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)

Microphthalmia transcription factor (Mitf) is a nuclear protein involved in the development of melanocytes and the regulation of melanin synthesis. Recent studies have suggested that Mitf may be a more sensitive and specific melanocyte marker than S-100 protein and gp100. However, there is insufficient knowledge on the specificity of Mitf, and a systematic examination of its use for the recognition of desmoplastic melanoma has not yet been performed. In this study, we compared the expression of Mitf with S-100 protein, gp100, and tyrosinase in 20 desmoplastic melanomas by using the antibodies D5 (anti-Mitf), anti-S100P, HMB-45 (anti-gp100), and T311 (anti-tyrosinase). All 20 melanomas were positive for S-100 protein, 7 were positive for Mitf, 6 for gp100, and 11 for tyrosinase. To examine the specificity of Mitf, a panel of normal tissue and 386 samples of miscellaneous tumors, including dermal and subcutaneous spindle cell lesions relevant for the differential diagnosis of desmoplastic melanoma, were examined by immunohistochemistry. Furthermore, normal tissue samples were tested for Mitf mRNA by reverse transcriptase polymerase chain reaction (rt-PCR). Immunoreactivity for Mitf was seen not only in melanocytes of normal skin, but also in macrophages, lymphocytes, fibroblasts, Schwann cells, and smooth muscle cells at various sites, and tumors derived thereof. Our results indicate that the antibody D5 lacks sufficient sensitivity and specificity for widespread diagnostic use. Especially in re-excisions, when immunohistochemistry is often needed to distinguish an inflamed scar tissue from tumor, the presence of immunopositive inflammatory cells and fibroblasts limits the diagnostic use of D5.
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PMID:Analysis of microphthalmia transcription factor expression in normal tissues and tumors, and comparison of its expression with S-100 protein, gp100, and tyrosinase in desmoplastic malignant melanoma. 1117 68

The aim of this study was to develop a highly sensitive two-marker assay for the detection of circulating melanoma cells in patients' blood using a reverse transcriptase-polymerase chain reaction (RT-PCR). We analysed the usefulness of two different sets of markers: tyrosinase and MUC-18 (TYR/MUC-18), and tyrosinase and MART 1 (TYR/MART 1). Total cellular RNA was isolated from 337 blood samples from 80 melanoma patients at different stages of the disease. All patients had undergone primary surgery. Assay sensitivity and specificity were confirmed using three different melanoma cell lines and two different fibroblast lines. In addition, blood from 47 healthy subjects and 10 patients with non-melanoma cancer was used as a negative control. We found that two-marker analysis is more accurate than the single tyrosinase assay. The frequency of melanoma cell detection in patients' blood was about 10% higher when the TYR/MART 1 two-marker assay was used. Using this assay we did not find any statistical correlation between the molecular markers and the UICC stage of disease or the Breslow thickness or Clark level of the primary melanoma. The frequency of melanoma cell detection with the TYR/MUC-18 two-marker assay was even higher than the TYR/MART 1 assay, but unfortunately the MUC-18 transcript was also present in about 20% of healthy subjects. Therefore we do not recommend the use of MUC-18 as a standard value marker.
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PMID:Detection of circulating melanoma cells in peripheral blood by a two-marker RT-PCR assay. 1125 17

Recently, reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of circulating tumor cells has been suggested as a potential technique for staging cancer. In this report, 43 melanoma patients (including 4 in situ melanoma patients) were tested for tyrosinase mRNA in blood by RT-PCR. All patients had melanoma thinner than 1.5 mm (stage I). Circulating melanoma cells were detected in 8 (18.6%) out of 43 MM patients tested: 5 (16.1%) of 31 patients with melanoma thinner than 0.76 mm and 3 (42.8%) out of 7 patients with melanoma thicker than 0.76 mm. Moreover, in the tyrosinase-negative group we found only 4/31 patients (13%) with histologic signs of regression, but in the tyrosinase-positive group, 3 out of 8 patients (37.5%) showed, at histologic examination, signs of regression. At the time of this analysis all the patients enrolled (tyrosinase-negative and tyrosinase-positive ones) were free of disease, probably due to the short median time of follow-up after the inclusion in the study. The presence of regression is an important cause of melanoma understaging and the tyrosinase test could represent an effective tool in order to achieve a realistic staging in this subgroup of melanoma patients. Probably, maximum sensitivity of the diagnostic RT-PCR approach to monitor MM patients with either localized or advanced disease could be achieved by using additional markers expressed with high frequencies in melanoma. We propose that one such marker could be the sign of regression.
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PMID:Detection of circulating malignant cells by RT-PCR in long-term clinically disease-free I stage melanoma patients. 1126 78

Little is known about the mechanism(s) underlying hyperpigmentation in lentigo senilis. We have previously reported that keratinocyte-derived endothelins are intrinsic paracrine mitogens and melanogens for human melanocytes and that they play an essential role in stimulating ultraviolet-B-induced melanogenesis. In this study, we have used immunohistochemistry and reverse transcriptase polymerase chain reaction analysis to clarify the role of the endothelin cascade, including endothelin production, processing by endothelin-converting enzyme, and expression of the endothelin B receptor, in the hyperpigmentary mechanism(s) involved in lentigo senilis. The number of tyrosinase immunopositive melanocytes in lentigo senilis lesional skin was increased 2-fold over the perilesional epidermis. Immunohistochemistry using antibodies to endothelin-1 demonstrated relatively stronger staining in the lesional epidermis than in the perilesional epidermis. Reverse transcriptase polymerase chain reaction analysis concomitantly demonstrated accentuated expression of transcripts for endothelin-1 and for the endothelin B receptor in lentigo senilis lesional skin, which was accompanied by a similar accentuated expression of tyrosinase mRNA compared with the perilesional control. The endothelin-1-inducible cytokine, tumor necrosis factor alpha, was consistently upregulated in the lentigo senilis lesional epidermis as determined at the transcriptional level and by immunostaining, whereas interleukin-1alpha was downregulated. In contrast, endothelin-converting enzyme 1alpha mRNA was not substantially increased in the lesional epidermis. These findings suggest that an accentuation of the epidermal endothelin cascade, especially with respect to expression of endothelin and the endothelin B receptor, plays an important role in the mechanism involved in the hyperpigmentation of lentigo senilis.
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PMID:The role of the epidermal endothelin cascade in the hyperpigmentation mechanism of lentigo senilis. 1128 25

The melanosome is a unique secretory granule of the melanocyte in which melanin pigments are synthesized by tyrosinase gene family glycoproteins. Melanogenesis is a highly regulated process because of its inherent toxicity. An understanding of the various regulatory mechanisms is important in delineating the pathophysiology involved in pigmentary disorders and melanoma. We have purified and analyzed the total melanosomal proteins from B16 mouse melanoma tumors in order to identify new proteins that may be involved in the control of the melanogenesis process. Melanosomal proteins were resolved by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, a predominant spot (27 kDa with isoelectric point 5.8-6.4) was excised and digested with cyanogen bromide, and the fragments were sequenced. Synthetic oligonucleotide primers were synthesized corresponding to the peptide sequences, and reverse transcriptase polymerase chain reaction amplification of total RNA from B16 cells was carried out. Sequencing of one of the polymerase-chain-reaction-mediated clones demonstrated 80%-97% sequence homology of 200 bp nucleotide with GTP-binding proteins at the 3'-untranslated region. GTP-binding assay on two-dimensional gels of melanosomal proteins showed the presence of several (five to six) small GTP-binding proteins, suggesting that small GTP-binding proteins are associated with the melanosome. Among the known GTP-binding proteins with similar molecular weight and isoelectric point ranges, rab3, rab7, and rab8 were found to be present in the melanosomal fraction by immunoblotting. Confocal immunofluorescence microscopy showed that rab7 is colocalized with the tyrosinase-related protein 1 around the perinuclear area as well as, in part, in the perikaryon, thereby suggesting that rab7 might be involved in the intracellular transport of tyrosinase-related protein 1. Tyrosinase-related protein 1 transport was blocked by the treatment of B16 cells with antisense oligonucleotide to rab7. We suggest (i) that rab7 is a melanosome-associated molecule, (ii) that tyrosinase-related protein 1 is present in late-endosome delineated granules, and (iii) that rab7 is involved in the transport of tyrosinase-related protein 1 from the late-endosome delineated granule to the melanosome.
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PMID:Identification of rab7 as a melanosome-associated protein involved in the intracellular transport of tyrosinase-related protein 1. 1144 53

For the molecular detection of rare tumour cells in clinical samples, real-time reverse transcription-polymerase chain reaction (RT-PCR) offers two important advantages over conventional RT-PCR assays: the results are quantitative and, perhaps more importantly, it facilitates exact sensitivity controls on a per sample basis as well as exact comparison of different assay protocols. We report here on quantitative results obtained with different protocols for RNA isolation and cDNA synthesis for amplification of beta2-microglobulin transcripts using the light cycler system. Furthermore, housekeeping gene-specific PCRs were compared with PCRs specific for an artificial transcript (internal standard) detected simultaneously at a level comparable to the wild-type sequence. Artificial tyrosinase transcripts derived from a vector construct stably transfected into a human lymphoma cell line were used as a model to test the usefulness of artificial internal standards as an alternative to housekeeping genes. The highest RNA yields were obtained using a combination of phenol-chloroform extraction and the High Pure RNA Isolation Kit. Analysing beta2-microglobulin transcript-specific RT-PCRs, the highest sensitivity was obtained for cDNAs generated with Omniscript reverse transcriptase and oligo-p(dT)15 primer. Regarding patient blood samples, RT-PCRs specific for beta2-microglobulin, porphobilinogen deaminase and artificial tyrosinase transcripts provided quantitative data for all, for 18 out of 21, and for 10 out of 21 samples, respectively. Quantification of beta2-microglobulin transcripts by the light cycler system defined the protocol revealing the highest cDNA quality. Comparisons of quantitative data from RT-PCRs specific for beta2-microglobulin, porphobilinogen deaminase and artificial tyrosinase transcripts enabled us to determine a close range for crossing points within which sufficient cDNA quality can be guaranteed, even for the detection of rare transcripts. PCRs specific for the artificial internal standard are ideally suited for cDNA quality assessment on a per sample basis.
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PMID:Reliability of PCR-based detection of occult tumour cells: lessons from real-time RT-PCR. 1147 25

Melanoma frequently metastasizes to the central nervous system (CNS). The diagnosis of CNS metastases typically is made following the onset of clinical symptoms. Thus, more sensitive diagnostic approaches are needed to identify subclinical CNS metastases. Currently, standard cytologic analysis of the cerebrospinal fluid (CSF) is limited by its poor sensitivity. A more sensitive assay was therefore developed using multiple reverse transcriptase-polymerase chain reaction (RT-PCR) markers. CSF was collected and assessed by RT-PCR for three known melanoma-associated markers (MAGE-3, MART-1, and tyrosinase) to detect occult metastatic melanoma cells in the CSF of 37 American Joint Committee on Cancer (AJCC) stage IV melanoma patients. Cytologic analysis of CSF was performed on all patients, and immunohistochemistry (IHC) analysis was performed on 33 CSF samples using anti-S100 and anti-HMB-45 antibodies. Only one patient (3%) had tumor-positive CSF cytology and IHC upon entry into the study, whereas 12 patients (32%) were positive for at least one RT-PCR marker. The correlation between CSF RT-PCR positivity of MART-1 and/or MAGE-3 and the development of CNS metastases at 3 mo was significant (p = 0.04). Fifteen of 37 patients (41%) had either positive MRI and/or positive RT-PCR results. Multimarker RT-PCR is more informative and sensitive than cytology/IHC in assessing the CSF of melanoma patients.
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PMID:Molecular detection of metastatic melanoma cells in cerebrospinal fluid in melanoma patients. 1151 19

Increasingly, reverse transcriptase polymerase chain reaction (RT-PCR) is used to detect clinically significant tumour cells in blood or bone marrow. This may result in a redefinition of disease-free and clinical relapse. However, its clinical utility may be limited by lack of automation or reproducibility. Recent studies have suggested nucleic acid sequence-based amplification of target RNA may be more robust. In this study, nucleic acid sequence-based amplification was established to detect melanoma, colorectal and prostate cancer cells. Nucleic acid sequence-based amplification and RT-PCR both successfully amplified target RNA in peripheral blood samples from patients with melanoma and colorectal cancer, but only RT-PCR detected PSA in blood samples from patients with prostate cancer. There was relatively good agreement between sample replicates analyzed by RT-PCR (Kappa values of one for tyrosinase, 0.67 for CK-20 and one for PSA), but less agreement when analyzed by nucleic acid sequence-based amplification. This may limit the routine use of NASBA for the detection of clinically significant disease. In summary, RT-PCR appears at present to be the most reliable and reproducible method for the detection of low-level disease in cancer patients, although prospective studies are warranted to assess the clinical utility of different molecular diagnostic methods.
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PMID:Comparison of the RNA-amplification based methods RT-PCR and NASBA for the detection of circulating tumour cells. 1185 20

The aim of this study was to evaluate the correlation between the detection of circulating melanoma cells and prognostic criteria for malignant melanoma such as clinical stage, tumor thickness and histological type of the primary tumor. Using a reverse transcriptase-polymerase chain reaction (RT-PCR) technique, melanoma cells were identified by detecting tyrosinase mRNA in peripheral blood from 58 patients with malignant melanoma classified according to the American Joint Committee on Cancer guidelines. The results of the RT-PCR assay for tyrosinase were related to two prognostic markers typically used to evaluate these tumors: clinical stage and thickness. Positive PCR results were more frequent in primary tumors measuring > 4 mm (83%) than in thinner tumors (1.1-4.0 mm, 74%; < or = 1.0 mm, 23%) (P = 0.005). No statistical correlation was found between the PCR results and histological appearance of the primary tumor. Although further studies are necessary, our results suggest the possible application of the PCR assay for tyrosinase mRNA in clinical evaluation of the prognosis of malignant melanoma.
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PMID:Reverse transcriptase-polymerase chain reaction detection of circulating tumor cells in patients with melanoma: correlation with clinical stage, tumor thickness and histological type. 1203 Oct 85

We previously described a transgenic mouse line (TG-3) that spontaneously develops pigmented cutaneous melanoma. The generation of several albino mice that developed amelanotic melanoma has also been reported. In this report, we describe an unanticipated result with crosses between C57BL/6-c2j and TG-3 mice. C57BL/6-c2j has the same genetic background as TG-3 (C57BL/6), except for a single base mutation (nucleotide 291) in the tyrosinase locus, resulting in albino coat colour. Only albino F2 mice generated from (TG-3 x C57BL/6-c2j) F1s were selected for further studies. Mice that contained the transgene showed a very high incidence of tumor development as early as 4-6 weeks of age. Raised amelanotic tumors developed on the ear pinnae and perianal region in young F2 albino mice, similar phenotypes as those described earlier for the other albino inbred strains. However, with time, these amelanotic tumors not only increased in size, but unexpectedly developed foci of dark pigmentation. DNA sequence analysis on reverse transcriptase-polymerase chain reactions (RT-PCRs) of tyrosinase mRNA showed that the original tyrosinase mutation was still present in the tumors, indicating that no reversion at this nucleotide had occurred in the tumors. Two different tyrosinase activity assays were used and tyrosinase activity was detected in most tumor samples. Furthermore, Western blot analysis demonstrated various levels of tyrosinase protein in ear tumor samples. These results suggest that tyrosinase and/or melanin are not directly involved in the establishment of melanoma, but that late events occurring within the tumors may generate some tyrosinase activity and production of melanin.
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PMID:Progressive appearance of pigmentation in amelanotic melanoma lesions. 1210 Apr 94


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