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:3.4.22.56 (caspase-3)
35,750 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transduction of cancer cells with herpes simplex virus thymidine kinase gene (HSVtk) followed by prodrug ganciclovir (GCV) treatment has been shown to induce apoptosis. In this study, four murine tumors including B16F10 melanoma, NG4TL4 sarcoma, H6 hepatoma and 1MEA 7R.1 hepatoma were found to vary in sensitivity to this gene therapy strategy in vitro but, at effective doses of GCV, the HSVtk-transduced cells of all four tumors showed similar kinetics of early rise in p53 protein levels, then cell cycle S-/G2-phase arrest and finally signs of apoptosis. Immunoblot analyses revealed that Fas (CD95/APO-1), Fas ligand (FasL) and two downstream mediators, RIP and caspase-3, (CPP32, YAMA, Apopain) were increased in GCV-treated HSVtk-transduced tumor cells the cell cycle arrest and before apoptosis. Increased expression of FasL could also be observed in vivo in HSVtk-transduced tumors induced to regress by GCV treatment. Enzyme measurements using specific substrate showed that the caspase-3 activation followed kinetically the FasL expression. More than half of the HSVtk/GCV-induced cell death could be abrogated by addition to the cell culture medium of a specific antisense oligonucleotide to block FasL synthesis, a recombinant Fas/Fc chimeric protein to compete with Fas receptor for FasL binding, or cell-permeable specific tetrapeptide inhibitors of caspase-3 or caspase-8.
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PMID:Involvement of Fas (CD95/APO-1) and Fas ligand in apoptosis induced by ganciclovir treatment of tumor cells transduced with herpes simplex virus thymidine kinase. 1043 92

There is a need to enhance the efficacy of genetic prodrug activation therapy using herpes simplex virus thymidine kinase (tk) and ganciclovir (GCV) following disappointing results in early clinical trials. tk/GCV has been shown to lead to the activation of caspase-3, a potent executor of apoptosis. We demonstrate that co-expression of pro-caspase-3 with tk/GCV leads to enhanced cell death in ovarian carcinoma cells in vitro. Following transfection with recombinant adenoviral vectors encoding tk, GCV treatment leads to greater cell death in pro-caspase-3-expressing clones of SKOV3 and IGROV1 than control cells, as well as more rapid activation of caspase-3 and more rapid cleavage of PARP. Flow cytometry suggests that there is a greater degree of S-phase block in the pro-caspase-3-expressing clones than in control cells following treatment with tk/GCV. None of these effects is seen following transfection with a control adenovirus that does not encode tk. The increased cell death, early caspase-3 activation and PARP cleavage, and flow cytometric changes seen in pro-caspase-3-expressing cells can be partially inhibited by treatment with benzyloxycarbonyl-val-ala-asp fluoromethylketone, a synthetic caspase inhibitor. Our data suggest that co-expression of pro-caspase-3 may lead to a significant enhancement of the efficacy of tk/GCV therapy.
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PMID:Herpes simplex virus thymidine kinase/ganciclovir-induced cell death is enhanced by co-expression of caspase-3 in ovarian carcinoma cells. 1139 84

Human non-small cell lung cancer (NSCLC) cells were transfected with recombinant prodrug herpes simplex virus type I thymidine kinase (HSV-tk) cDNA, and the selected clones underwent apoptosis in response to induction by antiviral ganciclovir (GCV). The efficiency of GCV-induced growth inhibition and the extent of the bystander effect were associated with the expression level of HSV-TK in stable transfectants. Development in the HSV-tk/GCV system toward cell death was initiated with cell-cycle accumulation at S and G(2)/M phases, immediately followed by the appearance of sub-G(0)/G(1) cells after drug exposure. To investigate the regulation of cell-cycle modulators during drug treatment, we analyzed release of the apoptosis initiator cytochrome c and activation of the downstream effectors caspase-9, caspase-3 and poly(ADP-ribose)polymerase 16 hr after GCV sensitization, followed by transient escalation of tumor-suppressor p53 and cell-cycle modulators cyclin A and B(1) before committing to programmed cell death. Furthermore, tumor regression was proportional to the degree of ectopic expression of the transferred HSV-tk gene. Our results demonstrate that the HSV-tk/GCV system effectively inhibits the proliferation of NSCLC cells in vitro and in vivo through potent induction of apoptosis, thus providing a rationale for further development.
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PMID:Ectopic expression of herpes simplex virus-thymidine kinase gene in human non-small cell lung cancer cells conferred caspase-activated apoptosis sensitized by ganciclovir. 1240

Liver fibrosis is the result from a relative imbalance between synthesis and degradation of matrix proteins. Following liver injury of any etiology, hepatic stellate cells undergo a response known as activation, which is the transition of quiescent cells into proliferative, fibrogenic, and contractile myofibroblasts. Upon this cellular transdifferentiation the effector cell becomes the major source of fibrillar and non-fibrillar matrix proteins resulting in excessive scar formation and cirrhosis, the end stage of fibrosis. Concomitant with progressive liver fibrosis, the tissue inhibitor of metalloproteinases-1 (TIMP-1) is strongly activated in hepatic stellate cells. We have developed a recombinant replication-defective adenovirus in which the TIMP-1 promoter is coupled to the herpes simplex virus thymidine kinase gene rendering activated hepatic stellate cells susceptible to ganciclovir. This novel targeted suicide gene approach was validated in a culture model considered to reflect an accelerated time course of the cellular and molecular events that occur during liver fibrosis. We demonstrate that transfer of the suicide gene to culture-activated hepatic stellate cells results in a strong expression of the respective transgene as assessed by Northern blot and Western blot analyses. The enzyme catalyzed the proper conversion of its prodrug subsequently initiating programmed cell death as estimated by caspase-3 assay and Annexin V-Fluos staining. Altogether, these results indicate that induction of programmed cell death is a promising approach to eliminate fibrogenic HSC.
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PMID:Induction of cell death in activated hepatic stellate cells by targeted gene expression of the thymidine kinase/ganciclovir system. 1504 99

We investigated the effectiveness of in vivo electrogene transfer as a means of therapy in rat urinary bladder carcinoma and in mammary carcinoma models in both athymic and syngeneic mice using the herpes simplex virus 1 thymidine kinase (HSVtk) or IL-12 genes in combination with ganciclovir (GCV). A significant increase in the levels of tissue apoptosis and necrosis was induced with a single injection of HSVtk vector directly into bladder and mammary tumors followed by in vivo transfection and a regimen of intraperitoneal GCV injection. This procedure induced significant selective tumor cell death, characterized by marked inflammation and peripheral macrophage influx. Active caspase-3 was also strongly expressed in areas of cell death, indicating the initiation of apoptosis. This result was confirmed in corollary in vitro studies on a mouse bladder carcinoma cell line in which elevated caspase-3, -8, and -9 activities and decreased mitochondrial membrane potential were observed as a result of transfection with HSVtk and addition of GCV to the medium. In the syngeneic mouse mammary cancer model, we additionally found both tumor volume and metastasis to lymph nodes and lungs to be significantly reduced throughout the 2-month experiment. However, in contrast to their syngeneic counterparts, HSVtk/GCV therapy did not effectively inhibit mammary tumor growth/metastasis in an athymic mouse model, leading us to believe that T-cell-mediated immune responses may participate via the bystander effect in HSVtk/GCV experimental therapy. We subsequently evaluated the antitumor activity of IL-12, which can activate T-cell-mediated immune responses involving macrophages, in the syngeneic mammary tumors and found that IL-12 also significantly suppressed mammary tumor growth and metastasis. We thus suggest that in vivo electrogene transfer is a useful transfection tool in cancer gene therapy and, in addition, we show that T-cell-mediated immune responses may be a critical factor in cancer gene therapy using HSVtk/GCV and IL-12.
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PMID:Experimental gene therapy in mammary and urinary bladder cancer using electrogene transfer. 1561 46

To improve the effectiveness of herpes simplex virus (HSV) thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy, the replication-defective HSV vector TOIkappaB expressing both HSV-TK and a mutant form of the NF-kappaB inhibitor IkappaBalpha (IkappaBalphaM) was developed. TOIkappaB was constructed by recombining the IkappaBalphaM gene into the U(L)41 locus of a replication-defective lacZ expression vector, TOZ.1. Expression of IkappaBalphaM was confirmed by Western blotting, and the ability of the mutant protein to inhibit NF-kappaB nuclear translocation was examined by electrophoretic mobility shift assay. In human glioblastoma U-87MG cells, the p50/p50 dimer of NF-kappaB was already translocated to the nucleus without receptor-dependent signaling by TNF-alpha. Following infection with TOIkappaB, nuclear translocation of NF-kappaB in U-87MG cells was significantly inhibited and caspase-3 activity increased compared with TOZ.1-infected cells. The cytotoxicity of TOIkappaB for U-87MG cells was investigated by colorimetric MTT assay. At an MOI of 3, TOIkappaB infection killed 85% of the cells compared to 20% killed by TOZ.1 infection. In the presence of GCV, these numbers increased to 95-100% for TOIkappaB and 80-85% for TOZ.1. TOIkappaB neurotoxicity measured on cultured murine neurons was relatively low and similar to that of TOZ.1. The survival of nude mice implanted into the brain with U-87MG tumor cells was markedly prolonged by intratumoral TOIkappaB injection and GCV administration. Survival of TOIkappaB+GCV group was significantly longer (P<.02, Wilcoxon test) than for the control groups (TOZ.1 or TOIkappaB only, PBS or PBS+GCV). These results suggest that IkappaBalphaM expression may be a safe enhancement of replication-defective HSV-based suicide gene therapy in vitro and in vivo.
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PMID:Combination gene therapy for glioblastoma involving herpes simplex virus vector-mediated codelivery of mutant IkappaBalpha and HSV thymidine kinase. 1569 8

Herpesvirus tegument protein VP22 has been shown to have biotherapeutic potential in tumor gene therapy. Some studies indicate that VP22 may enhance the transfer efficiency of therapeutic proteins by delivering them to more cells while trafficking. Our previous study showed that bovine herpesvirus VP22 (BVP22) enhanced equine herpesvirus thymidine kinase-ganciclovir (Etk-GCV) suicide gene therapy by an unknown intracellular effect. In this study, the interaction between BVP22 and host tumor cells was studied in neuroblastoma NXS2 cells. Cell cycle analysis was performed to determine whether BVP22 possesses biotherapeutic potential by altering the cell cycle, making cells more sensitive to therapeutic genes. As a result, the cell cycle was not affected by the transfection of BVP22 into NXS2 cells. However, cytotoxicity induced by BVP22 was observed in NXS2 cells on the second and third days after transient transfection. Further, analyses of caspase-3 activity and apoptosis suggested that BVP22 induces apoptosis in host tumor cells by upregulating the expression ratio of Bax to Bcl-2.
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PMID:Bovine herpesvirus VP22 induces apoptosis in neuroblastoma cells by upregulating the expression ratio of Bax to Bcl-2. 1570 93

Overexpression of NF-kappa B reportedly plays anti-apoptotic roles in the growth of AML cells. Control of AML cell growth was attempted using a replication-defective herpes simplex virus-1 vector, T0I kappa B alpha, overexpressing mutant I kappa B alpha to inhibit NF-kappa B in vitro. T0I kappa B alpha displays defective ICP4/ICP22/ICP27, isogenic thymidine kinase, and mutant I kappa B alpha. T0Z.1 expressing lacZ instead of I kappa B was used for controls. Infection of T0I kappa B alpha at 15 multiplicity of infection (MOI) with cells of AML lines, HL60, K562, and NB4 displaying >90% infection efficiency and tumor killing in vitro. Use of 10 microM of Ara-C alone was clinically equivalent to high-dose Ara-C, displaying 11% tumor killing. Neither ganciclovir (GCV) nor Ara-C enhanced T0I kappa B- alpha mediated tumor killing. Attenuation of NF-kappa B by T0I kappa B alpha was confirmed by EMSA. T0I kappa B alpha induced caspase-3 activity, with subsequent apoptosis confirmed by colorimetric and TUNEL assays. Fresh AML cells from 8 patients were infected with T0I kappa B alpha at 3 MOI, with or without GCV or 10 microM of Ara-C in vitro. Infection efficiency was 10%. T0I kappa B alpha displayed 8-15% tumor killing, superior to Ara-C in 6 of the 8 patients. Administration of Ara-C enhanced tumor killing in 5 of these 6 cases. Our results suggest that T0I kappa B alpha-mediated gene therapy induces apoptosis of AML cells in vitro.
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PMID:I kappa B-mediated apoptotic gene therapy against acute myelogenous leukemia using replication-defective HSV-1 vector expressing TK and mutant I kappa B alpha. 1617 66

Because the efficacy of genetic prodrug activation therapy (GPAT) using herpes simplex virus thymidine kinase (tk)/ganciclovir (GCV) or Escherichia coli cytosine deaminase (cd)/5-fluorocytosine (5-FC) is not satisfied in early clinical trials and the mechanism of both the GPATs have been shown to lead to the activation of cell apoptotic pathway, we hypothesized that coexpression of procaspase-3, a central downstream executioner of apoptotic pathways, with cd-tk gene leads to enhanced cell death in ovarian cancer cells in vitro. Following transfection with the vectors encoding cd and tk, 5-FC and GCV treatments lead to greater cell death in procaspase-3-expressing clones of 3AO (3AO-caspase-3) than control cells (3AO-pcDNA3), as well as more rapid activation of caspase-3 and more rapid cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP). There is a greater degree of cell apoptotic rate in the procaspase-3-expressing clones than in control cells following the treatment with cd-tk/5-FC + GCV, and apoptosis is the main cell death form. None of these effects is seen following transfection with a control vector that does not encode tk and cd (pBTdel-279). The results strongly suggest that coexpression of procaspase-3 may lead to a significant enhancement of the efficacy of cd-tk/5-FC + GCV, and this strategy would be a novel and promising approach for the treatment of ovarian cancer.
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PMID:Procaspase-3 enhances the in vitro effect of cytosine deaminase-thymidine kinase disuicide gene therapy on human ovarian cancer. 1644 27

Use of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system is one of the promising approaches in the rapidly growing area of gene therapy. The "bystander effect," a phenomenon in which HSV-tk+ cells exposed to GCV are toxic to adjacent HSV-tk- cells, was reported to play an important role in suicide gene therapy. However, the mechanism by which HSV-tk/GCV induces the bystander effect is poorly understood. We monitored the activation of caspase-3 in living cells induced by the HSV-tk/GCV system using a genetically encoded fluorescence resonance energy transfer (FRET) probe CD3, , a caspase-3 recognition site fused with a cyan fluorescent protien (CFP) and a red fluorescent protein (DsRed) which we reported and named in a previous paper. Fluorescence protein (FP)-based multicolor cellular labeling, combined with the multichannel fluorescence imaging and FRET imaging techniques, provides a novel and improved approach to directly determine whether the activation of caspase-3 involved in the HSV-tk/GCV system induces cell apoptosis in tk gene-expressing cells and their neighboring cells. FRET ratio images of CD3, and fluorescence images of the fusion protein of thymidine kinase linked with green fluorescent protein (TK-GFP), indicated that HSV-tk/GCV system-induced apoptosis in human adenoid cystic carcinoma (ACC-M) cells was via a caspase-3 pathway, and the activation of caspase-3 was not involved in the bystander effect of HSV-tk/GCV system.
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PMID:Activation of caspase-3 noninvolved in the bystander effect of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system. 1860 33


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