Explore the vast range of titles available.

Background/Aims: Herpes-simplex-virus-associated erythema multiforme (HAEM) is characterized by lesional skin expression of the viral protein Pol and localized inflammation. The objective of this study is to examine the mechanism whereby Pol induces localized inflammation. Methods: A431 cells transfected with Pol or an empty vector and lesional skin from HAEM or drug-induced erythema multiforme patients were examined for expression of the transcription factor SP1 and SP1-regulated genes by immunoblotting, immunohistochemistry and immunofluorescence. Results: SP1, TGF-β, p21 waf1 and Hsp27 were upregulated in A431 cells transfected with Pol but not the empty vector. Expression was further increased by exposure to IFN-γ. Pol+ HAEM lesional skin expressed SP1, Hsp27, TGF-β and p21 waf1 . Normal skin and drug-induced erythema multiforme lesional skin were negative. Conclusion: The data indicate that Pol activates SP1, causing upregulation of SP1 target genes (notably TGF-β) involved in localized inflammation. Upregulation is potentiated by IFN-γ.

Identification of targets and delivery platforms for gene therapy of neurodegenerative disorders is a clinical challenge. We describe a novel paradigm in which the neuroprotective gene is the herpes simplex virus type 2 (HSV-2) antiapoptotic gene ICP10PK and the vector is the growth-compromised HSV-2 mutant ΔRR. ΔRR is delivered intranasally. It is not toxic in rats and mice. ICP10PK is expressed in the hippocampus of the ΔRR-treated animals for at least 42 days in the absence of virus replication and late virus gene expression. Its expression is regulated by an AP-1 amplification loop. Intranasally delivered ΔRR prevents kainic acid-induced seizures, neuronal loss, and inflammation, in both rats and mice. The data suggest that ΔRR is a promising therapeutic platform for neurodegenerative diseases.

The newly cloned small heat shock protein H11 evidences cell-type specific silencing by aberrant DNA methylation in 5/9 melanoma cell lines. In these cells, the de-methylating agent Aza-C causes H11 overload and triggers growth arrest and apoptosis. Apoptosis is blocked by antisense, but not sense, H11 oligonucleotides, indicating that it is induced by H11. Aza-C treatment did not induce H11 expression nor trigger growth arrest/apoptosis in cultured normal melanocytes. To identify the mechanism of growth arrest/apoptosis induced by H11 overload, melanoma cells were stably transfected with H11 under the control of a tetracycline sensitive promoter and examined for altered gene expression and cell survival before and after treatment with Doxycycline (Dox). Dox (2 μg/ml) caused a time-dependent increase in H11 expression, reaching maximal levels on day 3 post treatment. H11 overload was accompanied by growth arrest (first seen on days 1-2 post Dox), activation of caspases-9 and-3, seen as early as day 1 post Dox and a time-dependent increase in the% TUNEL+ (apoptotic) cells (70-85% on days 3-4 post Dox). Immunoprecipitation/immunoblotting and immunocomplex PK assays indicated that H11 bound and activated TAK1. TAK1 did not bind and was not activated by the H11 mutant, W51C which is dominant negative for apoptosis. Caspase activation was inhibited with the dominant negative TAK1 mutant K63W or the p38MAPK pharmacologic inhibitor SB203580, indicating that apoptosis is caused by H11-mediated activation of the TAK1/p38MAPK pathway. H11-mediated growth arrest was also caused by activated TAK1, through phosphorylation of b-catenin and inhibition of MITF and CDK2, which are required for melanoma cell proliferation. H11 did not bind nor activate TAK1 in normal melanocytes. Studies of the highly tumorigenic A2058 and LM melanoma cultures in a mouse xenograft model, indicated that tumor growth was significantly (p<0.001) reduced by treatment with Aza-C (5mg/kg) given by ip injection on days 6 and 15 after initiation of the xenografts (3 doses at 3hr intervals). Decreased tumor growth was due to H11 induced apoptosis as evidenced by H11/TUNEL co-localization in residual tumor cells. The data suggest that H11 is a promising target for chemogene therapy of melanoma and identify additional members of the H11-mediated pro-apoptotic cascade.

Background: The heat shock protein H11 is silenced in melanoma cell lines, where its forced expression by demethylation with Aza-C triggers apoptosis. Objective: To examine whether H11 is silenced by aberrant DNA methylation in melanoma as compared to nevi and normal skin tissues. Methods: Cell suspensions from benign intradermal nevi, atypical nevi and malignant melanoma tissues were used in reverse-transcriptase PCR and methylation-specific PCR. Paraffin-embedded tissues were stained with H11 antibody. Results: H11 is methylated in 60–75% of melanoma and atypical nevi, but not in normal skin or most benign nevi. Methylation is inversely correlated with H11 expression. Conclusion: The heat shock protein H11 is silenced by aberrant DNA methylation in melanoma, but not benign melanocytic lesions or normal skin melanocytes. The data suggest that H11 is a promising target for the molecular therapy of melanoma.

Molecular therapeutics is a recognized promising approach for melanoma, but relevant target genes remain elusive. We report that overload of the recently cloned H11/HspB8 induces apoptosis in 55% of examined melanoma cultures. Apoptosis was determined by activation of caspases-9 and -3 and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL), and was not seen in normal melanocytes. It was associated with H11/HspB8 complexation with transforming growth factor- β- activated kinase (TAK) 1 and activation of TAK1 and p38 mitogen activated protein 3 kinases. TAK1 was not bound, nor activated by the H11/HspB8 mutant W51C, which has dominant antiapoptotic activity. β -Catenin was phosphorylated by activated TAK1, inhibiting its nuclear accumulation and mictophthalmia-associated transcription factor and cyclin dependent kinase 2 expression. The dominant-negative TAK1 mutant K63W inhibited β -catenin phosphorylation and caspase activation. The data indicate that H11/HspB8 overload causes melanoma growth arrest and apoptosis through TAK1 activation and suggest that H11/HspB8 is a promising molecular therapy target.

Apoptosis is a widely accepted component of the pathogenesis of Parkinson's disease (PD), a debilitating neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra. However, additional death programs were implicated, and current understanding of the cycle of intracellular events that leads to the demise of these neuronJis limited. Gene therapy strategies were proposed to inhibit apoptosis, but they have met with relatively limited success. Here we report that the antiapoptotic herpes simplex virus type 2 gene ICP10PK protects neuronally differentiated PC12 cells from death caused by 1-methyl-4-phenylpyridinium ( in vitro PD model) through inhibition of calpain I activation and the resulting inhibition of Bax translocation to the mitochondria, apoptosis-inducing factor release and caspase-3 activation. Neuroprotection is through ICP10PK-mediated activation of the PI3-K/Akt survival pathway and upregulation/stabilization of the antiapoptotic protein Bcl-2 and the cytoprotective chaperone heat-shock protein 70.

Malignant melanoma is a highly aggressive and drug-resistant cancer. Virotherapy is a novel therapeutic strategy based on cancer cell lysis through selective virus replication. However, its clinical efficacy is modest, apparently related to poor virus replication within the tumors. We report that the growth compromised HSV-2 mutant ΔPK has strong oncolytic activity for melanoma largely caused by a mechanism other than replication-induced cell lysis. The ratio of dead cells (determined by trypan blue or ethidium homodimer staining) to cells that stain with antibody to the major capsid protein VP5 (indicative of productive infection) was 1.8-4.1 for different melanoma cultures at 24-72hrs p.i. Cell death was due to activation of calpain as well as caspases-7 and -3 and it was abolished by the combination of calpain (PD150606) and pancaspase (zVAD-fmk) inhibitors. Upregulation of the autopahgy protein Beclin-1 and the pro-apoptotic protein H11/HspB8 accompanied ΔPK-induced melanoma oncolysis. Intratumoral ΔPK injection (106-107 pfu) significantly reduced melanoma tumor burden associated with calpain and caspases-7 and -3 activation, Beclin-1 and H11/HspB8 upregulation and activation of caspase-1 related inflammation. Complete remission was seen for 87.5% of the LM melanoma xenografts at 5 months after treatment termination. The data indicate that ΔPK is a promising virotherapy for melanoma that functions through virus-induced programmed cell death (PCD) pathways.

Gene therapy of neurodegenerative diseases is of significant current interest, but the identification of appropriate gene targets and effective delivery platforms has proven to be a major stumbling block. We constructed a novel, growth compromised HSV-2 based vector, ΔRR, which utilizes the viral anti-apoptotic gene ICP10PK to protect neurons from excitotoxic injury in animal models of temporal lobe epilepsy. In this model, intranasally delivered ΔRR evidenced no toxicity and the anti-apoptotic gene ICP10PK was expressed in tissues along the olfactory bulb route as determined by immunoblotting and/or immunohistochemistry. In the hippocampus, expression began on day 2 after ΔRR administration and was independed of virus replication, as evidenced by plaque assays and the failure to detect the viral protein, VP5, the expression of which requires viral DNA replication. ICP10PK expression lasted for at least 42 days, and it prevented kainic acid (KA) induced neuronal cell loss in the hippocmapus. An HSV-2 vector deleted in ICP10PK(ΔPK) failed to protect from KA induced seizures and neuronal loss. Studies of organotypic hippocampal cultures (OHC) indicated that protection depended on ΔRR induced activation of the MEK/ ERK and PI3K/Akt survival pathways, which override KA-induced apoptosis. Brains from ΔRR treated animals were also protected from KA induced inflammation, including microglial expression of TNFa, and astrocyte upregulation of glial fibrillary protein (GFAP). This may reflect the anti-inflammatory activity of ΔRR, because the levels of TNFα and RANTES in supernatants from ΔRR treated primary microglia cultures (114.4 pg/ml and 34.2 pg/ml, respectively) were lower than those in supernatants from ΔPK treated microglia (586.8 pg/ml and 321.7 pg/ml, respectively). In addition, ΔRR treated microglia induced neuroprotective factors. Thus, neurons exposed to the excitotoxin NMDA (5uM; 3hrs) were protected from apoptosis when co-cultured with ΔRR treated microglia as evidenced by double immunofluorescence with FITC-dUTP (TUNEL) and Texas red labeled NeuN antibody. Protection was mediated by a soluble factor, because similar results were obtained in cultures in which the ΔRR treated microglia were physically separated from the neurons using the Co-Star transwell insert co-culturing system. Protection was not seen with ΔPK or PBS treated microglia (10.4±3.3%, 53.2±6.5%, and 60±4.3%, TUNEL+cells for ΔRR, ΔPK, and PBS respectively, p<0.001). ΔRR (but not ΔPK) also induced expression of the neuroprotective transforming growth factorβ (TGFβ) and nerve growth factor (NGF) in primary astrocyte cultures. Collectively, the data indicate that ΔRR protects neurons from excitotoxic cell death by overriding apoptotic pathways, as well as modulating support cells in the CNS to create a more favorable microenviornment for neuron survival.