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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The dissemination of cancer cells is a prerequisite in the development of micrometastases and solid metastases. Our previous examinations of these cells were based on immunocytological staining of tumor-associated antigens and cytokeratins. We have now developed a highly sensitive and specific detection method based on a nested reverse-transcriptase-polymerase-chain reaction (RT-PCR) of cytokeratin-20 (CK-20) mRNA. Using this method, we examined the bone marrow of 57 patients with colorectal cancer and detected increasing numbers of CK-20-positive samples, depending on the UICC stage. Some 35% of all bone-marrow samples tested positive for CK-20: none were found in colorectal cancer stage 1, 24% were in stage II, 31% in stage III and 71% in stage IV. Investigation of bone-marrow specimens of patients with pancreatic cancer showed that 4 out of 11 patients were positive for CK-20 mRNA. To confirm that sample positivity for CK-20 expression was due to disseminated tumor cells, we examined bone marrow from a control group (n = 16) without apparent carcinoma. In this group, 15 out of 16 donors were CK-20-negative, while one donor with familial adenomatous polyposis showed a CK-20-specific signal.
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PMID:The detection of disseminated tumor cells in bone marrow from colorectal-cancer patients by a cytokeratin-20-specific nested reverse-transcriptase-polymerase-chain reaction is related to the stage of disease. 879 68

A two-step reverse-transcriptase-based polymerase chain reaction (PCR) with nested primer pairs was developed to amplify sensitive and specific cytokeratin-19 (CK-19) mRNA sequences from human breast cancer cells. No CK-19 pseudogene interference was seen. The larger DNA-derived amplification products could be clearly discriminated from mRNA-derived products. The CK-19 message was not amplified from bone marrow or blood of healthy volunteers and patients with haematological malignancies nor from myeloid and lymphoid cell lines. Breast cancer cells were diluted in buffy coat cells up to 10(-6) and CK-19 mRNA sought by PCR. The CK-19 message was detected in 14 of 26 blood samples and 14 of 24 marrow samples but in neither of two peripheral blood stem cell samples taken from 35 breast cancer patients. By sequence-analysis control of two of these samples and two cell lines, the amplified DNA fragments were confirmed to be homologous with the CK-19 sequence. The CK-19 message was further sought in matched blood/marrow samples taken from 13 untreated women in the same cohort at the time of diagnosis. In 3 of these, CK-19 RNA was detected in blood and marrow and, in 3 others, only in blood, but never in marrow alone. The results show that CK-19 assay by reverse transcriptase/PCR is a sensitive and specific technique for the detection of cancer cells in bone marrow and blood. It could be helpful in diagnosis and monitoring of metastatic breast cancer and detection of micrometastases. This should be evaluated on larger numbers of patients, with different clinical samples and epithelial malignancies.
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PMID:Reverse transcriptase/polymerase chain reaction detection of cytokeratin-19 mRNA in bone marrow and blood of breast cancer patients. 889 79

The presence of axillary lymph node metastasis in patients with breast cancer is a major prognostic factor and also determines the use of adjuvant chemotherapy. Micrometastasis has been arbitrary defined as deposits of < 2 mm dimension. Earlier studies of micrometastases failed to demonstrate prognostic relevance. However, when larger numbers of patients were followed up for longer periods, micrometastasis was shown to be a significantly poor prognostic parameter with patients having a survival rate similar to those with macrometastasis or nodal disease. There are no compelling reasons to retain the term "micrometastasis" in the light of these findings and our understanding of tumor biology. Routine histological examination of axillary lymph nodes is a notoriously inaccurate method for the detection of metastases. When serial or multilevel sectioning and/or immunohistochemical staining for cytokeratin were employed, detection rates increased by as much as 33%. Reverse transcriptase-polymerase chain reaction and Southern blotting for CK19 may be a more accurate method of examination. However, there are inherent technical problems associated with this method, and the recent finding of a pseudogene with great homology to CK19 in normal peripheral blood nucleated cells further emphasises the need for caution in this approach. It is not cost-effective to employ serial sectioning and immunohistochemistry when examining the axillary contents. However, the introduction of sentinel-node biopsy may allow detailed examination of the single node most likely to harbour a metastatic tumor.
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PMID:The prognostic dilemma of nodal micrometastases in breast carcinoma. 1089 73

examination. The patient died 10 months after surgery. Histologically, the tumor was composed of predominantly large epithelioid cells with foci of anaplasia mimicking metastatic carcinoma. Immunohistochemically, the tumor cells stained with anti-cytokeratin, EMA, desmin, and NSE antisera. Electron microscopy showed secretory lumina, desmosomes, cell processes with microtubules and electron-dense granules, and focal whorls of intermediate filaments. Reverse transcriptase-polymerase chain reaction performed on paraffin block-retrieved tissue demonstrated the EWS/WT-1 fusion transcript characteristic of the t(11;22)(p13;q12). This case illustrates a less common histological pattern of DSRCT, i.e., diffuse large cells, thus supporting the view that this tumor presents a wider morphological spectrum than that previously recognized.
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PMID:Desmoplastic small round-cell tumor: a case report on the large cell variant with immunohistochemical, ultrastructural, and molecular genetic analysis. 1107 72

Reverse transcriptase polymerase chain reaction (RT-PCR) is often used for sensitive detection of micrometastasis in peripheral blood, lymph nodes and bone marrow. While the utility of this method has been documented, it also has limitations in the detection of micrometastasis. The mRNA of target genes can be detected in healthy donors or in samples used for negative control, therefore the non-quantitativeness of conventional RT-PCR has been called into question. We analyzed the expression level of cytokeratin (CK) 18 mRNA in established esophageal and gastrointestinal carcinoma cell lines and non-epithelial cells, using quantitative RT-PCR, based on real time 'TaqMan TM' technology. CK 18 mRNA is more highly expressed in carcinoma cells than in non-epithelial cells. However, the expression level in non-epithelial cells was easily detected using conventional RT-PCR and agarose gel electrophoresis. In an analysis of CK 18 mRNA expression in peripheral venous blood in 13 healthy volunteers, we found that CK 18 mRNA was much less expressed than in cancer cell lines. However, the expression in all samples was at a level which was also detected using conventional RT-PCR. It would thus seem that not only qualitative, but also quantitative analysis, of the target mRNA is important to detect micrometastasis. Quantitative RT-PCR methods will make comparisons of the possible differences in expression levels of the target gene. For clinical applications, much further study is needed.
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PMID:Application of quantitative RT-PCR using "TaqMan" technology to evaluate the expression of CK 18 mRNA in various cell lines. 1114 32

In this study amplification of cytokeratin-19 mRNA by Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect circulating tumor cells in the peripheral blood of patients with cancer of the head and neck before, during and after radiation therapy. Detection of cytokeratin-19-positive cells coincided with local failure, distant metastasis and anemia.
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PMID:Epithelial cells in the peripheral blood of patients with cancer of the head and neck: incidence, detection and possible clinical significance. 1132 51

A natural animal model for human head and neck squamous cell carcinoma (H/N SCC) has not been described. The domestic cat has a high spontaneous occurrence of oropharyngeal SCC, which is similar to the human disease in aggressiveness and incurability. We have developed a cell line (SCCF1) from a laryngeal SCC of a cat. Keratinocytes were maintained in culture for greater than 50 passages. SCCF1 had strong cytokeratin immunohistochemical staining, weak vimentin staining, and no p53 staining. Ultrastructual features included cytokeratin filaments and desmosomes, as well as features of anaplasia (irregular cytoplasmic and nuclear margins, surface filopodia, and abnormal intermediate filament production). Karyotype analysis revealed aneuploidy, with a stemline chromosomal number of 34. The cells grew logarithmically for 6 d until confluency. SCCF1 expressed parathyroid hormone-related protein (PTHrP) messenger ribonucleic acid (mRNA) and protein, and secreted the protein into the medium. Treatment of SCCF1 with transforming growth factor-beta increased PTHrP production but did not affect PTHrP mRNA stability. Reverse transcriptase-polymerase chain reaction was used to amplify a 282-base pair region of feline PTHrP mRNA, encoding portions of the pre-pro and coding regions. The complementary deoxyribonucleic acid (cDNA) was cloned and sequenced. The cDNA and the predicted amino acid sequences had a high degree of homology to human and canine PTHrP. RT-PCR was used to confirm alternate splicing of PTHrP mRNA for translation of PTHrP 1-139 and PTHrP 1-141. The SCCF1 cell line will permit mechanistic experiments on genetic dysregulation in neoplastic keratinocytes of the feline oropharynx, and development of an in vitro model for H/N cancer.
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PMID:Feline head and neck squamous cell carcinoma cell line: characterization, production of parathyroid hormone-related protein, and regulation by transforming growth factor-beta. 1177 73

Several reverse-transcriptase polymerase chain reaction (rtPCR) assays have been designed for the detection of disseminated cancer cells. The specificity of these discussed molecular approaches is controversial. Biological interference of the cytokeratin-20 and mammaglobin rtPCR assays has been investigated. Cell lines of different lineages and bone marrow and peripheral stem cells from patients without epithelial cancer have been examined for the transcription of the cytokeratin-20 (CK20) and mammaglobin messages prior to and after stimulation with different cytokines in a total of 370 liquid cultures. Amplification of both messages from clinical samples prior to stimulation does not support the high specificity for the detection of disseminated epithelial cancer cells as reported. Cytokeratin-20 was amplified from the chronic myeloic leukemia (CML)-derived line K562. Transcription was not influenced by cytokines, either in cell-line experiments or in clinical samples. The thesis of a low-level background transcription in granulocytes is supported. Mammaglobin was induced in cell lines significantly by GM-CSF and in clinical samples additionally by several more cytokines. These results indicate that under certain conditions involving cytokine production, the use of mammaglobin rtPCR for the detection of epithelial cancer cells could be limited. In conclusion, the mechanism of interference of both rtPCR assays are completely different and further research is necessary before the cytokeratin-20 or mammaglobin rtPCR could become standard methods for the detection of disseminated epithelial cancer cells. These factors leading to so-called false-positive results have to be considered in future applications of rtPCR for the detection of minimal residual disease.
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PMID:Interference of cytokeratin-20 and mammaglobin-reverse-transcriptase polymerase chain assays designed for the detection of disseminated cancer cells. 1177 68

The rhabdoid cell, which is typically observed in malignant rhabdoid tumor (MRT) and other malignant neoplasms, has an eosinophilic cytoplasm containing a spheroid perinuclear inclusion body. This distinct cell is known to act as a highly aggressive indicator in many types of malignant tumors and is characterized by aggregates of intermediate filaments, comprising both vimentin and cytokeratin (CK) 8, which is mainly expressed in simple-type epithelium such as liver and intestine. To clarify the cause of the inclusion body formation, we analyzed the alteration of the complete human CK8 gene (KRT 8: 1724 base pairs) in seven samples of MRT (three from frozen materials and four from cultured cell lines) by reverse-transcriptase polymerase chain reaction, followed by direct sequencing. In addition, the two cell lines, Huh7 and HeLa, which lacked rhabdoid feature, six pediatric malignant tumors, including three cases of primitive neuroectodermal tumor (PNET) and three of Wilms' tumor; and 15 normal liver tissue (as a control) were also analyzed. All MRT samples had missense mutations in the human KRT 8 gene, i.e., Arg89 --> Cys (5/7); Arg --> Cys251 (3/7); Glu267 --> Lys (6/7); Ser290 --> Ile, Met; (7/7) and Arg301 --> His(4/7), none of which was detected in any control samples. Among these mutations, the most noteworthy findings were that Arg89 belongs to the H1 subdomain of the head domain and that Arg251 belongs to the short nonhelical linker segment, or L1-2. Both these mutations are noted for their relationships to lateral protofilament-protofilament interactions. In addition, Ser290 has been previously reported to be a phosphorylation site, which has been recognized to play an important role in filament organization, leading to conformational change of the CK8 filaments. In conclusion, mutated codons of CK8 gene in MRT were located in the important region involved in the conformational change of intermediate filament.
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PMID:Mutation analysis of human cytokeratin 8 gene in malignant rhabdoid tumor: a possible association with intracytoplasmic inclusion body formation. 1185 May 43

Rhabdomyosarcomas (RMSs) are classified into embryonal (ERMS), alveolar (ARMS), and pleomorphic (PRMS) subtypes. ERMS, including botryoid variants, typically occurs in young children, ARMS typically occurs in older children and young adults, and PRMS occurs in older adults. Although ARMSs show thin fibrous bands separating nests of cells, abundant extracellular matrix production is rare in RMS. In the course of reviewing hyalinizing sarcomas we discovered a distinctive RMS in adults that closely mimicked osteosarcoma or chondrosarcoma because of the extensive matrix production. Four RMSs with hyalinized matrix were retrieved from our files. These cases were evaluated with respect to patient age and sex, tumor site and size, growth pattern, nuclear grade, cellularity, mitotic figures/20 high power fields, vascular invasion, necrosis, the presence of rhabdomyoblasts, multinucleated cells, and alveolar growth pattern. Immunohistochemistry for desmin, myogenin, MyoD1, actin, cytokeratin, S-100 protein, collagen II, and CD99 was performed. Reverse transcriptase polymerase chain reaction for the ARMS-associated PAX3/FKHR and PAX7/PKHF was also performed on three cases. The cases involved the forearm, hand, orbit, and nasopharynx of a 40-year-old woman, a 50-year-old man, an 18-year-old man, and a 21-year-old man, respectively. The tumors ranged from 3.7 to 8 cm and consisted of lobules and infiltrating cords of small round malignant cells embedded in a densely hyalinized matrix having both a chondroid and osteoid-like appearance. No definite lacunae or matrix calcification was present. An alveolar pattern was only present focally, and tumor giant cells were not present. One case had a single focus of rhabdomyoblastic differentiation with strap cells. Mitotic activity was >20 mitotic figures/20 high power fields in three of four cases. Immunohistochemically, one case strongly expressed desmin, whereas three cases expressed it focally, with a dot-like pattern. Myogenin was only focally positive, but MyoD1 was present in nearly every cell of each case. Two cases expressed actin and one expressed CD99. No case expressed cytokeratin, S-100 protein, or collagen II. Only one case contained adequate RNA for reverse transcriptase polymerase chain reaction, and this case was negative for the ARMS-associated gene fusions. Follow-up showed one patient to be dead of metastatic disease at 60 months despite intensive therapy, another patient to be disease free at 26 months, and the third patient to be disease free at 5 months. The fourth case is recent. These cases are a distinctive-appearing rhabdomyosarcoma easily mistaken for variants of chondrosarcoma, osteosarcoma, or even sclerosing epithelioid fibrosarcoma because of their hyalinizing appearance compounded by their typically focal and dot-like desmin expression. These four cases are essentially identical to the three unusual RMSs recently reported by Mentzel and Katenkamp as "sclerosing, pseudovascular rhabdomyosarcoma in adults." Although the focal alveolar architecture and the primitive cytologic appearance of these hyalinizing RMS suggest a relationship with ARMS, the presence of abundant strap cells in one case, the predominant expression of MyoD1 rather than myogenin, and the absence of ARMS-associated fusions genes point more strongly toward a variant of ERMS. However, the late adult age in two cases is unusual for both EMRS and ARMS, suggesting that sclerosing RMS may prove to be a distinct subtype of RMS. Study of additional cases will be necessary to more fully elucidate its place among RMS and its prognostic significance.
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PMID:Sclerosing rhabdomyosarcoma in adults: report of four cases of a hyalinizing, matrix-rich variant of rhabdomyosarcoma that may be confused with osteosarcoma, chondrosarcoma, or angiosarcoma. 1221 74


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