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)

This study was performed to detect circulating melanoma cells in peripheral blood using a novel method based on magnetic-activated cell separation (MACS) followed by a nested reverse transcriptase-polymerase chain reaction (RT-PCR) for tyrosinase and MART-1 mRNA. Samples to be tested were enriched for tumour cells either by isolating melanoma cells using two anti-melanoma antibodies (MART-1 and HMB-45) or by CD45 depletion of the non-melanoma cell fraction. The tumour cell-enriched fractions were subjected to mRNA isolation using oligo-deoxythymidylate (oligo-dT) magnetic beads followed by a nested RT-PCR. Sensitivity was assessed by spiking experiments and compared with a commonly used total RNA isolation system previously established in our department. Positive isolation of melanoma cells showed insufficient sensitivity, whereas negative isolation by depletion of leukocytes showed a detection limit of at least one melanoma cell per millilitre of whole blood. In further experiments, the depletion assay was applied to 25 peripheral blood samples of melanoma patients. The preliminary data obtained from the new method indicate a comparable detection rate to the established total RNA extraction method. However, not all the results were concordant. Therefore, future experiments need to be performed with a statistically greater number of patients.
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PMID:Magnetic bead RT-PCR: establishment of a new method for detecting circulating melanoma cells. 1217 Jan 79

In this study, we used melb-a melanoblasts as a model to study mechanisms involved in stimulating melanocyte function in vitiliginous skin following exposure to 8-methoxypsoralen (8MOP). Melanin content and tyrosinase activity increased 3- and 7-fold, respectively, in melanoblasts treated with 8MOP for 6 d compared with untreated controls. The intracellular signal pathways involved in 8MOP-induced effects on melanoblasts were investigated, particularly the roles of protein kinase A and protein kinase C. Forskolin, a protein kinase A activator, mimicked and enhanced the 8MOP stimulation of melanoblast pigmentation whereas a protein kinase C activator, 1-oleoyl-2-acetylglycerol, had no effect, indicating that the protein kinase A pathway is involved rather than the protein kinase C pathway. Those observations were confirmed using inhibitors of the protein kinase A or protein kinase C pathways. Western blot and semiquantitative reverse transcriptase polymerase chain reaction were performed to assess the protein and mRNA expression levels of microphthalmia-associated transcription factor and tyrosinase in melanoblasts treated with 8MOP for 3 h, 6 h, 1 d, 3 d, or 6 d. Incubation with 8MOP stimulated microphthalmia-associated transcription factor protein and mRNA levels within 3 h, but, in contrast, tyrosinase mRNA and protein levels did not increase following 8MOP treatment until 1 d after treatment. The proteasome inhibitor lactacystin blocked the proteasome-mediated proteolysis of tyrosinase, and its effect on proteasomal function was enhanced by 8MOP. Taken together, these results show that 8MOP functions by initially stimulating levels of microphthalmia-associated transcription factor expression via activation of the protein kinase A pathway, which thereby stimulates tyrosinase expression and function and eventually leads to dramatic increases in melanin production by melanoblasts.
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PMID:Stimulation of melanoblast pigmentation by 8-methoxypsoralen:the involvement of microphthalmia-associated transcription factor, the protein kinase a signal pathway, and proteasome-mediated degradation. 1248 19

Sequence comparisons and functional analysis of the 5' upstream regions of tyrosinase genes have revealed the importance of cis-regulatory elements acting to control the spatiotemporal expression of tyrosinase in the melanocytes and retinal pigmented epithelium of developing embryos. To date there are no reports addressing the control of tyrosinase gene transcription in zebrafish, a vertebrate model organism of increasing importance. To exploit the tyrosinase gene as a marker in zebrafish we set out to clone its promoter and analyse its regulation during embryogenesis. Amplification of a zebrafish tyrosinase complementary DNA fragment by reverse transcriptase polymerase chain reaction allowed us to isolate and sequence a 1041 nt genomic DNA fragment that includes a transcription initiation site and 73 nt of the open reading frame. Bioinformatic analysis of this genomic sequence revealed five E-box motifs, including one CATGTG type E-box present in a putative initiation region. These are conserved positive regulatory elements in vertebrate tyrosinase promoters. We show that a region of 814 nt upstream from the translation start site of the zebrafish tyrosinase gene can drive expression in retinal pigmented epithelium in transiently transgenic zebrafish embryos but that its activity is not restricted to melanin-producing cells. This region is unable to drive transcription in human melanoma cell lines. Ectopic expression from this zebrafish tyrosinase promoter fragment is probably due to the absence of positive and negative cis-regulatory elements, such as a tyrosinase distal element, which is known to function as a pigment cell-specific enhancer.
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PMID:Expression analysis of a tyrosinase promoter sequence in zebrafish. 1262 88

The effect of androgens on human melanocytes has not been well clarified. We studied the effects of androgens on normal human melanocytes in the presence or absence of sex-hormone-binding globulin (SHBG), which complexes with those hormones. Immunohistochemically, testosterone and SHBG co-localized on the cell membrane. Androgens such as testosterone, 5alpha-dihydrotestosterone, and methyltrienolone (R1881, a potent synthetic androgen), reduced intracellular cAMP levels after treatment with SHBG, but hydrocortisone had no effect. We also found that testosterone and R1881 slightly suppressed tyrosinase activity in melanocytes when treated with SHBG, although they had no effect on the expression of tyrosinase at the transcriptional or translational level, as measured by semi-quantitative reverse transcriptase-polymerase chain reaction and by Western blot analysis, respectively. Our results suggest that androgens may modulate tyrosinase activity at the posttranslational level through the cell membrane signaling pathway.
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PMID:The inhibitory effect of androgen and sex-hormone-binding globulin on the intracellular cAMP level and tyrosinase activity of normal human melanocytes. 1275 85

Although reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of melanocyte-associated mRNA can detect sentinel node melanoma metastases, most published assays are semi-quantitative methods of unknown sensitivity and precision, unsuitable for clinical use. We describe a single-step real-time quantitative RT-PCR assay for MART-1 and tyrosinase mRNAs, suitable for sentinel node analysis in a clinical setting. Using serial dilutions of melanoma cell line SK-MEL-28 RNA in water as a calibrator, we obtained linear calibration curves covering the range 0.5 to 10,000 arbitrary units (SK-MEL-28 melanoma cell equivalents). The sensitivity limit was 0.32 (MART-1) and 5 (tyrosinase) arbitrary units. Analytical imprecision was between 11% and 34%. MART-1 PCR efficiency was unaffected when samples were diluted with negative lymph node RNA rather than water, whereas tyrosinase PCR efficiency was halved. To evaluate the clinical suitability of our assay, we quantified melanocyte mRNAs in sentinel nodes with histologically verified micrometastases (n = 10) and benign nevus inclusions (n = 10), and in sentinel nodes without evidence of intranodal melanocytes (n = 10). We found significant differences in median melanocyte-derived mRNA levels comparing the three types of lymph nodes, suggesting that this quantitative molecular protocol may increase assay precision and be useful for the clinical evaluation of sentinel nodes.
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PMID:Quantification of melanoma mRNA markers in sentinel nodes: pre-clinical evaluation of a single-step real-time reverse transcriptase-polymerase chain reaction assay. 1526 3

Melanoma lesions that develop in the same patient at different times or simultaneously at different locations may differ antigenically, because malignant melanoma is heterogeneous in terms of its biological, immunological and metastatic properties. The objective of this study was to characterize the molecular profiles of melanoma cells in peripheral blood, lymph nodes and metastatic tissues, employing the messenger RNA (mRNA) expression of tyrosinase, melanoma-inhibiting activity (MIA) and melanoma antigen recognized by T cells-1 (MART-1) as markers. Samples of cells propagated from metastatic sites were obtained from 17 stage III/IV melanoma patients and assayed by reverse transcriptase-polymerase chain reaction (RT-PCR), using specific primers for each marker. In eight patients, marker profiles were analysed in simultaneously obtained specimens of peripheral blood, lymph nodes and metastatic tissues originating from the same patient. Tyrosinase, MIA and MART-1 were expressed in 59%, 76% and 76% of the metastases, respectively. Simultaneously obtained specimens of peripheral blood, lymph nodes and metastatic tissues showed a high degree of homogeneity: 60%, 75% and 20% for tyrosinase, MIA and MART-1, respectively. Our findings suggest that the rather homogeneous expression pattern found in different tumour sites analysed in the same patient is of potential prognostic and therapeutic importance. Furthermore, melanoma lesions may be negative for the expression of antigens such as MART-1, and discrepancies in expression patterns between peripheral blood and metastatic tissues may occur, especially for this marker. Finally, our findings support the notion that molecular screening using an RT-PCR approach is appropriate in this kind of investigation.
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PMID:Molecular detection of MART-1, tyrosinase and MIA in peripheral blood, lymph nodes and metastatic sites of stage III/IV melanoma patients. 1545 91

This prospective study was performed to determine the prognostic value of tyrosinase mRNA detection in sentinel lymph nodes (SLN) of melanoma patients. About 847 SLNs from 322 consecutive patients were assessed by histopathology and immunohistochemistry as well as tyrosinase-reverse transcriptase-polymerase chain reaction (RT/PCR) for the presence of micrometastases. The results were correlated with the prognostic parameters employing a multivariate analysis after a median follow-up of 37 months. Histopathological analysis revealed metastases in 34/322 patients (10.6%). Among the 288 patients with histopathologically negative SLN, tyrosinase-mRNA was detected in 39 patients. A relapse of the tumour occurred in 44.1% of the patients with histopathologically positive SLN, in 25.6% with histopathologically negative, but tyrosinase-RT/PCR-positive SLN, and 8.0% with "double-negative" SLN. A multivariate analysis identified tumour thickness, the histopathological SLN status, and the ulceration of the primary tumour as independent prognostic factors. Thus, by assessing tyrosinase mRNA in the SLN of melanoma patients, we identified a subgroup with histopathologically negative, but Tyr-RT-PCR-positive SLN who have a high risk of disease relapse.
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PMID:Prognostic significance of detecting micrometastases by tyrosinase RT/PCR in sentinel lymph node biopsies: lessons from 322 consecutive melanoma patients. 1557 65

The sentinel lymph node (SLN) is the first draining node from the area in which a tumour is located. The presence or absence of SLN micrometastasis is an important prognostic factor for melanoma. As the first dissemination route for melanoma is lymphatic and we know that the immune system plays an important role in melanoma response, we hypothesize that melanoma and its corresponding SLN should constitute an immunological unit. Small portions of 54 SLNs from 37 patients undergoing selective lymphadenectomy were subjected to quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) to quantify messenger RNA (mRNA) transcripts of the following genes: tyrosinase, telomerase, cyclooxygenase-1 (COX-1), COX-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), interferon-gamma (IFN-gamma), IL-4, IL-10 and IL-12. In addition, 11 non-sentinel lymph nodes (NSLNs) were excised from 11 of the 37 patients and the same study was performed. Immunohistochemistry with different antibodies against dendritic cells (DCs) was performed in 10 pairs of SLNs and NSLNs. Significantly higher mRNA expression of COX-2, GM-CSF, IFN-gamma and IL-10 was found in SLNs compared with NSLNs in the overall group. DCs, as labelled by S-100 and CD1a, were significantly decreased in NSLNs compared with SLNs. These data suggest that the initial increase in GM-CSF observed in SLNs could lead to the attraction of a high number of DCs to SLNs. However, the presence of certain immunosuppressive molecules, such as IL-10 and COX-2, could block their maturation and their ability to become efficient antigen presenters.
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PMID:Cytokine expression and dendritic cell density in melanoma sentinel nodes. 1584 42

This study was conducted to examine the prognostic impact of four biomarkers [tyrosinase and MART-1 messenger RNA (mRNA), S100beta protein and lactate dehydrogenase (LDH)] in patients with metastatic melanoma, together with established clinical factors. Tyrosinase and MART-1 mRNA were measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). S100beta was measured using a commercially available immunoassay, and LDH was analysed conventionally. All markers were measured in blood samples before interleukin-2-based immunotherapy in 85 patients with metastatic melanoma. LDH, S100beta, tyrosinase, number of metastatic sites, location of metastatic sites and performance status were all significant factors for survival in univariate analyses. In multivariate analysis, tyrosinase [hazard ratio (HR)=1.6; 95% confidence interval (CI), 1.1-2.6; P=0.04] and LDH (HR=2.0; 95% CI, 1.1-3.5; P=0.02) were both independent prognostic factors for survival. A combination variable of tyrosinase and LDH remained independently associated with survival (P=0.04) after adjusting for the American Joint Committee on Cancer (AJCC) stage IV classification in a multivariate analysis involving both models. It can be concluded that tyrosinase mRNA and elevated LDH are independent prognostic factors for poor survival in this group of 85 patients. Additional studies are needed before the prognostic value of tyrosinase mRNA in metastatic melanoma can be firmly established. Further evaluation of the combined measurement of tyrosinase mRNA and LDH is warranted.
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PMID:Tyrosinase messenger RNA in peripheral blood is related to poor survival in patients with metastatic melanoma following interleukin-2-based immunotherapy. 1617 68

Tyrosinase-based reverse transcriptase-polymerase chain reaction (RT-PCR) is a method for the detection of circulating melanoma cells in peripheral blood. To our knowledge, no long-term studies on the prognostic impact of tyrosinase PCR in uveal melanoma have yet been reported. In this prospective, non-randomized, observational cohort study, we included 41 patients with uveal malignant melanoma. RT-PCR for tyrosinase was performed in each patient before and after treatment. A clinical follow-up was performed for each patient for at least 5 years, including chest X-ray, serum liver enzyme determination, ultrasound of the liver and bone scintigraphy. The PCR results, age of the patients, tumour size, tumour location, tumour therapy, internal reflectivity, histology, development of distant metastasis and survival rate during follow-up were analysed. At the time of diagnosis, tyrosinase messenger RNA (mRNA) in peripheral blood, suggesting the presence of circulating melanoma cells, was detected in 16 of the 41 patients. Sixty-nine percent of the PCR samples with a positive result prior to therapy revealed a negative result after therapy. The internal reflectivity of the tumour (P=0.021) and the 5-year survival (P=0.023) showed a statistically significant association with positive PCR. It can be concluded that tyrosinase RT-PCR is a sensitive method for the detection of melanoma cells in peripheral blood. This study indicates that the presence of tumour cells in peripheral blood correlates with 5-year survival. Our results suggest a prognostic value of this method. Nevertheless, prospective analysis of a larger cohort is needed to determine the ultimate value of RT-PCR for tyrosinase in blood testing.
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PMID:Five-year results of prognostic value of tyrosinase in peripheral blood of uveal melanoma patients. 1631 35


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