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 cyclin-dependent kinase 4 (cdk4) inhibitor (p16INK4/MTS1/CDKN2) gene has been recently identified as a putative tumor suppressor gene because of the high frequency of homozygous deletion observed in numerous human tumor cell lines, including leukemias. However, results obtained from uncultured tumor samples have led to discussion of the relevance of these findings. Using reverse transcriptase polymerase chain reaction (RT-PCR) and Southern blot analysis, we have investigated p16INK4A gene at both RNA and genomic levels in various types of leukemias: acute myeloid leukemia (AML) (n = 23); acute lymphocytic leukemia (ALL) (n = 22) and B cell chronic lymphoproliferative disorders (CLPD) (n = 33). p16INK4A mRNA expression was not found in only 1/20 AML and 2/23 CLPD samples. Conversely, p16INK4A mRNA was not detected in 5/17 ALL cases, and intensity of PCR products were barely detectable in seven additional cases, possibly related to the contamination by normal cells in some cases. By Southern blotting, a homozygous deletion of p16INK4A gene was found in 6/17 ALL cases (35%) among which 4/6 were negative or weakly positive by RT-PCR assay. None of the five AML and 20 CLL samples studied had p16INK4A deletion. Sequence analysis of p16INK4A exon 2 did not show point mutation in two of these cases lacking mRNA expression. Our data provide further evidence that among hematological malignancies, ALL are the most likely to be associated with p16INK4A inactivation, mainly by homozygous gene deletion. Since most hematological malignancies-except ALL-are infrequently associated with p16INK4A and retinoblastoma (Rb) gene alteration it seems worthwhile to explore cdk4 and cdk6 expression to determine whether or not the disruption of the p16INK4A/Rb/cdk4/cdk6 regulatory loop might play a role in their pathogenesis.
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PMID:Alterations of cyclin-dependent kinase 4 inhibitor (p16INK4A/MTS1) gene structure and expression in acute lymphoblastic leukemias. 763 Jan 99

A decrease in the intracellular concentrations of the transcripts for some tumor suppressor genes has been found during murine lung tumorigenesis; for p15INK4b and p16INK4a, this was due to homozygous deletions. We report here a decrease in the mRNA levels of the mutated in colorectal cancer (Mcc) and adenomatous polyposis coli (Apc) genes in mouse lung tumors and some neoplastic cell lines. This was assessed both by northern blotting and reverse transcriptase-polymerase chain reaction of RNA isolated from lung tumors that had been induced by urethane, N-nitrosodiethylamine, or 3-methylcholanthrene in (A/J x C57BL/6) F1 or A/J mice. A reduced amount of both Mcc and Apc messages was also seen when two neoplastic cell lines, a spontaneous transformant (E9) and a line derived from a chemically induced solid tumor (82-132), were compared with two independently derived nontumorigenic cell lines (E10 and C10); E9 was derived from E10, and all of these lines are probably of alveolar type 2 cell origin. A cell line derived from a chemically induced papillary lung tumor probably of bronchiolar Clara cell origin (LM2) had Mcc mRNA levels similar to those of C10 and E10 but reduced Apc mRNA levels. A line (p53-823) derived from a papillary tumor that arose in a mouse with a mutated p53 transgene had a reduced amount of the Mcc gene product only. These differential changes in the relative amounts of Apc and Mcc messages in LM2 and p53-823) cells may serve as useful models for studying the regulation of their expression. Both messages had half-lives of 6-9 h in normal E10 and neoplastic E9 cells, so decreased message stability does not account for these reductions. This is the first report of estimated degradation rates of these mRNAs. Apc and Mcc message content did not vary as a function of growth status of the cell lines. Single-strand conformation polymorphism analysis did not reveal mutations in Apc coding regions known to have a high mutation frequency in human colon tumors. Loss of heterozygosity of Apc and Mcc was not found in tumors that developed in the F1 mice, implying a lack of allelic deletions. These changes in tumor suppressor gene expression may contribute to the development and maintenance of neoplasia in lung epithelium.
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PMID:Decreased expression of the adenomatous polyposis coli (Apc) and mutated in colorectal cancer (Mcc) genes in mouse lung neoplasia. 947 70

Cyclin A is a cell cycle regulatory protein that functions in mitotic and S phase control in mammalian cells. However, in contrast to other G1 phase regulatory proteins, such as cyclin D, retinoblastoma protein and p16INK4A, cyclin A seems not to be commonly involved in tumorigenesis. Recently, a second human cyclin A--cyclin A1--has been identified. In contrast to cyclin A which is expressed throughout embryonic development and in adult tissue, the expression of cyclin A1 has been reported to be restricted to embryonic and germ line cells. We have confirmed the absence of cyclin A1 mRNA from normal peripheral blood leukocytes of seven healthy donors by single step reverse transcriptase-polymerase chain reaction (RT-PCR). Furthermore, we have examined the expression of cyclin A1 mRNA in 173 peripheral blood samples of 162 patients with various hematological malignancies. Cyclin A1 mRNA was detectable in 11 of 11 patients with acute myeloid leukemia, three of three patients with acute biphenotypic leukemia, eight of eight patients with myelodysplastic syndrome, 59 of 69 patients with chronic myelogenous leukemia (CML) at diagnosis, 13 of 15 patients with CML in blastic transformation, 10 of 18 patients with chronic lymphocytic leukemia, two of nine patients with essential thrombocythemia, and only two of 10 patients with acute lymphoblastic leukemia (ALL) with both cyclin A1 RT-PCR positive ALL leukemias being undifferentiated relapses. In addition, cyclin A1 mRNA was found in one of six leukapheresis products, harvested from individuals without hematological disorders. Taken together, cyclin A1 is expressed in the majority of myeloid and undifferentiated hematological malignancies as well as in normal hematopoietic progenitor cells. We conclude that cyclin A1, a protein potentially involved in G1/S phase progression of immature cells, might be necessary for proliferation of early hematopoietic progenitor cells and their leukemic counterparts being blocked at that stage of differentiation.
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PMID:Cyclin A1 is predominantly expressed in hematological malignancies with myeloid differentiation. 963 17

Two proteins, p16INK4A and p14ARF, originating from the same gene locus CDKN2A, use different promoters and alternative reading frames. p16INK4A is translated from alpha transcript and p14ARF is from beta transcript. These two proteins, which are inactivated in some human malignancies, are possible tumour suppressor candidates. In this study, we investigated the expression of p16INK4A and p14ARF mRNAs in haematological malignancies. We studied eight normal bone marrow samples, three reactive granulocytic hyperplasia patients, and 21 haematological malignancy patients, including seven acute myelogenous leukaemia, four acute lymphoblastic leukaemia, five myelodysplastic syndrome, five chronic myelogenous leukaemia (CML). p16INK4A and p14ARF mRNA expression was assayed by reverse transcriptase polymerase chain reaction. Normal bone marrows and reactive granulocytic hyperplasia showed barely detectable expression of either mRNA. In contrast, p16INK4A and p14ARF mRNA expression was abnormally increased in patients with haematological malignancies. Especially in CML, overexpression of p16INK4A and p14ARF mRNAs was more frequent than in controls (80 and 60%, respectively, P < 0.05). In conclusion, p16INK4A and p14ARF mRNA expression was frequently increased in haematological malignancies, especially in CML. We suggest that overexpression of these mRNAs may be related to the pathogenesis of haematological malignancies.
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PMID:Overexpression of p16INK4A and p14ARF in haematological malignancies. 1527 71

Hemizygous deletions in genomic DNA appear to play an important role in tumorigenesis. The loss or inactivation of tumour suppressor genes (TSGs) is of critical importance in most malignancies, and has been shown to affect response to therapy. Here, we report a quantitative real-time polymerase chain reaction (qPCR) designed to detect two TSGs at the CDKN2A locus, p16(INK4A) and p14(ARF) that allows the detection of hemizygous deletions. Testing by qPCR of 18 bone marrow specimens from paediatric acute lymphoblastic leukaemia (ALL) patients at diagnosis revealed nine to be GG, six to be GD and three to be DD for exon 2 of p14(ARF)/p16(INK4A), concordant with Southern blotting analysis. A panel of 13 ALL cell lines was investigated for deletions at the CDKN2A locus and one of the lines, typed as GD for all exons, was further assessed by fluorescence in situ hybridisation, confirming the qPCR findings. The expression levels of p16(INK4A) and p14(ARF) were measured in all cell lines and these quantitative reverse transcriptase PCR results also agreed with the typing by qPCR. The qPCR method described is suitable for detection of hemizygous loss in primary patient material and the accuracy of the method was verified by three independent techniques.
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PMID:Detection of hemizygous deletions in genomic DNA from leukaemia specimens for the diagnosis of patients. 1560 65

Fetal cardiomyocytes have been proposed as a potential source of cell-based therapy for heart failure. This study examined cellular senescence in cultured human fetal ventricular cardiomyocytes (HFCs). HFCs were isolated and identified by immunocytochemistry and RT-PCR. Cells were found to senesce after 20-25 population doublings, as determined by growth arrest, morphological changes and senescence-associated beta-galactosidase activity. Using the telomeric repeat amplification protocol assay, telomerase activity was undetectable in primary HFCs. Cells were transduced to express the human reverse transcriptase subunit (hTERT) of telomerase. This resulted in greatly increased telomerase activity, but no significant lifespan extension. Analysis of telomere length in primary HFCs revealed that the senescent phenotype was not accompanied by telomere shortening. Telomeres in hTERT-positive cells were elongated in comparison with primary cells, and elongation was retained in senescent cells. Levels of the tumor suppressor protein p16INK4A increased in all senescent cells whether telomerase-positive or -negative. Senescence was accompanied by a decline in transcript levels of the polycomb gene Bmi-1, Ets1 and Ets2 transcription factors, and Id1, Id2 and Id3 helix-loop-helix proteins, suggesting roles for these genes in maintenance of cardiomyocyte proliferative capacity. In addition to offering novel insights into the behavior of human fetal cardiomyocytes in culture, these findings have implications for the development of a cell-based therapy for cardiac injury using primary fetal heart tissue.
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PMID:Telomere-independent cellular senescence in human fetal cardiomyocytes. 1565 10

The molecular mechanisms of acute hepatitis C virus (HCV) infection, end-stage hepatitis (cirrhosis), and hepatocellular carcinoma have been extensively studied, but little is known of the changes in liver gene expression during the early stages of liver fibrosis associated with chronic HCV infection, that is, the transition from normal liver (NL) of uninfected patients to the first stage of liver fibrosis (F1-CH-C). To obtain insight into the molecular pathogenesis of F1-CH-C, we used real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to study the mRNA expression of 240 selected genes in liver tissue with F1-CH-C, in comparison with NL. The expression of 54 (22.5%) of the 240 genes was significantly different between F1-CH-C and NL; 46 genes were upregulated and 8 were downregulated in F1-CH-C. The most noteworthy changes in gene expression mainly affected the transcriptional network regulated by interferons (IFNs), including both IFN-alpha/beta-inducible genes (STAT1, STAT2, ISGF3G/IRF9, IFI27, G1P3, G1P2, OAS2, MX1) and IFN-gamma-inducible genes (CXCL9, CXCL10, CXCL11). Interesting, upregulation of IFN-alpha/beta-inducible genes (but not IFN-gamma-inducible genes) was independent of histological scores (grade and stage of fibrosis) and HCV characteristics (hepatic HCV mRNA levels and the HCV genotype), and was specific to HCV (as compared to hepatitis B virus (HBV)). Other genes dysregulated in F1-CH-C, albeit less markedly than IFN-alpha/beta- and IFN-gamma-inducible genes, were mainly involved in the activation of lymphocytes infiltrating the liver (IFNG, TNF, CXCL6, IL6, CCL8, CXCR3, CXCR4, CCR2), cell proliferation (p16/CDKN2A, MKI67, p14/ARF), extracellular matrix remodeling (MMP9, ITGA2), lymphangiogenesis (XLKD1/LYVE), oxidative stress (CYP2E1), and cytoskeleton microtubule organization (STMN2/SCG10). Thus, a limited number of signaling pathways, and particularly the transcriptional network regulated by interferons, are dysregulated in the first stage of HCV-induced liver fibrosis. Some of the genes identified here could form the basis for new approaches aimed at refining IFN-based therapies for chronic HCV infection.
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PMID:Molecular profiling of early stage liver fibrosis in patients with chronic hepatitis C virus infection. 1566 Nov 46

We have established a spontaneously immortalized chicken embryo fibroblast (CEF) cell line (SC-1) that has been in continuous culture for more than three years. This is only the second report of a spontaneously immortalized reverse transcriptase (RT)-negative chicken cell line. The SC-1 cells emerged from crisis (at about passage 29-31) with a slower growth rate than primary cells. Passage 50 SC-1 cells expressed similar levels of p53 mRNA, but slightly lower levels of p53 protein than passage 6 CEF cells. By passage 120, p53 mRNA levels were significantly decreased in the SC-1 cells, while protein levels were slightly increased compared to passage 6 CEF cells. However, functional analysis of p53 revealed reduced activity in later passage SC-1 cells. Other p53-related genes including p21WAF1, p27Kip1, MDM-2, and the p16INK4a alternate reading frame (ARF) sequence showed similar patterns of differential mRNA expression. Levels of p15INK4b mRNA and protein were dramatically decreased in SC-1 cells, suggesting that the Rb pathway also has been compromised. Telomerase expression was undetectable in SC-1 cells. Fluorescence-activated cell sorting analysis showed that SC-1 and primary cells contained a similar proportion of G0/G1 phase cells, unlike the only other spontaneously immortalized chicken cell line (DF-1). The present study suggests that alterations in the p53 and Rb pathways cause fluctuations in expression levels of important cell-cycle regulatory genes during crucial transition periods as the SC-1 spontaneously immortalized chicken fibroblast cells progress toward becoming a fully committed cell line.
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PMID:Modulation of p53 expression and its role in the conversion to a fully immortalized chicken embryo fibroblast line. 1631 5

Although inorganic arsenate (iAs(V)) or arsenite (iAs(III)) is clearly a human carcinogen, it has been difficult to produce tumors in rodents. In the present study, we orally administered iAs(V) to A/J mice to examine arsenic carcinogenicity in rodent. A/J mice (male, n = 120) assigned to four groups were given drinking water containing 0, 1, 10, and 100 ppm iAs(V) for 18 months. At the end of experiment, the complete lungs were removed and used for examining histopathology and extracting RNA and DNA. Epigenetic effects of iAs(V) on DNA methylation patterns of p16INK4a and RASSF1A genes were determined by methylation-specific polymerase chain reaction. Changes of p16INK4a and RASSF1A at mRNA and protein levels were examined by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Arsenic was accumulated dose dependently in the lung tissues of iAs(V)-exposed mice. Increase in lung tumor number and lung tumor size was observed in iAs(V)-exposed mice compared to the control. Histopathological examination showed that the rate of poorly differentiated lung adenocarcinoma was much higher in iAs(V)-exposed mice than in the control. Methylation rates appeared to be higher in a dose-related tendency in lung tumors from iAs(V)-exposed mice compared to the control. Lower or loss of p16INK4a and RASSF1A expression was found in lung tumors from iAs(V)-exposed mice, compared to that in nontumor lung tissues from both control and iAs(V)-exposed mice, and this reduced or lost expression was in accordance with hypermethylation of the genes. In conclusion, iAs(V) exposure increased lung tumor incidence and multiplicity in A/J mice. Epigenetic changes of tumor suppressor genes such as p16INK4a and RASSF1A are involved in the iAs(V)-induced lung carcinogenesis.
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PMID:Chronic oral exposure to inorganic arsenate interferes with methylation status of p16INK4a and RASSF1A and induces lung cancer in A/J mice. 1654 96

In the present study, we investigated methylation status of the CpG islands of some major tumor suppressor genes both in human hepatocellular carcinoma and liver cancer cell lines and examined whether demethylation by arsenic trioxide (As2O3) could restore their expression in the cell lines. HepG2 and Huh-7 cells were treated with 2 to 10 micromol/L of AS2O3 and/or 1 micromol/L of 5-aza-2'-deoxycytidine for 24, 48, and 72 hours. The methylation status of the CpG island around the promoter regions of p161NK4a, RASSF1A, E cadherin, and GSTP1 was detected by a methylation-specific polymerase chain reaction (MSP). The messenger RNA (mRNA) and protein levels of these genes were determined by quantitative real-time reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemical analyses. The DNA methyltransferase (DNMT) mRNA levels and enzyme activity were also examined. The hypermethylated status of the promoter regions of p16INK4a, RASSF1A, E cadherin, and GSTP1 was observed in 10 (40%), 14 (56%), 6 (24%), and 12 (48%) of 25 patients with hepatocellular carcinoma, respectively. CpG methylation of the p16INK4a, RASSF1A, E cadherin, and GSTP1 genes was correlated to the reduction of mRNA levels in the cell lines, and mRNA expression of these 4 genes were indeed restored by low concentrations (2-6 micromol/L) of As2O3 through demethylation, as well as 1 micromol/L of 5-aza-2'-deoxycytidine. Western blot and immunohistochemical analyses confirmed that each protein was markedly enhanced after treatment with a low concentration of As2O3. In contrast, As2O3 at a high concentration (10 micromol/L) damaged cell membranes and remarkably suppressed these 4 protein levels. As2O3 decreased the mRNA expression of DNMT 1 and also dose-dependently inhibited DNMT activity. In conclusion, a low concentration of As2O3 induces CpG island demethylation of tumor suppressor genes by inhibition of DNMT and reactivates the partially/fully silenced genes in liver cancer cells.
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PMID:Arsenic trioxide inhibits DNA methyltransferase and restores methylation-silenced genes in human liver cancer cells. 1661 25


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