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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1Recent molecular cloning studies have identified six members in the multidrug-resistance protein (MRP) gene family. However, the regulation of expression of these genes is largely unknown. We previously reported that expression of MRP1, encoding multidrug-resistance associated protein, and gamma-GCSh, which encodes the heavy subunit of gamma-glutamylcysteine synthetase (gamma-GCS), could be up-regulated by prooxidants [Yamane et al., J Biol Chem 1998;273:31075-85]. In the present study, we investigated whether different members of the MRP family exhibit different responses to induction by prooxidants, and whether p53 status influences the levels of induction. A panel of colorectal cancer cell lines with different p53 status, i.e. HCT116 containing wild-type p53, and HT29, SW480, and Caco2 containing mutant p53, was treated with tert-butylhydroquinone (t-BHQ) and pyrrolidinedithiocarbamate (PDTC). MRP1 and gamma-GCSh mRNA levels were determined by the RNase protection assay, using gene-specific probes. We report here that induction of MRP1 and gamma-GCSh expression by these prooxidants varied among the different cell lines, and p53 mutations were not always associated with elevated levels of induction. These results suggest that the effects of p53 on the induced expression of MRP1 and gamma-GCSh depend on the environment of the cell and/or nature of p53 mutations. In an isogenic HCT116 cell line containing p53(-/-) alleles, we demonstrated that, as for MRP1, expression of MRP2 and MRP3 was induced by the prooxidants, whereas expression of MRP4 and MRP5 was not. MRP6 mRNA was not detectable. Induction of MRP2 expression by prooxidants seemed to be independent of p53 status. Our results demonstrated the differential regulation of the MRP gene family by p53 mutation under oxidative stress.
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PMID:Differential sensitivities of the MRP gene family and gamma-glutamylcysteine synthetase to prooxidants in human colorectal carcinoma cell lines with different p53 status. 1123 98

The E6 and E7 oncogenes of human papillomavirus type 16 (HPV-16) are sufficient for the immortalization of human genital keratinocytes in vitro. The products of these viral genes associate with p53 and pRb tumor suppressor proteins, respectively, and interfere with their normal growth-regulatory functions. The HPV-16 E6 protein has also been shown to increase the telomerase enzyme activity in primary epithelial cells by an unknown mechanism. We report here that a study using reverse transcription-PCR and RNase protection assays in transduced primary human foreskin keratinocytes (HFKs) shows that the E6 gene (but not the E7 gene) increases telomerase hTERT gene transcription coordinately with E6-induced telomerase activity. In these same cells, the E6 gene induces a 6.5-fold increase in the activity of a 1,165-bp 5' promoter/regulatory region of the hTERT gene, and this induction is attributable to a minimal 251-bp sequence (-211 to +40). Furthermore, there is a 35-bp region (+5 to +40) within this minimal E6-responsive promoter that is responsible for 60% of E6 activity. Although the minimal hTERT promoter contains Myc-responsive E-box elements and recent studies have suggested a role for Myc protein in hTERT transcriptional control, we found no alterations in the abundance of either c-Myc or c-Mad in E6-transduced HFKs, suggesting that there are other or additional transcription factors critical for regulating hTERT expression.
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PMID:Transcriptional activation of the telomerase hTERT gene by human papillomavirus type 16 E6 oncoprotein. 1128 2

The neurotoxin 6-hydroxydopamine has been used to induce selective dopaminergic cell death in animal models of Parkinson's disease. The response of neurons to this toxin has been shown to be greatly influenced by astrocytes. Our laboratory reported previously that human neuroblastoma SH-SY5Y cells became more resistant to the toxicity of 6-hydroxydopamine when co-cultured with mouse astrocytes. This enhanced tolerance required direct and specific adhesion between SH-SY5Y cells and astrocytes. We hypothesized that this interaction led to biochemical changes in SH-SY5Y cells, thereby protecting these cells from toxicity. To study these changes, we again co-cultured SH-SY5Y cells with astrocytes and treated them with 6-hydroxydopamine. An optimized condition of trypsin treatment was employed to separate SH-SY5Y cells from astrocytes quickly. Western blot analysis demonstrated that 6-hydroxydopamine significantly increased p53 protein in monolayer SH-SY5Y cells grown in either regular medium or conditioned medium from astrocytes. This change, however, was not observed in the group co-cultured with astrocytes. Data obtained from the ribonuclease protection assay indicated that similar changes also occurred at the transcriptional level. The enhanced resistance of the co-cultured SH-SY5Y cells to the toxicity of 6-hydroxydopamine is attributed to the ability of astrocytes to prevent the increase of p53 induced by this toxin. This study demonstrates the significance of the interaction between astrocytes and neurons when they are exposed to neurotoxins.
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PMID:Inhibition of 6-hydroxydopamine-induced p53 expression and survival of neuroblastoma cells following interaction with astrocytes. 1131 93

A long-standing issue concerns the extent to which fragile sites predispose to cancer-associated chromosomal rearrangements. The FHIT gene at chromosome 3p14.2 spans the most common fragile site, FRA3B, in the human genome. Although the FHIT gene is altered in many human cancers, its status as a tumor suppressor gene has remained controversial, particularly since functional studies provided contradictory results. It had been suggested the FHIT alterations result from FRA3B induction promoted by the interference of carcinogens with DNA replication. Here we investigated the effect of FRA3B induction on FHIT expression. Common fragile sites were induced by treatment with aphidicolin and scored cytogenetically. FHIT transcription was analysed by RT--PCR and RNase protection analysis. Unexpectedly, FHIT transcription proceeded unchanged after fragile site induction. Aberrant FHIT transcripts lacking one or more exons were not observed. Moreover, Western blots revealed that the levels of FHIT prior to and following fragile site induction was unchanged, whereas p53 was found at elevated levels after induction. FRA3B induction thus has no direct effect on FHIT transcription and translation.
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PMID:Induction of the common fragile site FRA3B does not affect FHIT expression. 1131 27

The p53 protein activates promoters containing p53 binding sites, and it represses other promoters. We examined the effect of p53 on bcl-2 expression in both the DHL-4 B cell line and the K562 erythroleukemia line. Transient transfection analyses revealed that wild-type p53 repressed the bcl-2 full-length promoter. The region of the bcl-2 promoter that was responsive to p53 was mapped to the bcl-2 P2 minimal promoter region, and we showed that p53 and the TATA binding protein bound to the bcl-2 TATA sequence. The TATA binding protein, p53, histone deacetylase-1 and mSin3a could be co-immunoprecipitated from K562 cell nuclear extract. The TATA binding protein and mSin3a could be recovered in a complex at the bcl-2 promoter TATA sequence, however, the formation of this complex was not dependent on the presence of p53. Treatment of K562 cells with the histone deacetylase inhibitor, trichostatin A, resulted in an increase in bcl-2 promoter activity whether p53 was present or not. Therefore, we demonstrated that p53 and the histone deacetylases repress the bcl-2 promoter independently. Similar results were obtained when endogenous bcl-2 mRNA or protein levels were measured in response to either p53 or trichostatin A, and p53 expression resulted in enhanced apoptosis. RNase protection assays demonstrated that transcription from the endogenous 3' bcl-2 promoter was decreased by p53. The regions of p53 that were required for repression of the bcl-2 promoter were defined. We conclude that the TATA sequence in the bcl-2 P2 minimal promoter is the target for repression by p53, and that the interaction between p53 and TBP is most likely responsible for the repression. Mutation of p53 may play a role in the up-regulation of bcl-2 expression in some B cell lymphomas.
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PMID:Negative regulation of bcl-2 expression by p53 in hematopoietic cells. 1131 51

One of the main functions of the tumor suppressor p53 is the induction of programmed cell death. Here we investigated in detail the molecular mechanisms that underlay p53 transactivation-dependent apoptosis in the human colon cancer cell line DLD-1. Although p53 upregulated the death receptors Fas, TRAIL-R1 and TRAIL-R2 in this cell line, p53-induced cell death occurred without detectable caspase-8 activation whereas, activation of caspase-9 and caspase-3 was readily observed. In addition to the upregulation of death receptors, p53 induced the pro-apoptotic Bcl-2 family members Bik and Bak and downregulated the anti-apoptotic Bcl-xL protein. Moreover, in RNase protection assay analyses as well as in reporter gene analyses we found a p53-dependent upregulation of the death receptor-inhibitory protein cFLIP. Together, these data argue for a p53-mediated activation of the mitochondrial pathway of apoptosis. In contrast to recently published data obtained in different cellular systems, there was no evidence for an essential role of NF-kappaB in p53-induced cell death. Moreover, induction of p53 interfered with TNF-induced NF-kappaB activation independently from apoptosis-induction.
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PMID:p53 upregulates cFLIP, inhibits transcription of NF-kappaB-regulated genes and induces caspase-8-independent cell death in DLD-1 cells. 1131 89

Zinc-alpha(2)-glycoprotein (Znalpha(2)gp) is widely distributed in body fluids and epithelia. Its expression in stratified epithelia increases with differentiation. We previously showed that Zn alpha(2)gp has ribonuclease activity, and that squamous tumor cells grown on a matrix of Znalpha(2)gp were growth-inhibited. Here we demonstrate, both by adding Znalpha(2)gp to the culture medium and, more unequivocally, by stably transfecting SiHa cells with Znalpha(2)gp cDNA, that the introduction of Znalpha(2)gp into SiHa tumor cells reduces proliferation. In response to Znalpha(2)gp, we find an accumulation of the cell population in G(2)/M by flow cytometry, paralleling the reduction of proliferation. In order to distinguish growth inhibition by cell cycle arrest from that produced by apoptosis or differentiation, we examine by RT-PCR how Znalpha(2)gp affects the expression of genes commonly used as markers of these properties. No changes are observed for PCNA, p53, c-myc, or bcl-2. Only cdc2 expression responds to Znalpha(2)gp, with a reduction of up to over a factor of two. Cdc2 is the only cyclin-dependent kinase regulating the G(2)/M transition without redundancy and is required as a rate-limiting step in the cell cycle. Its increased expression has been directly linked to increased proliferation and decreased differentiation of advanced tumors; conversely, its downregulation by Znalpha(2)gp might hinder tumor progression. J. Cell. Biochem. Suppl. 36: 162-169, 2001.
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PMID:Zinc-alpha(2)-glycoprotein hinders cell proliferation and reduces cdc2 expression. 1145 81

Osteoblastic metastases are common in lethal prostate cancer. Effective therapy for bone metastases is lacking. Thus, developing an appropriate in vitro screening system is critical to prioritize which of the newly developed agents should undergo additional expensive and time-consuming in vivo evaluation in bone metastases animal models. In the past, such in vitro screening evaluated the response of prostate cancer cells to chemotherapeutic agents in monoculture without the presence of osteoblasts. In such monoculture, prostate cancer cells have a high (i.e., >90%) proliferative growth fraction. In contrast, the growth fraction (i.e., mean: 7.1 +/- 0.8%; median: 3.1%) in 117 metastatic sites of prostate cancer obtained from 11 androgen ablation failing patients at "warm" autopsy was found to be >10-fold lower. To better mimic the lower growth fraction observed clinically, LNCaP human prostate cancer cells were cocultured with membrane-separated hFOB human osteoblasts. Such coculturing significantly lowered the growth fraction of the LNCaP cells (i.e., from >90 to <30%) without enhancing their low rate (i.e., <5%) of apoptosis. This lowering of the growth fraction was documented using flow cytometry, Ki-67 immunohistochemistry, and 5-bromo-2-deoxyuridine incorporation. Using RNase protection assays, it was documented that coculture with osteoblasts causes enhanced p53, p27, and p21 expression leading to a decrease in the number of LNCaP cells entering the cell cycle (i.e., enhanced number of LNCaP cells in G(0)-G(1) and a decrease in S and G(2)-M and thus the growth fraction). This osteoblast-induced enhanced G(0)-G(1) checkpoint control affected the chemosensitivity of LNCaP cells. This was documented by coculturing LNCaP cells with hFOB cells to condition the medium for 3 days to lower the growth fraction to <30% before exposing the LNCaP cells for 48 h to various concentrations of Taxol, doxorubicin, or thapsigargin (TG). In standard high (i.e., >90%) growth fraction cultures (i.e., cultures in the absence of osteoblast-conditioned medium), there was a dose-dependent and significant (P < 0.05) increase in apoptosis of LNCaP cells exposed to Taxol or doxorubicin. In contrast, even the highest dose of Taxol (1 microM) did not enhance apoptosis of lower growth fraction LNCaP cells cultured in osteoblast-conditioned medium. Similarly, only the highest concentration of doxorubicin (1 microM) enhanced apoptosis in lower growth fraction cells. In contrast, 100 nM TG induced high levels of apoptosis in both lower and high-growth fraction LNCaP cultures. These results demonstrate that the osteoblast/LNCaP coculture system is a better in vitro screen than monoculture to identify proliferation-independent agents for the treatment of prostate cancer bone metastases, and TG is such an agent.
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PMID:Therapeutic implications of enhanced G(0)/G(1) checkpoint control induced by coculture of prostate cancer cells with osteoblasts. 1152 28

Despite many efforts to alter the relentlessly aggressive progression of tumors of neural origin, individuals bearing these tumors exhibit poor prognosis for long-term survival. In an attempt to find an effective treatment, we examined the efficacy of the non-steroidal anti-inflammatory drug, flurbiprofen, to suppress the growth of tumor cell lines derived from medulloblastoma and glioblastoma multiforme. Results from cell proliferation assays have revealed that flurbiprofen effectively inhibits the growth of various tumor cells in a dose-dependent manner and causes a noticeable change in the progression of cells through cell cycle stages. Treatment of tumor cells with flurbiprofen reduced the number of cells in G1 and G2, and significantly increased their numbers in S phase, suggesting that, flurbiprofen accelerates G1/S entry, and/or delays cell exit from S to G2/M stages. Results from RNase protection assay and Western blot analysis showed that while treatment of cells with flurbiprofen causes a minor change in the RNA level of different cyclins, there is a significant decrease in the level of cyclin B protein upon flurbiprofen treatment. Examination of tumor suppressors by RNase protection technique showed a subtle increase in the levels of several tumor suppressors upon flurbiprofen treatment. Interestingly, at the protein level, p53 tumor suppressor was substantially increased upon flurbiprofen treatment, yet the level of p21, a downstream target for p53 remained unchanged. Curiously, treatment of the cells with flurbiprofen enhanced the level of COX-2 expression. Results from co-immunoprecipitation showed association of COX-2 with p53 in tumor cells. These observations suggest that the interaction of COX-2 with p53 may cause p21-independent suppression of tumor cell growth upon flurbiprofen treatment.
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PMID:Inhibition of human brain tumor cell growth by the anti-inflammatory drug, flurbiprofen. 1168 65

The death receptor 5 (DR5) is a receptor for tumor necrosis factor-related apoptosis-inducing ligand and is able to induce apoptosis in various tumor cells. The expression of DR5 is up-regulated at the transcriptional level by p53, genotoxic stress and so on. To investigate the structure of the DR5 gene promoter, we screened and sequenced a genomic clone containing the 5'-flanking region of the DR5 gene. RNase protection assays showed two major transcription start sites around -122 and -137 upstream of the translation initiation codon ATG. Transient transfections with serial 5'-deletion mutants identified the minimal promoter element spanning -198 to -116. Site-directed mutagenesis demonstrated that the DR5 gene promoter has no typical TATA-box, but has two Sp1 sites responsible for the basal transcription activity of the DR5 gene promoter.
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PMID:Promoter structure and transcription initiation sites of the human death receptor 5/TRAIL-R2 gene. 1169 76


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