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 expression and biological function of interleukin-6(IL-6), and its receptor mRNA, were studied in a human megakaryocytic cell line (CMK). IL-6 possessed stimulatory effects on the DNA synthesis as well as colony formation of CMK cells. The IL-6 receptor mRNA could be detected by the method of reverse transcriptase polymerase chain reaction (RT-PCR) but not Northern blotting. On the contrary, IL-6 mRNA was detected by the method of RT-PCR, and its expression induced by the addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) could be clearly shown by Northern blotting. These findings indicate that IL-6 and its receptor mRNA should be analyzed by both methods, and the growth and differentiation of CMK cells may be controlled by an IL-6 autocrine loop. Next, the expression and biological role of low molecular GTP-binding proteins (smg p21A and -B) mRNAs were examined in CMK cells. Both the smg p21A and -B mRNAs were detected in CMK cells using Northern blotting, and their levels were markedly elevated by TPA treatment. The mRNA level of glycoprotein IIb, a typical marker of the megakaryocytes, was increased by TPA, but the time course of the increase in the smg p21 mRNA levels was more rapid that that in the GPIIb mRNA level. These findings suggest that smg p21s play an important role during the TPA-induced differentiation of CMK cells.
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PMID:[Expression and detection of platelet specific genes in human megakaryocytic cells]. 177 68

Human continuous bone marrow cultures were established from intraoperative marrow specimens and infected with amphotropic murine leukemia virus (Am-MuLV) pseudotypes of Kirsten or Harvey murine sarcoma virus, and the biologic effects were compared with mouse continuous bone marrow cultures. Cultures were tested for production of total nonadherent granulocytes and granulocyte-macrophage progenitor cells (GM-CFUc); virus replication by supernatant reverse transcriptase activity; percentage of adherent and nonadherent cells and GM-CFUc that released virus by infectious center assay; and for synthesis of Harvey ras p21 protein. High-efficiency, stable Am-MuLV infection of over 90% of human marrow-culture nonadherent and adherent cells and both seven- and 14-day GM-CFUc were detected as Kirsten or Harvey pseudotype virus release by infectious center assay. Synthesis of Harvey ras p21 was detected in the adherent and nonadherent cell populations of human as well as mouse continuous marrow cultures infected with Kirsten or Harvey pseudotype virus. In contrast to data with mouse cultures, cumulative production of GM-CFUc and differentiated granulocytes in human cultures was not detectably altered by Harvey or Kirsten virus infection, and all cultures ceased to produce hematopoietic cells by 20 weeks. Of 54 virus-infected cultures in ten separate experiments, 13 produced a second peak of nonadherent cells (greater than 10(5) per flask) after 20 weeks, significantly more frequently than did control uninfected cultures (one of 32). When subcultured, these harvests produced permanent Epstein-Barr virus (EBV)-transformed pre-B cell lines that released the original inoculating pseudotype virus. Thus, Am-MuLV is a potentially valuable vector for inserting genetic sequences by recombinant techniques into human hematopoietic and stromal cells in culture; however, activation of EBV may be a significant complication.
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PMID:Amphotropic retrovirus vector transfer of the v-ras oncogene to human hematopoietic and stromal cells in continuous bone marrow cultures. 257 39

The primary structure analysis of the gag gene products of human T-cell leukemia virus (HTLV)-ICR has been nearly completed. A comparison of the amino acid sequences with the published nucleotide sequence of HTLV-IATK established that i) p19 which is known to share antigenic determinants with a protein present in normal thymic epithelium, is nevertheless virally coded. ii) The gene order and complete primary structure of the gag precursor (Pr55) which has been shown to be myristylated (My) at its N-terminus is My-p19-p24-p15-OH; and iii) the Pr55gag amino acid sequences of HTLV-ICR and HTLV-IATK are nearly identical showing only a single residue difference in the C-terminal region of p15. Antibodies to synthetic peptides inferred from the nucleotide sequence of the env gene of HTLV-IATK were also raised and used to identify and purify env precursor gPr62-68, surface glycoprotein gp46-51 and transmembrane protein p21. While most of the peptide sera were shown to be subgroup specific some of them detected antigenic determinants shared between protein homologs of viruses of subgroups I and II. Partial or complete amino acid sequences of both the gag and env gene coded proteins of bovine leukemia virus (BLV) structural proteins have also been determined. These extensive protein data together with nucleotide sequences confirm and extend our initial finding that HTLV and BLV are structurally and antigenically related and may have originated from common ancestor. The structural and immunological studies revealed also relationships between HTLV and a number of type C and type D retroviruses studied. One of the highly conserved sequences is shared by the transmembrane proteins of these retroviruses which have been implicated in immunosuppression. It is conceivable that these common regions have common biological function. Two previously unidentified proteins of BLV have also been purified and structurally characterized. Nucleotide sequences capable of coding for related products are present in HTLV. The nature and possible biological functions of these new BLV proteins and the putative HTLV gene products will be discussed. The size and complexity of the genome of the replication competent retroviruses are similar but not identical. The 35S RNA of all replication competent helper viruses is divided into three genes encoding the viral structural proteins: the gag (group-specific antigen) gene codes for the internal structural proteins, the pol (polymerase) gene codes for the enzymes protease, reverse transcriptase and endonuclease and the env (envelope) gene codes for the proteins of the viral envelope.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Structural and antigenic characterization of the proteins of human T-cell leukemia viruses and their relationships to the gene products of other retroviruses. 610 Jun 35

A retroviral vector system was developed to transduce a K-ras antisense construct efficiently into human cancer cells. A 2-kb fragment of K-ras gene DNA in antisense orientation was linked to a beta-actin promoter and inserted into retroviral vector LNSX in two different orientations. The constructs were transfected into amphotropic packaging cell line GP+envAm12 followed by alternating transduction between the ecotropic packaging cell line psi-2 and GP+envAm12. Titers up to 9.7 x 10(7) colony-forming units (cfu)/ml were achieved without detectable replication-competent virus. The human large cell lung carcinoma cell line H460a, which has a homozygous codon 61 K-ras mutation, was transduced with an efficiency of 95% after five to seven repeated transductions. DNA polymerase chain reaction (PCR) and genomic DNA Southern blot analysis showed that the retroviral construct was integrated into the genome of H460a cells. K-ras antisense RNA expression was detected in the cells by Northern analysis, slot blot hybridization, and reverse transcriptase-PCR. Translation of the mutated K-ras p21 protein RNA was specifically inhibited, whereas expression of other p21 species was unchanged. Proliferation of H460a cells was suppressed 10-fold following transduction by the antisense construct. Colony formation in soft agarose and tumorigenicity in an orthotopic lung cancer model in nu/nu mice were dramatically reduced in H460a cells expressing antisense K-ras. We conclude that an antisense construct for K-ras can be expressed effectively in a retroviral vector that can efficiently transduce human cancer cells.
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PMID:Retroviral vector-mediated transduction of K-ras antisense RNA into human lung cancer cells inhibits expression of the malignant phenotype. 839 92

Cell cycle progression requires activation of different cyclin-dependent kinases (CDKs) which are positively regulated by cyclins and negatively regulated by CDK inhibitors. Growth inhibition of the Calu-1 lung carcinoma cells induced with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, is associated with G2/M arrest and induction of expression of a novel, faster-migrating form of p21(WAF1/CIP1/SDI1) (p21) protein, an inhibitor of cyclin-dependent kinases. This faster-migrating p21 protein was also expressed in TPA-treated A549 lung carcinoma cells which also exhibited G2/M arrest but not in TPA-treated U937 leukemia cells, which only expressed a slower-migrating form of p21 protein. However, reverse transcriptase-polymerase chain reaction and Southern analysis demonstrated no evidence of novel splice in TPA-treated Calu-1 cells. On the other hand, immunoblotting analysis demonstrated that the faster-migrating p21 protein could be detected only by peptide antibody directed against the N terminus but not the C terminus, suggestive of truncation of the latter or protein modification that results in the loss of the C-terminal epitope. Correlation of G2/M arrest with expression of the faster-migrating p21 protein suggests that this novel form of p21 protein may be a mediator of G2/M arrest and growth inhibition.
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PMID:Novel form of p21(WAF1/CIP1/SDI1) protein in phorbol ester-induced G2/M arrest. 893 83

Continuous antiprogestin administration to hormone replaced, castrate monkeys inhibits estrogen-induced endometrial proliferation through mechanisms which remains unclear. To elucidate the molecular mechanisms of RU486-induced endometrial suppression, we treated six intact female cynomolgus monkeys on cycle days 2-22 sequentially with placebo, RU486 (1 mg/kg/day) and levonorgestrel (LNG) (2 microg/kg/day) intramuscularly (i.m.), with uterine wedge sections and endometrial biopsies collected on day 22 of each cycle. The uterine sections were evaluated for morphology, mitosis and proliferating cell nuclear antigen (PCNA) immunohistochemistry. Changes in the mRNA levels of ER, PR, cyclin-B and tumour suppressor gene p21 were assessed using co-amplification with beta-actin by reverse transcriptase-polymerase chain reaction (RT-PCR). Administration of RU486 uniformly resulted in characteristic suppression of endometrium with few mitosis, dense stroma and simple glands, whereas the effects of LNG were less uniform. Following RU486 administration, the levels of endometrial ER and PR mRNA were comparable to proliferative phase endometrium, and significantly higher than those seen in the secretory endometrium, indicating that some of the biological actions of E2 were not inhibited during RU486 treatment. Despite scarce mitosis, PCNA was readily detectable in all samples. Curiously, in comparison to secretory phase controls, the levels of cyclin-B, but not p21, mRNA were markedly increased following RU486. The effects of LNG on the levels of these mRNA species varied, with mean levels falling between those of the secretory phase controls, and RU486-treated specimens. The increase in cyclin-B mRNA and lack of mitosis suggests that anti-proliferative actions of RU486 in the primate endometrium might be associated with a cell-cycle block at the G2-M interphase. Whether mechanisms similar to these are associated with the beneficial clinical effects of RU486 seen in the treatment of various hormone dependent maladies remains to be determined.
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PMID:Endometrial effects of RU486 in primates--antiproliferative action despite signs of estrogen action and increased cyclin-B expression. 901 Mar 33

The tumor suppresser p53 is a cell cycle checkpoint protein that contributes to the preservation of genetic stability by mediating either a G1 arrest or apoptosis in response to DNA damage. p53 causes growth arrest through transcriptional activation of the cyclin-dependent kinase inhibitor p21. During p53-mediated suppression of cell proliferation, p21 is important for coordinating cell cycle progression, DNA replication, and repair of damaged DNA. The purpose of this study is to investigate the expression of p53 and p21 mRNA in association with DNA damage and normal repair in acute immune complex alveolitis in mice. Male ICR mice were injected intravenously with IgG antibodies against oval albumin, aerosolized with oval albumin solution, and killed at 4, 6, 12, 24, and 48 hours and 1 week after aerosolization. We assessed the expression of p53 and p21 mRNA by reverse transcriptase (RT)-PCR and by RT in situ PCR. We also assessed DNA damage by terminal deoxynucleotidyl transferase mediated biotin-dUTP nick-end-labeling (TUNEL) and by gel electrophoresis of DNA extracted from lung tissues. The results of RT-PCR and RT in situ PCR showed that p53 and p21 mRNA were concurrently up-regulated at 4 to 48 hours after aerosolization in alveolar epithelial cells. Bronchial and alveolar epithelial cells were positively stained by TUNEL in this period but not at 1 week after aerosolization or in control mice. The result of electrophoretic analysis of DNA was compatible with that of TUNEL. These studies suggest that the responses of p53 and p21 mRNA are associated with physiologic processes of DNA damage and repair in acute immune complex alveolitis in mice.
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PMID:P53 and p21 (Waf1/Cip1) mRNA expression associated with DNA damage and repair in acute immune complex alveolitis in mice. 904 52

Human T-cell leukemia virus type 1 (HTLV-1) infection is associated with adult T-cell leukemia and HTLV-associated myelopathy/tropical spastic paraparesis. Inhibition of HTLV-1 transmission is important to prevent the above HTLV-1-associated diseases. We used the antisense oligodeoxynucleotides (oligos) complementary to the first splice junction, rex responsive site, gag, env, tax, rex, and p21 and evaluated the effects on the syncytium formation between HTLV-1 producing human T-cell line, C9/PL cells, and HTLV-1-uninfected human glioma cell line, U251-MG cells. The syncytium formation was significantly inhibited the virion production assayed by antisense oligos to env, tax, gag, p21, and rex, with antisense oligo to env being the most inhibitory. Antisense oligos to env and tax also inhibited reverse transcriptase activity. Antisense oligo to env may have a potential as a preventive measure of HTLV-1 replication and transmission in vivo.
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PMID:Inhibition of human T-cell leukemia virus type 1 replication by antisense env oligodeoxynucleotide. 947 88

The inhibitor of cyclin-dependent kinases WAF1/p21 has been shown to mediate cell cycle arrest by p53 and other factors. We have studied its expression in urothelial carcinoma. Immunohistochemistry of paraffin-embedded tissues revealed no detectable p21 protein in normal mucosa, whereas 8 of 17 (47%) carcinomata in situ, 41 of 62 (66%) pTa, 14 of 30 (47%) pT1 and 5 of 15 (33%) muscle-invasive tumours stained positive, usually with a heterogeneous pattern. Expression of p21 was associated with low grade tumours. In contrast, the frequency of p53 accumulation increased with grade and stage as did the frequency of staining for the proliferation marker Ki67. The level of WAF1 mRNA was determined relative to beta-actin mRNA by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in 15 freshly frozen invasive tumours. In eight samples obtained from normal bladder mucosa, the values ranged from 0.93 to 2.19 arbitrary units (AU) (mean 1.54+/-0.37 AU), but varied widely from non-detectable to 16.21 AU (mean 3.02+/-4.44 AU) in the tumour specimens. In accord with the immunohistochemical findings, WAF1 mRNA expression was elevated over the range found in normal mucosa in 5 of 15 advanced tumours. In addition, RNA analysis revealed a decrease in expression in six tumours. No mutations were observed in the WAF1/p21 gene in these tumours, but two were heterozygous for the codon 31 polymorphism. These data indicate that p21 is frequently expressed in superficial, well differentiated urothelial carcinomas, but less often in muscle-invasive urothelial carcinomas, irrespective of their p53 status. The expression of p21 and its prevalence in low-stage tumours may reflect residual growth-regulatory influences potentially impeding but not necessarily inhibiting tumour development.
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PMID:Frequent and heterogeneous expression of cyclin-dependent kinase inhibitor WAF1/p21 protein and mRNA in urothelial carcinoma. 948 5

Recent methodological developments allow expression measurement of many genes simultaneously, thereby revealing patterns of gene expression that can be related to phenotype. We hypothesized that through the use of such methods we could identify patterns of gene expression associated with the malignant phenotype in human bronchial epithelial cells (BEC). To test this hypothesis, a recently developed quantitative reverse transcriptase polymerase chain reaction method was used to assess simultaneously expression of 15 genes mechanistically associated with cell-cycle control (c-myc, E2F-1, p21, rb, PCNA, cyclin D2, cyclin D3, cyclin E, cdc2, CDK2, CDK4, mad, max p21, max p22, and p53) in normal cell cultures from five individuals and in nine different malignant BEC lines. Relative to the mean expression levels in cultured normal cell populations, expression of c-myc, E2F-1, PCNA, cyclin E, and CDK4 messenger RNA (mRNA) were significantly increased and expression of p21 and p53 mRNA were significantly decreased in one or two, but not all three subtypes (squamous, adenocarcinoma and small cell) of carcinoma cell lines evaluated. No single cell-cycle control gene discriminated all three subtypes from normal cell populations. In contrast, the gene expression index c-myc x E2F-1/p21 separated all carcinoma cell lines from all normal cell populations initially evaluated. This malignancy index was validated in an additional three cultured normal BEC and three carcinoma cell lines, as well as three pairs of matched primary normal bronchial epithelial and primary bronchogenic carcinoma samples, and three pairs of matched primary normal lung parenchyma and primary bronchogenic carcinoma tissue. Again, the c-myc x E2F-1/ p21 index successfully discriminated all cultured and primary normal from malignant samples and thereby had a predictive value of 1 (no false positives and no false negatives). We hypothesize that because of functional mutations in cell-cycle regulatory genes (e.g., p53 and/or rb), cells lose the ability to maintain a pattern of gene expression mechanistically associated with normal, division-limited homeostatic equilibrium. Because the c-myc x E2F-1/p21 gene expression index has high specificity for malignant tissue, it will allow confirmation that there is a significant amount of tumor tissue present in small (e.g., fine-needle) biopsy specimens prior to evaluating them for expression of other genes, such as those involved in chemoresistance or radioresistance. In addition, the goal of most gene therapy efforts is to alter levels of gene expression quantitatively. This index and others derived in a similar manner may better define potential gene therapy targets as well as response of targeted genes to therapy.
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PMID:The gene expression index c-myc x E2F-1/p21 is highly predictive of malignant phenotype in human bronchial epithelial cells. 965 Nov 76


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