Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Buffy coats from 31 patients with a diagnosis of leukemia and 16 normal donors were tested for the presence of a viral-like reverse transcriptase. Eighty-five percent of fresh leukemic buffy coats were positive. Also tested were spleens from 16 patients with hematological disorders and 5 spleens from patients without history of hematological malignancy. The 5 normal spleens were negative. Also negative were 4 spleens from patients with Hairy cell leukemia. From the remaining 12 spleens 7 were positive. Reverse transcriptase measurements can be used to distinguish leukemic from normal buffy coats.
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PMID:On the presence of reverse transcriptase in myelo- and lymphoproliferative disorders. 8 54

2-Chloro-2'-deoxyadenosine (cladribine [CldAdo]) represents one of the most promising therapeutic agents for the treatment of pediatric leukemias and adult hairy cell leukemia. We examined whether CldAdo incorporation into DNA inhibited subsequent transcription in vitro using purified phage RNA polymerases. Control (Ade-containing) and 2-chloroadenine (ClAde)-substituted DNA strands that contained a RNA polymerase promoter sequence were synthesized by a modified asymmetric polymerase chain reaction. Complementary (+) and (-) strands were annealed, incubated with phage RNA polymerase, and analyzed with denaturing PAGE. When ClAde was present in both strands, the yield of full-length transcripts (approximately equal to 100 bases) was reduced by approximately equal to 90% relative to control DNA. Transcription was also reduced to a slightly lesser degree when substitutions occurred in only one of two strands. The observed low transcript levels on ClAde-containing DNA were due in part to the presence of the analogue within the promoter region. With gel shift binding assays, we demonstrated that RNA polymerase did not bind as well to ClAde-containing promoters. Polymerase/DNA complex formation was decreased by approximately equal to 80% compared with that on control unsubstituted promoters. In addition, on binding to the substituted promoter, RNA polymerase had an altered conformation that led to enhanced proteolytic clipping by endoproteinase Glu-C. Transcript sequence analysis indicated that SP6 RNA polymerase read through template ClAde residues with no apparent misincorporation into RNA. Our results provide insight into a novel effect of this nucleoside analogue that may explain its cytotoxicity in nondividing cells.
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PMID:In vitro transcription of DNA containing 2-chloro-2'-deoxyadenosine monophosphate. 747 21

To extent our knowledge on the cytokines possibly involved in the pathophysiology of B-cell chronic lymphocytic leukemia (B-CLL), the mRNA expression of a panel of 10 cytokines was investigated on purified B-CLL cells using a reverse-transcriptase polymerase chain reaction method. Whereas negative RT-PCR signals were recorded for interleukin-1 alpha (IL-1 alpha), IL-2, IL-3, IL-4, IL-5, IL-7, tumor necrosis factor beta (TNF beta), and granulocyte-macrophage colony-stimulating factor, we detected the expression of IL-1 beta, IL-6 and TNF alpha. Furthermore, the constitutive expression of IL-8 mRNA was observed in all 17 B-CLL samples analyzed. mRNA expression was associated with the capacity of the leukemic cells to release IL-8 both constitutively (4.6 +/- 8.1 SD ng/mL) and, to a further extent, after stimulation (14.5 +/- 19.4 ng/mL). The circulating levels of IL-8 were also evaluated in 12 untreated B-CLL sera samples and the overall mean level was significantly higher (P < .01) than in normal sera. In addition, supernatants of purified B-CLL cells cultured in the presence of 12-O-tetradecanoylphorbol-13-acetate showed chemotactic activity towards neutrophils; this activity was neutralized in the presence of an anti-IL-8 antiserum. The mRNA for IL-8 was absent in five B-cell preparations from hairy cell leukemia cases and in four B-cell lines. Normal tonsil CD5+ B cells showed a low expression of IL-8 mRNA only in two of the nine preparations tested and the overall quantity of IL-8 released by these cells after 3 days' incubation was significantly lower compared with that released by B-CLL cells (0.4 +/- 0.3 and 1.6 +/- 0.9 ng/mL under basal and stimulated conditions, respectively). These findings point to an involvement of a member of the proinflammatory chemokine supergene family in human CD5+ B lymphocytes. The different IL-8 behavior observed between B-CLL cells and their normal counterpart is likely to reflect an activation state of the leukemic population.
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PMID:Cytokine gene expression in B-cell chronic lymphocytic leukemia: evidence of constitutive interleukin-8 (IL-8) mRNA expression and secretion of biologically active IL-8 protein. 751 9

The nucleoside analog 2-chloro-2'-deoxyadenosine (CldAdo; cladribine) is effective in the treatment of hairy cell leukemia and chronic lymphocytic leukemia. CldAdo is phosphorylated and incorporated into cellular DNA but is not an absolute chain terminator. We demonstrated by in vitro gel-shift assays that binding interactions of the human TATA box-binding protein (TBP) were disrupted on 2-chlorodeoxyadenosine monophosphate (CldAMP)-substituted TATA box consensus sequences. We hypothesized that human RNA polymerase II (pol II) transcriptional processes would therefore be affected by 2-chlorodeoxyadenosine triphosphate (CldATP) incorporation into a promoter TATA element. Double-stranded DNA templates containing the adenovirus major late promoter and coding sequences were enzymatically synthesized as control or with site-specific CldAMP residues, incubated with HeLa extract, and the synthesis of radiolabeled 44-base transcripts was assessed. With increasing amounts of HeLa extract, CldAMP substitution for dAMP within the TATA box decreased in vitro pol II transcription by approximately 35% compared with control substrates. Time-course studies showed that transcript production increased in a linear fashion on control substrates. In contrast, transcription on CldAMP-substituted TATA sequences reached a plateau after 20 min. Furthermore, CldAMP-substituted promoter sequences trapped or sequestered TBP, preventing its dissociation from DNA and subsequent binding to additional TATA elements to reinitiate transcription. CldAdo thus represents the first example of a nucleoside analog that acts as a transcriptional antagonist. CldATP incorporation into gene regulatory sequences may provide a novel strategy to modulate specific protein/DNA interactions.
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PMID:The antileukemia drug 2-chloro-2'-deoxyadenosine: an intrinsic transcriptional antagonist. 1472 55