Administration Of Trichostatin A Research Articles

6 EFFECT OF TRICHOSTATIN A ON HISTONE ACETYLATION AND METHYLATION, EMBRYO DEVELOPMENT, AND IN VIVO VIABILITY OF INTER-SPECIES BLACK FOOTED CAT CLONED EMBRYOS

Aberrant DNA methylation and histone acetylation patterns following somatic cell nuclear transfer (SCNT) may disrupt the expression of imprinted and pluripotency-related genes, resulting in improper embryo development and fetal abnormalities. Treatment of mouse, pig and rabbit intra-species cloned embryos with the histone deacetylase inhibitor trichostatin A (TSA) has been reported to improve embryo development to the blastocyst stage (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Zhang et al. 2007 Cloning Stem Cells 9, 357–363; Shi et al. 2008 J. Anim. Sci. 86, 1106–1113). TSA has also increased the number of mouse live offspring after embryo transfer (Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). We have evaluated the effect of TSA on the ability of domestic cat (DSH, Felis catus) cytoplasts to support nuclear reprogramming and embryo development using black-footed cat (BFC, Felis nigripes) donor nuclei. To observe the effect of TSA on the covalent modification of histone H3, BFC-DSH reconstructed embryos were treated with 0, 5, 50, and 100 nm concentrations of TSA for 2 h before activation and 4 h and 16 h following activation. Levels of trimethylation and acetylation at lysine 9 of histone 3 (H3K9) were assessed by immunofluorescence and confocal microscopy. Average nuclear staining intensity was calculated for each antibody and compared between TSA treatments for each time point. Two to three oocytes were analyzed per group. H3K9 methylation was low or non-detectable for all treatments with or without TSA. H3K9 acetylation levels were not affected at 2 h for each of the TSA concentrations v. control. At 4 h and 16 h post-activation, H3K9 acetylation increased with exposure to TSA in a concentration-dependent manner. Next, BFC-DSH cloned embryos were treated with 50 or 100 nm TSA for 6 h or 24 h to determine the effect of TSA on in vitro development to the blastocyst stage (n = 157) and in vivo development after embryo transfer into domestic cat recipients (n = 255). TSA exposure did not enhance embryo cleavage, and among TSA treated embryos, blastocyst development was observed only in cybrids that were treated with 50 nm TSA for 24 h. The development rate of 2.8% for this group (n = 38) was similar to that of untreated embryos (2.0 to 3.3%, n = 502; P > 0.05). One pregnancy was established after transfer of TSA treated embryos, using a 24 h, 50 nm TSA treatment. The pregnancy rate (50%) and percentage of embryos that implanted following transfer (4.4%, n = 68) corresponded to those of untreated BFC-DSH cloned embryos (57.1% and 4.2%, n = 213; P > 0.05). Similar to untreated controls, all TSA treated embryos that implanted in DSH recipients developed an amorphous fetal mass and started reabsorption by Day 30 of gestation. In summary, administration of TSA did have an effect on the acetylation level of histone H3K9, but did not improve embryo development to the blastocyst stage or in vivo viability of inter-species black-footed cat cloned embryos.

The histone deacetylase inhibitor trichostatin A upregulates regulatory T cells and modulates autoimmunity in NZB/W F1 mice

We sought to determine if the histone deacetylase inhibitor (HDI), trichostatin A (TSA), would alter systemic lupus erythematosus (SLE) in NZB/W mice. Fourteen to sixteen-week-old female NZB/W F1 mice were given TSA (1.0 mg/kg body weight (BW)) intraperitonealy (i.p.) daily, TSA (1.0 mg/kg BW) i.p. + anti-CD25 (250 mg/mouse) i.p. every third day, only anti-CD25 (250 mg/mouse) i.p., DMSO or isotype IgG. Disease progression was assessed as they aged. Mice were sacrificed at 26 or 38 weeks of age, tissues collected and evaluated. At 36 weeks, TSA-treated animals had decreased anti-double stranded DNA (dsDNA) autoantibodies and decreased protein excretion compared to controls. Spleen size and the percentage of CD4 +CD69 + cells were decreased, with an increase in CD4 +CD25 + T cells in the TSA-treated mice. Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis of T cells showed a decrease in IL-6 production but an increase in TGF-β1 and Foxp3 in the TSA-treated animals. Kidney analysis showed a decrease in IgG and C3 deposition, decrease in pathologic glomerular disease and renal MCP-1, MMP-9, and IL-6 mRNA expression. Anti-CD25-treated mice euthanized at 26 weeks of age showed decreased Foxp3 +CD4 +CD25 + T cells compared to TSA-treated mice. These data suggest TSA administration modulates lupus-like disease, in part, by increasing T regulatory cells.

Trichostatin A and Oncolytic HSV Combination Therapy Shows Enhanced Antitumoral and Antiangiogenic Effects

Oncolytic herpes simplex viruses (HSVs) possess direct oncolytic and antiangiogenic activities and are promising anticancer agents, but their efficacy, when used as single agents, leaves room for improvement. We investigated whether combination therapy of HSV with histone deacetylase inhibitor trichostatin A (TSA), an agent that also targets cancer cells and tumor vasculature, would result in enhanced efficacy. In vitro, TSA and G47Delta showed strong synergy of action against proliferating endothelial cells, varying degrees of synergistic action against most cancer cell lines, but no effect in quiescent, normal endothelial and prostate epithelial cells. Synergy is dependent on viral replication; however, it is not dependent on the dosing sequence of TSA and G47Delta, viral genetic alterations, infectivity, or replication kinetics of G47Delta. Using an isogenic cell system, we found that a high level of cellular cyclin D1 is also critically important for the interaction. Normal cells with low cyclin D1 levels were not subjected to toxicity by either agent. In tumor cells and proliferating endothelial cells, the combination treatment enhanced the inhibition of cyclin D1 and vascular endothelial growth factor (VEGF). Concurrent systemic TSA and intratumoral G47Delta administration resulted in enhanced antiangiogenesis and enhanced antitumoral efficacy in animal models. Therefore, combination treatment with TSA and oncolytic HSV provides a novel approach to cancer therapy.

The histone deacetylase inhibitor trichostatin A (TSA) upregulates regulatory T cells and modulates renal disease in NZB/NZW F1 mice

In our present studies we sought to determine if the histone deacetylase inhibitor (HDI), trichostatin A (TSA), would alter systemic lupus erythematosus (SLE) in NZB/W mice by regulating CD4+CD25+ T‐regulatory cells. Fourteen to sixteen‐week‐old female NZB/W mice were injected with TSA (1.0 mg/kg BW) intraperitoneally (i.p.) daily, anti‐CD25 (250 mg/mouse) i.p. weekly, both TSA (1.0 mg/kg BW) and anti‐CD25 (250 mg/mouse) i.p. bi‐weekly, or untreated. The mice were monitored for various disease parameters as they aged. Mice were sacrificed at either 26 or 36 weeks of age, tissues collected and assessed for various parameters of disease. Flow cytometric analysis of splenic tissue showed TSA treated mice exhibited increased FOXP3 expression and increased CD4+CD25+ T‐regulatory cells. At 36 weeks, TSA treated animals had decreased anti‐double stranded DNA autoantibodies and decreased protein excretion. Renal pathology showed a decrease in IgG and C3 deposition in renal tissue and a decrease in pathologic glomerular disease in the TSA treated mice. These data indicate TSA decreases lupus nephritis by upregulating FOXP3 expression and CD4+CD25+T cells in NZB/W mice. Taken together, these data show TSA administration modulates lupus in part by increasing T‐regulatory cell function, and warrants further investigation as to its potential in treating SLE.

Trichostatin A, a histone deacetylase inhibitor, suppresses synovial inflammation and subsequent cartilage destruction in a collagen antibody-induced arthritis mouse model

To investigate the effect of the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), on joint inflammation and cartilage degeneration in a collagen antibody-induced arthritis (CAIA) mouse model. CAIA mice were given daily subcutaneous injections of various concentrations of TSA (0, 0.5, 1.0, and 2.0 mg/kg) and various parameters were monitored for 14 days. On Day 15, the hind paws were examined histologically. To investigate the effects of TSA on the expressions of matrix metalloproteinase (MMP)-3, MMP-13, tissue inhibitor of MMP-1 (TIMP-1), and acetyl-H4 by chondrocytes, another group of mice was sacrificed on Day 6. In vitro direct effect of TSA was examined by real-time PCR for mRNA of type II collagen, aggrecan, MMP-3, and MMP-13 in murine chondrogenic ATDC5 cells after pro-inflammatory cytokine stimulation. In the TSA-treated group, clinical arthritis was significantly ameliorated in a dose-dependent manner. The severity of synovial inflammation and the cartilage destruction score were significantly lower in the TSA 2.0 mg/kg group compared to the other TSA-treated groups. On immunohistochemistry, the number of MMP-3 and MMP-13-positive chondrocytes was significantly lower in the TSA 2.0 mg/kg group than in the control group. In contrast, the number of TIMP-1-positive cells and acetyl-histone H4-positive cells was significantly higher in the TSA 2.0mg/kg group than in the control group. TSA suppressed interleukin 1-beta and tumor necrosis factor-alpha-stimulated up-regulation of MMP-3, but not MMP-13 mRNA expression by ATDC5. The systemic administration of TSA ameliorated synovial inflammation in CAIA mice. Subsequently cartilage destruction was also suppressed by TSA, at least in part, by modulating chondrocyte gene expression.

Histone deacetylase inhibitors and paclitaxel cause synergistic effects on apoptosis and microtubule stabilization in papillary serous endometrial cancer cells

The use of histone deacetylase (HDAC) inhibitors has shown promise for a variety of malignancies. In this investigation, we define the activity of this class of inhibitors in combination with traditional cytotoxic chemotherapy in endometrial cancer cells. Significant reductions in growth were observed in Ark2 and KLE endometrial cancer cells following treatment with paclitaxel, doxorubicin, carboplatin, or the HDAC inhibitor trichostatin A (TSA). However, only combined treatment with TSA/paclitaxel caused synergistic inhibition of cell growth. This combination also resulted in significant changes in cell morphology. Using cell cycle analysis, nuclear staining, and Western blot analysis for poly(ADP-ribose) polymerase and caspase-9 degradation products, TSA/paclitaxel showed the most dramatic activation of the apoptotic cascade. These effects were also observed when the HDAC inhibitors HDAC inhibitor-1 or oxamflatin were substituted for TSA. The anticancer properties of paclitaxel are known to result in part from inhibition of microtubule depolymerization, which results in apoptosis. We show that TSA administration also stabilizes microtubules via alpha-tubulin acetylation. Furthermore, using Western blot and immunohistochemical analysis, treatment with TSA/paclitaxel led to a significant increase in acetylated tubulin and microtubule stabilization. These effects were confirmed in a mouse xenograft model. Moreover, TSA/paclitaxel resulted in a 50% reduction in tumor weight compared with either agent alone. This study provides in vivo evidence of nonhistone protein acetylation as one possible mechanism by which HDAC inhibitors reduce cancer growth. The TSA/paclitaxel combination seems to hold promise for the treatment of serous endometrial carcinoma and other malignancies with limited sensitivity to paclitaxel.

Inhibition of histone deacetylation by trichostatin A intensifies the transcriptions of neuronal c- fos and c- jun genes after kainate stimulation

Kainate stimulation induces the expression of immediate early genes, c- fos and c- jun genes. Trichostatin A (TSA), a potent histone deacetylase (HDAC) enzyme inhibitor was used to test the role of histone hyperacetylation in the transcriptional regulation of c- fos and c- jun genes in neuronal cells in vivo and in vitro. Intraperitoneal administration of TSA increased histone H4 acetylation in hippocampi. Mice pretreatment with TSA were injected with kainic acid intraperitoneally and sacrificed over a time course of 12 h. Northern blot analysis and in situ hybridization showed that TSA pretreatment caused an increase in pre-existing basal levels of c- jun at 0 h and also intensified the maximal expression of both genes especially in the pyramidal layers of the hippocampus, thus demonstrating the inhibition of HDACs subsequently led to histone hyperacetylation to increase these genes expressions. TSA did not prolong the expression of c- fos or c- jun gene, in contrary to what we expected. Primary hippocampal neuron cell culture also displayed a similar pattern of c- fos and c- jun mRNA enhancement with trichostatin A pretreatment. This study demonstrated that inhibition of histone deacetylation by TSA in neuronal cells affect the expressions of c- fos and c- jun genes, suggesting histone acetylation might play a role in the regulation of both genes expressions after kainate stimulation.

Circadian and light-induced transcription of clock gene Per1 depends on histone acetylation and deacetylation.

Circadian clock genes are regulated through a transcriptional-translational feedback loop. Alterations of the chromatin structure by histone acetyltransferases and histone deacetylases (HDACs) are commonly implicated in the regulation of gene transcription. However, little is known about the transcriptional regulation of mammalian clock genes by chromatin modification. Here, we show that the state of acetylated histones fluctuated in parallel with the rhythm of mouse Per1 (mPer1) or mPer2 expression in fibroblast cells and liver. Mouse CRY1 (mCRY1) repressed transcription with HDACs and mSin3B, which was relieved by the HDAC inhibitor trichostatin A (TSA). In turn, TSA induced endogenous mPer1 expression as well as the acetylation of histones H3 and H4, which interacted with the mPer1 promoter region in fibroblast cells. Moreover, a light pulse stimulated rapid histone acetylation associated with the promoters of mPer1 or mPer2 in the suprachiasmatic nucleus (SCN) and the binding of phospho-CREB in the CRE of mPer1. We also showed that TSA administration into the lateral ventricle induced mPer1 and mPer2 expression in the SCN. Taken together, these data indicate that the rhythmic transcription and light induction of clock genes are regulated by histone acetylation and deacetylation.

Transcriptionally active drugs improve adenovirus vector performance in vitro and in vivo.

Cytomegalovirus (CMV) promoter is often present in recombinant adenovirus vectors (AdVs) suitable for gene therapy, ensuring high levels of transgene production in a wide range of hosts. Despite this characteristic, the presence of the AdV genome in target cells and tissues typically lasts longer than transgene production that may be rapidly extincted by ill-defined silencing mechanisms. In the present article, it is reported that transcriptionally active drugs, retinoic acid (RA) and histone deacetylase inhibitor trichostatin A (TSA), enhance AdV transgene expression in infected cells and tissues. The association of RA and TSA increased more than seven-fold above control the activity of AdVs encoding for LacZ or VEGF165. This effect was, at least in part, mediated by the direct activation of retinoic acid receptors. Finally, administration of RA and TSA alone at days 0 and 5 after infection prolonged transgene production up to 21 days after infection versus 6-8 days in untreated controls. These results indicate that transcriptionally active drugs improve AdV function and may represent a novel strategy to more efficiently design AdVs for gene therapy interventions.