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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To study the involvement of DNA topoisomerase (topo) II on nonhomologous (illegitimate) recombination, we examined the effect of topo II inhibitors on random integration of exogenous vectors into human chromosomes. We transfected human cell lines PA1, HeLa and EJ-1 with linearized plasmid pSV2neo by electroporation, treated with topo II inhibitors and determined the frequency of Geneticin-resistant (G418r) colonies. We found that three topo II inhibitors, etoposide (VP-16), ICRF-193 and amsacrine (m-AMSA), greatly enhanced the frequency of G418r colonies. These effects were maximally expressed by as little as 12 hrs treatment with the drugs. Similar enhancements were found with different vectors (closed-circular and linear), different cell types, or by different transfection methods (calcium precipitation and lipofection). In contrast, the inhibitor treatments did not affect the transient expression of chloramphenicol acetyltransferase and beta-galactosidase activity following transfection with pSV2CAT and pCH110, respectively. Southern blot analysis revealed that the integration pattern of transfected pSV2neo into PA1 chromosomes was random and not characteristic for each inhibitor. These results suggest that topo II inhibitors directly act at a nonhomologous recombination reaction, promoting the integration process of transfected vectors into human chromosomes. We discuss the enhancement mechanism with a special emphasis on DNA strand breaks induced by the inhibitors.
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PMID:DNA topoisomerase II inhibitors enhance random integration of transfected vectors into human chromosomes. 900 Jan 72

Hepatitis B virus (HBV) has a unique fourth open reading frame coding for a 16.5-kDa protein known as hepatitis B virus X protein (HBX). The importance of HBX in the life cycle of HBV has been well established, but the underlying molecular function of HBX remains controversial. We previously identified a proteasome subunit PSMA7 that interacts specifically with HBX in the Saccharomyces cerevisiae two-hybrid system. Here we demonstrate that PSMC1, an ATPase-like subunit of the 19 S proteasome component, also interacts with HBX and PSMA7. Analysis of the interacting domains among PSMA7, PSMC1, and HBX by deletion and site-directed mutagenesis suggested a mutually competitive structural relationship among these polypeptides. The competitive nature of these interactions is further demonstrated using a modified yeast two-hybrid dissociator system. The crucial HBX sequences involved in interaction with PSMA7 and PSMC1 are important for its function as a transcriptional coactivator. HBX, while functioning as a coactivator of AP-1 and acidic activator VP-16 in mammalian cells, had no effect on the transactivation function of their functional orthologs GCN4 and Gal4 in yeast. Overexpression of PSMC1 seemed to suppress the expression of various reporters in mammalian cells; this effect, however, was overcome by coexpression of HBX. In addition, HBX expression inhibited the cellular turnover of c-Jun and ubiquitin-Arg-beta-galactosidase, two well known substrates of the ubiquitin-proteasome pathway. Thus, interaction of HBX with the proteasome complex in metazoan cells may underlie the functional basis of proteasome as a cellular target of HBX.
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PMID:Structural and functional characterization of interaction between hepatitis B virus X protein and the proteasome complex. 1074 18

Our objective was to determine the efficacy of adenoviral-mediated gene therapy with wild-type p53 or p21 in human breast cancer cells and investigate interactions with radiation and chemotherapy. Two human breast cancer cell lines, MDA-MB-231 and MDA-MB-435, both with p53 mutations, were transduced with adenoviral vectors containing wild-type p53 (Ad5CMV-p53) or p21/WAF1/Cip1 (Ad5CMV-p21), and the effects on growth were determined. Infection was combined with low-dose (1.4 - 3.7 Gy) irradiation to see if this would improve transduction efficiency and enhance numbers of cells killed. Transduction with either vector resulted in expression of p21WAF1/cip1 and growth inhibition, although Ad5CMV-p53 transduction produced greater growth inhibition than did Ad5CMV-p21. The cell lines differed in sensitivity to the vectors. The Ad5CMV-p53 vector in a multiplicity of infection (MOI) of 125 resulted in 50% to 80% inhibition of MDA-MB-231, while MOI 250 of the same vector resulted in 27% inhibition of MDA-MB-435. Infection with Ad5CMV-p21 produced modest growth inhibition in both cell lines (< or = 40% at MOI 200), although protein expression was detected at lower viral doses. Low dose gamma-irradiation (1.4 to 3.7 Gy) was used to try and improve the rate of gene transfer. Modest increases in transduction efficiency and duration of expression of a vector containing beta-galactosidase occurred in irradiated breast cancer cells. Radiation 24 hr before transduction with Ad5CMV-p53 increased the proportions of apoptotic MDA-MB-231 cells. The cells transduced with Ad5CMV-p21 were arrested in G1, yet when they were irradiated before adenoviral transduction, the overexpression of p21 protected the cells from the cytotoxic effects of the radiation. Clonogenic assays showed that Ad5CMV-p21 reduced the sensitivity of MDA-MB-231 to VP-16 and paclitaxel. Combining these drugs with Ad5CMV-p53 did not consistently or significantly decrease clonogenic survival.
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PMID:Adenoviral-mediated gene therapy with Ad5CMVp53 and Ad5CMVp21 in combination with standard therapies in human breast cancer cell lines. 1104 64

Recently, it has been demonstrated that Etoposide, a topoisomerase II inhibitor, can induce apoptosis in MDM2-overexpressing tumor cells by inhibition of MDM2 synthesis. We have previously shown that E2F-1 overexpression induces apoptosis of MDM2-overexpressing sarcoma cells, which is related to the inhibition of MDM2 expression. Therefore, the present study was designed to investigate the in vitro and in vivo effect of combined treatment of adenovirus-mediated E2F-1 and topoisomerase II inhibitors on the growth inhibition and apoptosis in human sarcoma cells. Two human sarcoma cell lines, OsACL and U2OS, were treated with topoisomerase II inhibitors (Etoposide and Adriamycin), alone or in combination with adenoviral vectors expressing beta-galactosidase (Ad-LacZ) or E2F-1 (Ad-E2F-1). E2F-1 expression was confirmed by Western blot analysis. Ad-E2F-1 gene transfer at a low dose (multiplicity of infection, 2) markedly increased the sensitivity of human sarcoma cells to topoisomerase II inhibitor treatment. This cooperative effect of E2F-1 and topoisomerase II inhibitors was less marked in SAOS-2 cells (p53 and pRb null). Topoisomerase II inhibitors also cooperated with E2F-1 overexpression to enhance tumor cell killing in an in vivo model using xenografts in nude mice. When combined with Adriamycin or Etoposide, E2F-1 adenovirus therapy resulted in approximately 95% and 85% decrease in tumor size, respectively, compared to controls (P<.05). These results suggest a new chemosensitization strategy that is effective in MDM2-overexpressing tumors and may have clinical utility.
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PMID:Additive effect of adenovirus-mediated E2F-1 gene transfer and topoisomerase II inhibitors on apoptosis in human osteosarcoma cells. 1139 76

In this work, we described the proliferation of human non-small-cell-lung-cancer (NSCLC) cells H1437 harboring p53 alleles (proline-267) can be inhibited by low-dosage topoisomerase II inhibitor etoposide (VP-16) in vitro and in vivo. The cytotoxicity was demonstrated by prolonged cell arrest at G2-M checkpoint exhibiting senescence-like phenotype followed by apoptotic cell death that appeared on the sixth day of VP-16 treatment. The experimental in vivo evidence of growth suppression was also demonstrated in xenograft tumors. The appearance of senescence-like state during extended G2-M phase arrest was indicated by slow proliferation and loss of growth sensitivity in culture accompanied with cellular morphological changes, time-dependent regulation of beta-galactosidase staining as well as distinct reduction of telomerase activity upon protracted VP-16 exposure. Further molecular determinants leading to G2-M cell arrest was also characterized by the concerted up-regulation of cyclin-dependent kinase inhibitors, p16(INK4a) and p21(Waf1/Cipi), beginning 2 days later following drug exposure at both translational and transcriptional levels, while human telomerase reverse transcriptase (hTERT) activities reduced progressively. The clinically important therapeutic agent VP-16-mediated prolonged cell arrest at G2-M phase prior to apoptotic death offered a different perspective in restraining human cancer cells at low drug dosage, thereby serving as an effective telomerase inhibitor as well as an apoptosis effector. The overall results demonstrated that apoptosis can be regulated differently in human NSCLC cells with disrupted p53. Further effort in elucidating G2-M arrest before leading to apoptosis promises to provide an alternative insight in reversing tumorigenic phenotype of human cancers.
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PMID:Etoposide (VP-16) elicits apoptosis following prolonged G2-M cell arrest in p53-mutated human non-small cell lung cancer cells. 1589 59

Phosphorylation of histone H2AX is a sensitive marker of DNA damage, particularly of DNA double strand breaks. Using multiparameter cytometry we explored effects of etoposide and temozolomide (TMZ) on three glioblastoma cell lines with different p53 status (A172, T98G, YKG-1) and on normal human astrocytes (NHA) correlating the drug-induced phosphorylated H2AX (gammaH2AX) with cell cycle phase and induction of apoptosis. Etoposide induced gammaH2AX in all phases of the cell cycle in all three glioblastoma lines and led to an arrest of T98G and YKG-1 cells in S and G(2)/M. NHA cells were arrested in G(1) with no evidence of gammaH2AX induction. A172 responded by rise in gammaH2AX throughout all phases of the cycle, arrest at the late S- to G(2)/M-phase, and appearance of senescence features: induction of p53, p21(WAF1/CIP1), p16(INK4A) and beta-galactosidase, accompanied by morphological changes typical of senescence. T98G cells showed the presence of gammaH2AX in S phase with no evidence of cell cycle arrest. A modest degree of arrest in G(1) was seen in YKG-1 cells with no rise in gammaH2AX. While frequency of apoptotic cells in all four TMZ-treated cell cultures was relatively low it is conceivable that the cells with extensive DNA damage were reproductively dead. The data show that neither the status of p53 (wild-type vs. mutated, or inhibited by pifithrin-alpha) nor the expression of O(6)-methylguanine-DNA methyltransferase significantly affected the cell response to TMZ. Because of diversity in response to TMZ between individual glioblastoma lines our data suggest that with better understanding of the mechanisms, the treatment may have to be customized to individual patients.
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PMID:Diversity of DNA damage response of astrocytes and glioblastoma cell lines with various p53 status to treatment with etoposide and temozolomide. 1930 57

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