lymph node(s) draining the tumor (sentinel lymph node, SN) to the lymphatic system and that detection of melanoma cells in peripheral blood directly correlates with prognosis in melanoma. To identify lymph node metastases and circulating melanocytes, we developed a single-step reverse transcriptase-polymerase chain reaction assay (RT-PCR) for detection of two melanoma-specific markers: the tyrosinase gene, which encodes an enzyme associated with melanin synthesis, and melanoma antigen-related T-cells, which are present in tumor infiltrating T-lymphocytes. This method detects two tumor cells in a background of 10(7) lymphocytes. Thirty patients with stage I-IV cutaneous melanoma entered the study. Blood samples were taken preoperatively, one month after excision of the primary melanoma lesion and the SN or total lymphadenectomy, and before the start of chemotherapy and every three months thereafter in metastatic patients. SNs were collected from 22 patients, bisected and analyzed by RT-PCR and routine pathological and immunohistochemical tests. The preliminary results indicate that RT-PCR for melanoma markers is a sensitive and valuable method for the detection of micrometastases and for early diagnosis and staging of melanoma.
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PMID:Detection of melanoma cells in peripheral blood and sentinel lymph nodes by RT-PCR analysis: a comparative study with immunohistochemistry. 1101 21

The detection of circulating tumor cells (CTC) and bone marrow micrometastases (BMM) by reverse transcriptase polymerase chain reaction (RT-PCR) may help predict recurrence and survival in malignant melanoma (MM). Since the appearance of the original article by Smith et al. in 1991 (Lancet 338:1227), several groups have attempted the detection of CTC and BMM in MM using RT-PCR for melanocytic specific markers, mainly tyrosinase mRNA. Most studies show that tyrosinase is not present in the PB and BM of control individuals without MM. The PCR positivity rates in MM are extremely variable, ranging from 0% to 100%. There was a correlation between RT-PCR and stage in some but not all of the studies. These disparate findings could in part be explained by differences in RNA extraction and blood separation techniques, to unrecognized contamination leading to false positive results, or differences in patient selection. Despite these discrepancies, we and others have shown that RT-PCR for tyrosinase mRNA in PB is able to predict overall survival (OS) and disease-free survival (DFS) in a statistically significant manner. In AJCC stage II-IV patients rendered surgically free of disease, we found that blood tyrosinase positivity was an independent predictor of OS and DFS. We also found that BM tyrosinase positivity is an independent predictor of DFS in the same group of patients. RT-PCR may help identify subgroups of patients at high risk for early relapse for more aggressive adjuvant therapy. Large prospective studies and interlaboratory quality assurance initiatives are necessary to confirm the accuracy and prognostic value of these RT-PCR assays.
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PMID:Reverse transcriptase polymerase chain reaction (RT-PCR) detection of melanoma-related transcripts in the peripheral blood and bone marrow of patients with malignant melanoma. What have we learned? 1109 34

Reverse transcription (RT) of tyrosinase mRNA and specific cDNA amplification to facilitate the early detection of circulating tumor cells in melanoma patients have been reported. The significance and practical value of these procedures for the diagnosis of tumor dissemination in melanoma patients is, however, still unclear. We analyzed peripheral blood samples of melanoma patients of different clinical stages for the presence of tyrosinase mRNA by reverse transcriptase polymerase chain reaction (RT-PCR). In addition to a nested RT-PCR-based system, we evaluated the new PCR enzyme-linked immunosorbent assay tyrosinase system for sensitivity and specificity in detecting circulating melanoma cells. Our results showed a high sensitivity and specificity for this system in detecting one melanoma cell in 1 ml of whole blood. Using different methods of detection, no tyrosinase mRNA was detectable in blood samples of patients with primary melanoma and regional lymph node metastases. In a small number of patients with visceral metastases (10-30%), we found tyrosinase mRNA-positive results. Analyses of different blood samples taken at 2-h intervals indicate that tumor cells persist only transiently in the peripheral blood. Successful establishment of melanoma cell growth from tyrosinase mRNA-positive samples indicates that viable tumor cells exist in melanoma patients' peripheral blood. Our results indicate a low amount of tyrosinase-specific transcripts in a small subset of stage IV patients and suggest that the analysis of tyrosinase mRNA in peripheral blood samples is not helpful as a prognostic marker or monitoring tool in melanoma patients.
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PMID:Facts and pitfalls in the detection of tyrosinase mRNA in the blood of melanoma patients by RT-PCR. 1109 37

The reverse transcriptase polymerase chain reaction (RT-PCR) can be of clinical relevance in identifying malignant melanoma cells in blood or tissues of patients at risk for disseminated melanoma. The diagnostic value of this marker however, is still controversial. The objective of this study was to compare and quantify the difference in sensitivity of the nested RT-PCR for tyrosinase, with respect to the method utilized to produce the template c-DNA. We found a difference of a factor 10 in favor of a specific priming versus a random one. We concluded that this difference can be exploited in the analysis of blood samples. However, in the analysis of lymph node specimens, where the chance of positivity due to tyrosinase positive non-melanoma cells is much higher, the choice of a highly sensitive assay should be made with caution.
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PMID:Effect of specific or random c-DNA priming on sensitivity of tyrosinase nested RT-PCR: potential clinical relevance. 1113 60


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