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)

Transformation of hematopoietic cells by the p210bcr/abl tyrosine kinase appears to require the expression of a functional MYC protein, suggesting that simultaneous targeting of BCR-ABL and c-myc might be a rational strategy for attempting treatment of Phil-adelphia leukemia. To test this hypothesis, severe combined immunodeficiency mice injected with Philadelphia leukemic cells were treated systemically with equal doses of bcr-abl or c-myc antisense oligodeoxynucleotides (ODNs) or with both ODNs in combination. Compared with the mice treated with individual agents, the disease process was much slower in the group treated with both ODNs, as revealed by flow cytometry, clonogenic assay, and reverse transcriptase-polymerase chain reaction analysis to detect leukemic cells in mouse tissue cell suspensions, and by enumeration of liver metastases. The retardation of the disease process was positively correlated with a markedly increased survival of leukemic mice treated with both ODNs. These data demonstrate the therapeutic potential of targeting multiple cooperating oncogenes.
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PMID:Leukemia treatment in severe combined immunodeficiency mice by antisense oligodeoxynucleotides targeting cooperating oncogenes. 750 9

A sensitive assay of multiple mRNAs by reverse transcriptase-polymerase chain reaction was adopted to study the hormonally regulated expression of steroidogenic enzymes in primary rat granulosa cells in culture. As little as 15-60 ng total RNA prepared from cultured cells were reverse transcribed in the presence of pd(T)6, and polymerase chain reaction was conducted in the presence of specific oligonucleotide pairs designed to identify cDNAs of steroidogenic enzymes. In combination with Northern blot analysis of cholesterol side-chain cleavage cytochrome P450 (P450scc) message, it is shown that a novel protein kinase inhibitor, tyrphostin AG18, arrests the FSH-induced accumulation of P450scc mRNA. This inhibition is dose dependent (IC50, 15 microM) and reversible. The addition of 80 microM AG18 to cells containing high levels of P450scc mRNA caused a rapid decline of the cytochrome message (t 1/2, 5 h), similar to the effect of 30 micrograms/ml alpha-amanitin. However, concomitant addition of the two drugs did not accelerate the mRNA degradation process, suggesting that AG18 does not affect message stabilization. Tyrphostin AG18 did not affect mRNA species that are not FSH inducible, such as the ribosomal protein L19, or the constitutively expressed low levels of steroid 5 alpha-reductase mRNA. Moreover, even the extremely high levels of P450scc mRNA in granulosa-lutein cells, being cAMP independent and terminally differentiated a few hours after LH surge, were not affected by the addition of AG18 in culture. In contrast, two additional key and FSH-inducible steroidogenic enzymes, i.e. aromatase cytochrome P450 and 3 beta-hydroxysteroid dehydrogenase-I, were inhibited by AG18 at their mRNA levels. These results suggest that an as yet undetermined tyrosine kinase pathway is involved in the cAMP-dependent signal transduction pathway of FSH action, so that the presence of AG18 does not allow FSH induction of gene expression to occur.
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PMID:Tyrosine kinase inhibitor AG18 arrests follicle-stimulating hormone-induced granulosa cell differentiation: use of reverse transcriptase-polymerase chain reaction assay for multiple messenger ribonucleic acids. 751 96

Signal transduction of cytokine receptors is mediated by the JAK family of tyrosine kinases. Recently, the kinase partners for the interleukin (IL)-2 receptor have been identified as JAK1 and JAK3. In this study, we report the identification of splice variants that may modulate JAK3 signaling. Three splice variants were isolated from different mRNA sources: breast (B), spleen (S), and activated monocytes (M). Sequence analysis revealed that the splice variants contain identical NH2-terminal regions but diverge at the COOH termini. Analyses of expression of the JAK3 splice isoforms by reverse transcriptase-polymerase chain reaction on a panel of cell lines show splice preferences in different cell lines: the S-form is more commonly seen in hematopoietic lines, whereas the B- and M-forms are detected in cells both of hematopoietic and epithelial origins. Antibodies raised against peptides to the B-form splice variant confirmed that the 125-kDa JAK3B protein product is found abundantly in hematopoietic as well as epithelial cells, including primary breast cancers. The lack of subdomain XI in the tyrosine kinase core of the B-form JAK3 protein suggests that it is a defective kinase. This is supported by the lack of detected autokinase activity of the B-form JAK3. Intriguingly, both the S and B splice isoforms of JAK3 appear to co-immunoprecipitate with the IL-2 receptor from HUT-78 cell lysates. This and the presence of multiple COOH-terminal splice variants coexpressed in the same cells suggest that the JAK3 splice isoforms are functional in JAK3 signaling and may enrich the complexity of the intracellular responses functional in IL-2 or cytokine signaling.
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PMID:A kinase-deficient splice variant of the human JAK3 is expressed in hematopoietic and epithelial cancer cells. 755 33

Efficient replication of HIV-1 requires establishment of the proviral state, i.e., the integration of a DNA copy of the viral genome, synthesized by reverse transcriptase, into a chromosome of the host cell. Integration is catalyzed by the viral integrase protein. We have previously reported that phenolic moieties in compounds such as napthoquinones, flavones, caffeic acid phenethyl ester (CAPE), and curcumin confer inhibitory activity against HIV-1 integrase. We have extended these findings by examining the effects of tryphostins, tyrosine kinase inhibitors. The catalytic activities of HIV-1 integrase and the formation of enzyme-DNA complexes using photocross-linking were examined. Both steps of the integration reaction, 3'-processing and strand transfer, were inhibited by tyrphostins at micromolar concentrations. The DNA binding activity of integrase was inhibited at higher concentrations of tryphostins. Disintegration, an apparent reversal of the strand transfer reaction, catalyzed by an integrase mutant lacking the N-terminal zinc finger and C-terminal DNA binding domains is also inhibited by tyrphostins, indicating that the binding site for these compounds resides in the central catalytic core of HIV-1 integrase. Binding of tyrphostins at or near the integrase catalytic site was also suggested by experiments showing a global inhibition of the choice of attacking nucleophile in the 3'-processing reaction. None of the tryphostins tested inhibited eukaryotic topoisomerase I, even at 100 microM, suggesting selectivity for integrase inhibition. Molecular-modeling studies have revealed that, after energy minimization, several tyrphostins may adopt folded conformations. The similarity of the tyrphostin family to other families of inhibitors is discussed. Tyrphostins may provide lead compounds for development of novel antiviral agents for the treatment of acquired immunodeficiency syndrome based upon inhibition of HIV-1 integrase.
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PMID:Effects of tyrphostins, protein kinase inhibitors, on human immunodeficiency virus type 1 integrase. 757 25

Anaplastic large cell lymphoma (ALCL) and Hodgkin's disease (HD) have some pathologic and immunohistochemical similarities, and a histogenetic relationship between them has been suggested by some investigators. By cytogenetic study, the t(2;5)(p23;q35) translocation appears to be unique for ALCL. The breakpoints of the t(2;5)(p23;q35) have recently been cloned and are reported to involve a novel tyrosine kinase gene, anaplastic lymphoma kinase (alk), on chromosome 2 and the nucleophosmin gene (npm) on chromosome 5. Therefore, we studied the frequency of npm-alk translocation in ALCL using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. We also studied HD and a variety of reactive lymphoid lesions since there is contradictory information in the literature on the occurrence of the npm-alk rearrangement in HD. We detected npm-alk hybrid mRNA in 8 of 22 cases of ALCL (36%), but none of the 21 cases of HD or the 11 cases with reactive lesions contained amplifiable template. All positive ALCL had the T or indeterminate phenotype and occurred in young adults or children. There was very good correlation between a cytogenetically detectable t(2;5) and a positive signal by RT-PCR. Our results indicate a selective but relatively infrequent association between the t(2;5) and ALCL of T or indeterminate phenotype, not shared with HD or reactive hyperplasia.
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PMID:Transcripts of the npm-alk fusion gene in anaplastic large cell lymphoma, Hodgkin's disease, and reactive lymphoid lesions. 757 58

The BCR/ABL oncogenic tyrosine kinase is responsible for initiating and maintaining the leukemic phenotype of Philadelphia chromosome (Ph1)-positive cells. Phosphatidylinositol-3 (PI-3) kinase is known to interact with and be activated by receptor and nonreceptor tyrosine kinases. We investigated whether PI-3 kinase associates with and/or is regulated by BCR/ABL, whether this interaction is functionally significant for Ph1 cell proliferation, and, if so, whether inhibition of PI-3 kinase activity can be exploited to eliminate Ph1-positive cells from bone marrow. We show that the p85 alpha subunit of PI-3 kinase associates with BCR/ABL and that transient expression of BCR/ABL in fibroblasts and down-regulation of BCR/ABL expression using antisense oligodeoxynucleotides (ODNs) in Ph1 cells activates and inhibits, respectively, PI-3 kinase enzymatic activity. The use of specific ODNs or antisense constructs to downregulate p85 alpha expression showed a requirement for p85 alpha subunit in the proliferation of BCR/ABL-dependent cell lines and chronic myelogenous leukemia (CML) primary cells. Similarly, wortmannin, a specific inhibitor of the enzymatic activity of the p110 subunit of PI-3 kinase, inhibited growth of these cells. The growth of normal bone marrow and erythromyeloid, but not megakaryocyte, progenitors was inhibited by p85 alpha antisense [S]ODNs, but wortmannin, at the concentrations tested, did not affect normal hematopoiesis. The proliferation of two BCR/ABL- and growth factor-independent cell lines was not affected by downregulation of the expression of the p85 alpha subunit or inhibition of p110 enzymatic activity, confirming the specificity of the observed effects on Ph1 cells. Thus, PI-3 kinase is one of the downstream effectors of BCR/ABL tyrosine kinase in CML cells. Moreover, reverse transcriptase-polymerase chain reaction performed on single colonies to detect BCR-ABL transcripts showed that wortmannin was able to eliminate selectively CML-blast crisis cells from a mixture of normal bone marrow and Ph1 cells.
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PMID:Phosphatidylinositol-3 kinase activity is regulated by BCR/ABL and is required for the growth of Philadelphia chromosome-positive cells. 760 2

To assess the pathophysiological role of the RET protooncogene in sporadic pheochromocytomas, we examined the 2 regions of the gene in which molecular defects are specifically associated with the multiple endocrine neoplasias (MEN) type 2A (the cysteine-rich domain encoded by exons 10 and 11), and type 2B (the tyrosine kinase domain encoded by exon 16). The sequences of both regions were amplified by reverse transcriptase-polymerase chain reaction (PCR) or PCR from tumor RNA and/or leukocyte DNA. The amplified fragments were analyzed by denaturing gradient gel electrophoresis using chemical clamps. In 28 patients with unilateral sporadic tumors, 6 RET mutations were found, 3 in the MEN 2A region, 3 in the MEN 2B region. Five patients had missense mutations: 2 in the MEN 2A region (C634W and D631Y), and 3 in the MEN 2B region (M918T). Analysis of leukocyte DNA in 3 of these patients confirmed that RET mutations were only present in tumor DNA. The sixth patient had lost exon 10 in the tumor complementary DNA as a result of the deletion of the dinucleotide -AG- at the 3'splice acceptor site of intron 9; this molecular defect was only found in the tumor DNA. Thus RET mutations of the MEN 2A and 2B regions are also found in about 20% of sporadic pheochromocytomas. We describe new types of molecular defects of the RET protooncogene in the MEN 2A region that involve noncysteine residues and loss of exon 10. Further studies should be extended to analyze the entire RET protooncogene. These findings have a profound clinical impact for the management of patients with supposedly sporadic pheochromocytomas.
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PMID:The RET protooncogene in sporadic pheochromocytomas: frequent MEN 2-like mutations and new molecular defects. 855 Jul 89

Ws/Ws rats have a small deletion at the tyrosine kinase domain of the c-kit gene, and practically no mast cells were detectable when the tissues were stained with alcian blue. Because alcian blue stains proteoglycans, there is a possibility that immature mast cells that do not contain a sufficient amount of proteoglycans are not detectable by this method. We examined this possibility by using other markers of mast cells. The histamine content in the skin of Ws/Ws rats was 0.3% that of control normal (+/+) rats. Because the number of alcian blue-positive mast cells in the skin of Ws/Ws rats was also 0.3% that of +/+ rats, histamine in the skin seemed to be concentrated to alcian blue-positive mast cells. Mast cells in the skin of +/+ rats express messenger RNA of Fc epsilon RI beta-subunit and c-kit protein. Because c-kit messenger RNA was normally expressed at least in the brain of Ws/Ws rats despite the small deletion, we examined the expression of Fc epsilon RI beta-subunit and c-kit messenger RNA in the skin and stomach of Ws/Ws rats by reverse transcriptase modification of polymerase chain reaction. Expression of either Fc epsilon RI beta-subunit or c-kit messenger RNA in the skin and stomach of Ws/Ws rats was estimated to be less than 1% that of +/+ rats. Moreover no Fc epsilon RI beta-subunit-expressing and no c-kit-expressing cells were detectable in the skin of Ws/Ws rats by in situ hybridization histochemistry. The present result suggests the absence of immature mast cells in tissues of Ws/Ws rats.
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PMID:Absence of immature mast cells in the skin of Ws/Ws rats with a small deletion at tyrosine kinase domain of the c-kit gene. 768 55

Xenopus laevis and X. borealis were screened for tyrosine kinase genes using the polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR and 34 X. laevis and 23 X. borealis tyrosine genes were identified. Eighteen of the genes represented novel tyrosine kinase family members. The rest could be classified into known tyrosine kinase subfamilies, of which however only three have been previously identified in Xenopus. Eight clones, including bFGFR (xFGFR1) and potential Trk, Ins R., Fak, Fyn, and Abl homologs, were used to probe temporal and spatial gene expression in early development. Quantitative RT-PCR and whole-mount and in situ hybridization showed that most of these mRNAs were present throughout development and were broadly distributed, mainly in ectodermal and mesodermal derived tissues. At the blastula stage, bFGFR mRNA was detected within the ectoderm and a gradient of expression was noted within the invaginating mesoderm. The unexpected promiscuous expression of many tyrosine kinase genes in early development is discussed.
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PMID:An analysis of Xenopus tyrosine kinase genes and their expression in early development. 777 53

Prolactin (PRL) is involved in a wide range of physiological effects in several species and its immunoregulatory role has already been well documented. The PRL receptor has been cloned from various species. There are at least two receptor isoforms (short and long) in rats and mice, which differ only in their cytoplasmic domains, generated by alternative splicing of a single gene, although in human only the long form exists. Using the reverse transcriptase-polymerase chain reaction (RT-PCR), we detected transcripts encoding both forms of PRL receptor in all lymphoid tissues examined in human, mouse, and rat, but in mouse and rat the ratio between the two forms was variable from animal to animal. Concerning the transcript encoding the PRL itself, a clear signal was always found in human lymphocytes and occasionally in rat thymus. We also developed a quantitative PCR (Q-PCR) in order to measure the absolute number of transcripts in thymus and spleen from rats at two stages of estrous cycle. The level of expression of the two forms was about equal. Finally, we identified the tyrosine kinase JAK2, which is constitutively associated with the PRLR, using the Nb2 rat lymphoma cell line as a model system with which to study the action of PRL on cell mitogenesis. We also showed that, after stimulation by PRL, the dimerization process is a prerequisite step for the phosphorylation of the PRLR and JAK2, which represents the earliest event in the signal transduction pathway.
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PMID:Prolactin and the immune system. 784 46


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