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)

Both melanocytes and glial cells are derived embryologically from the neural ectoderm. Their malignant transformed counterparts, melanoma and glioma cells, respectively, may share common antigens. Numerous tumor-associated antigens have been identified in melanomas but only a few a gliomas. Using an established reverse transcriptase polymerase chain reaction plus Southern blot assay, we compared the mRNA expression of melanoma-associated antigens (MAAs) of melanomas to brain tumors primarily derived from glial cells. The MAAs studied included tyrosinase (Tyr), tyrosinase-related protein-1 and -2 (TRP-1 and TRP-2), gp100, human melanoma antigen-encoding genes 1 and 3 (MAGE-1 and MAGE-3), and melanotransferrin (p97). Glioblastoma multiforme (n = 21), anaplastic astrocytoma (n = 3), ependymoma (n = 2), meningioma (n = 3), oligodendroglioma (n = 1), and melanoma (n = 12) tumor specimens were assayed for MAA mRNA expression. Glioblastoma multiforme, astrocytoma, and melanoma cell lines were also assayed. We observed that individual MAA mRNAs were expressed in these brain tumors and cell lines at varying frequencies. The melanogenesis-pathway-related MAAs Tyr, TRP-1, TRP-2, and gp100 mRNAs were also expressed at different levels in normal brain tissues but at a much lower frequency than in glioblastoma multiforme and melanoma. MAGE-1 and MAGE-3 mRNA were expressed in different types of tumor specimens and cell lines but never in normal brain tissue. Tumor antigen p97 was expressed in all types of tumors and also in normal brain tissues. These studies demonstrate that melanomas and primary brain tumors express common MAAs and could be exploited in patients with malignant glioma by active specific immunotherapy against these common MAAs.
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PMID:Molecular detection of tumor-associated antigens shared by human cutaneous melanomas and gliomas. 917 5

Recent studies have characterized a number of the Ags that are recognized by melanoma-reactive T cells. Although the majority of tumor Ags appear to represent nonmutated gene products, a variety of epitopes have been shown to arise from either mutated or alternatively processed transcripts. Here, we report that the screening of a cDNA library with a HLA-A24-restricted melanoma-reactive T cell cloid derived from tumor infiltrating lymphocytes resulted in the isolation of a variant of the gp100 gene that had retained the entire fourth intron of this gene, termed gp100-in4. The gp100-in4 transcript could be detected by reverse transcriptase-PCR but could not be detected in Northern blots conducted with melanoma RNA, indicating that it represents a relatively rare transcript. Read-through of this transcript into the region corresponding to the fourth intron gave rise to an additional 35 amino acids not found in the normal gp100 glycoprotein, and a peptide within this region conforming to the HLA-A24 consensus motif (VYFFLPDHL) was shown to be recognized by the T cell cloid. The sequence of the intron was identical with that of a previously isolated genomic gp100 clone, and T cells that recognized the gp100-in4 gene product were found to recognize HLA-A24-matched allogeneic melanoma cell lines and melanocytes, demonstrating that this represents a nonmutated epitope. These results further extend the types of Ags that can be recognized by melanoma-reactive T cells to aberrant transcripts of melanosomal genes.
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PMID:The intronic region of an incompletely spliced gp100 gene transcript encodes an epitope recognized by melanoma-reactive tumor-infiltrating lymphocytes. 920 Apr 67

Experimental melanin-protein induced uveitis (EMIU) is a T-cell mediated autoimmune uveitis induced by immunization with bovine uveal melanin protein. Gp100, a melanocyte lineage-specific protein, is identified as a human melanoma antigen. A recombinant adenovirus construct encoding gp100 (Ad2CMV-gp100) has been used as a vaccine for cancer therapy. This study examines the effect of Ad2CMV-gp100 on EMIU. To induce EMIU, rats were injected intraperitoneally on day 7 before immunization with ad2CMV-gp100, control adenovirus encoding LacZ (Ad2CMV-LacZ), or no virus. On day 21 after immunization, the right eye was processed for histology and the left eye was analysed for cytokines by quantitative reverse transcriptase-polymerase chain reaction. Western blot analysis showed that uveal melanin-protein contains gp100. In three independent experiments, ocular inflammation was significantly suppressed, and expression of ocular IL-12p40 mRNA was much lower in the rats which received Ad2CMV-gp100 before immunization than in those that received Ad2CMV-LacZ or no virus. No abnormalities developed in rats which received Ad2CMV-gp100 or Ad2CMV-LacZ alone. Therefore, Ad2CMV-gp100 injection prevents the development of EMIU, at least in part, through cytokine regulation.
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PMID:Recombinant adenovirus encoding gp100 modulates experimental melanin-protein induced uveitis (EMIU). 965 89

Expression of HLA class I molecules is essential for the recognition of tumor cells by CD8+ T cells. In this study, 48 bioptic samples of 42 patients in all stages of melanoma were investigated after short-time cultivation of tumor cells. To confirm melanocytic origin of cultured cells, samples were screened for mRNA expression of melanoma markers gp100, tyrosinase, MAGE-3, MelanA, and MUC18 by reverse transcriptase-polymerase chain reaction. Surface expression of specific HLA-A and -B allospecificities on melanoma cells were analyzed with a standard microcytotoxicity assay after stimulation with interferon (IFN)-alpha and compared with the background found in peripheral blood mononuclear cells from the corresponding patients. Immunohistochemistry and flow cytometry confirmed specific losses in cases where the appropriate monoclonal antibodies were available. The level of expression of HLA-I, HLA-II, and intercellular adhesion molecule 1 antigens on melanoma cells cultured in the presence or absence of IFN-alpha and IFN-gamma was determined cytofluorometrically. All cell cultures tested were found to be positive for one or more melanocytic markers by reverse transcriptase-polymerase chain reaction. The specific HLA-I alleles on the cultured cells were detectable in 45 of 48 samples. In 11 cases a specific loss of one HLA-I allele was observed (2 x A2, B7, B8, B18, 4XB44, B47, B49). Ten of these samples were derived from locoregional lymphnode metastases or from distant metastatic tumors. Only one sample from a primary melanoma showed a specific loss of HLA-I (B47). IFN-alpha upregulated expression of HLA-I up to 4-fold. IFN-gamma enhanced the appearance of HLA-II up to 35-fold and the expression of intercellular adhesion molecule 1 up to 40-fold. Selective loss of HLA-I allospecificities might be a major step in tumor progression.
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PMID:Higher frequency of selective losses of HLA-A and -B allospecificities in metastasis than in primary melanoma lesions. 974 Feb 47

The aim of our study was to investigate the metastatic pathways of melanoma cells in sentinel and other regional lymph nodes. The term "sentinel lymph node" means that the first lymph node of the draining site of a primary tumor is never bypassed in malignant melanoma. In this case lymph node dissection would be necessary only when melanoma cells are detected in the sentinel node. Tyrosinase reverse transcriptase-polymerase chain reaction was applied to search for metastatic melanoma in the sentinel lymph node and in further lymph nodes of a complete lymph node basin in patients who underwent lymph node dissection. In 24 patients with malignant melanoma the draining site of the tumor was marked by lymphoscintigraphy and by intraoperative injection of patent blue V in the area around the primary tumor. The lymph nodes of the affected basin were excised and prepared for histopathologic, immunohistochemical, and molecular biologic examinations. Regarding the sentinel lymph node, 10 of 24 patients showed morphologic evidence for metastases, three additional patients showed only tyrosinase transcripts. In 11 of these 13 cases we found one or more nonsentinel lymph nodes with morphologically detectable melanoma cells and/or tyrosinase mRNA. Interestingly, in seven of 24 patients a positive tyrosinase reverse transcriptase-polymerase chain reaction was received in nonsentinel lymph nodes, whereas the sentinel lymph node was negative, not only for all histologic examinations but also by tyrosinase reverse transcriptase-polymerase chain reaction. In five of seven patients of the latter group, gp100 reverse transcriptase-polymerase chain reaction was carried out, showing also gp100 mRNA in nonsentinel lymph nodes only. Our data indicate that the concept of the sentinel lymph node may miss micrometastases. Whether such micrometastases cause a recurrence or a metastasis of malignant melanoma, or can be destroyed by the immune system, remains to be clarified.
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PMID:Detection of melanoma micrometastases in the sentinel lymph node and in nonsentinel nodes by tyrosinase polymerase chain reaction. 1050 40

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

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

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

An autologous melanoma cell line selected for loss of expression of the immunodominant MART-1 and gp100 antigens was initially used to carry out a mixed lymphocyte tumor culture (MLTC) in a patient who expressed the human leukocyte antigen (HLA)-AI and HLA-A2 class I major histocompatibility complex alleles. Ten clones identified from this MLTC seemed to recognize melanoma in an HLA-A1-restricted manner but failed to recognize a panel of previously described melanoma antigens. The screening of an autologous melanoma cDNA library with one HLA-Al-restricted melanoma-reactive T-cell clone resulted in the isolation of a cDNA clone called AIM-2 (antigen isolated from immunoselected melanoma-2). The AIM-2 transcript seemed to have retained an intronic sequence based on its alignment with genomic sequences as well as expressed sequence tags. This transcript was not readily detected after Northern blot analysis of melanoma mRNA, indicating that only low levels of this product may be expressed in tumor cells. Quantitative reverse transcriptase-polymerase chain reaction analysis, however, demonstrated a correlation between T-cell recognition and expression in HLA-A1-expressing tumor cell lines. A peptide that was encoded within a short open reading frame of 23 amino acids and conformed to the HLA-A1 binding motif RSDSGQQARY was found to represent the T-cell epitope. The AIM-2-reactive T-cell clone recognized a number of neuroectodermal tumors as well as breast, ovarian, and colon carcinomas that expressed HLA-A1, indicating that this represents a widely expressed tumor antigen. Thus, AIM-2 may represent a potential target for the development of vaccines in patients bearing tumors of a variety of histologies.
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PMID:Melanoma-Reactive CD8+ T cells recognize a novel tumor antigen expressed in a wide variety of tumor types. 1156 34

In malignant melanoma, tumor-infiltrating lymphocytes are frequently reactive with melanosomal antigens. Achieving complete remissions by peptide therapy is frequently hampered by metastases evading immune recognition. The tumor microenvironment seems to favor reduced expression of target antigens by melanoma cells. Among candidate factors, interferon-gamma (IFN-gamma) (10(2) to 10(3) U/ml) suppressed expression of antigens MART-1, TRP-1, and gp100 by M14 melanoma cells as shown by immunohistology and fluorescence-activated cell sorting analysis, reducing MART-1 expression by >65%. Northern blot analysis revealed that reduced expression was regulated at the transcriptional level, demonstrating a 79% reduction in MART-1 transcript abundance after 32 hours of IFN-gamma treatment. To evaluate consequences of IFN-gamma exposure for immune recognition, MART-1-responsive T cells were reacted with pretreated HLA-matched melanoma cells. Cytotoxicity was reduced up to 78% by IFN-gamma pretreatment, and was restored by addition of MART-1 peptide AAGIGILTV for 2 hours. Examination of melanoma lesions by quantitative reverse transcriptase-polymerase chain reaction revealed up to 188-fold more abundant IFN-gamma transcripts when compared to control skin. Laser capture microdissection and immunohistology localized most IFN-gamma-producing T cells to the tumor stroma. Reduced MART-1 expression was frequently observed in adjacent tumor cells. Consequently, IFN-gamma may enhance inflammatory responses yet hamper effective recognition of melanoma cells.
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PMID:Interferon-gamma reduces melanosomal antigen expression and recognition of melanoma cells by cytotoxic T cells. 1183 72


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