Die Spinale Muskelatrophie (SMA) ist mit einer Inzidenz von 1:6000 die zweithäufigste autosomal-rezessive Erbkrankheit im frühen Kindesalter. Die durch den Verlust des SMN- (survival of motoneuron) Gens reduzierte SMN Protein Expression führt zu einer Degeneration der spinalen Motoneurone mit proximal betonter Muskelschwäche. Deshalb zielen erste Therapieversuche darauf ab, diese zu erhöhen. Es war gezeigt worden, dass durch den Einsatz von Histon Deacetylase Inhibitoren (HDAC) in neuronalen Kontroll Zellen und in nicht neuronalen Zellen von SMA Patienten die SMN Protein Expression signifikant gesteigert werden konnte. In der vorgelegten Arbeit wurde untersucht, ob die HDAC Inhibitoren Valproat, SAHA und FK228 Einfluss auf die SMN Protein Expression in kortikalen neuronalen Vorläuferzellen (NSC), auf primär embryonale Fibroblasten (PMEF) und auf die morphologischen Veränderungen in primär kultivierten embryonalen Motoneuronen eines Mausmodells der SMA haben. Es konnte eine signifikante Steigerung der SMN Protein Expression durch den Einsatz von Valproat und FK228 in kortikalen neuronalen Vorläuferzellen nachgewiesen werden. Es ergab sich jedoch kein Einfluss auf die SMN Protein Expression in primär embryonalen Fibroblasten. Bei NSCs und primär kultivierten embryonalen Motoneuronen wirkten die HDAC Inhibitoren Valproat und FK228 konzentrationsabhängig toxisch auf das Überleben, die Länge der Axone und die Größe der Wachstumskegel. Es konnte kein positiver Einfluss auf die morphologischen Veränderungen des Mausmodells gesehen werden. Zusammenfassend zeigte sich in der vorgelegten Arbeit ein positiver Effekt auf die SMN Protein Expression durch den Einsatz von HDAC Inhibitoren, der jedoch mit einem toxischen Effekt auf die behandelten neuronalen Zellen einherging. / Spinal muscular atrophy (SMA) has an incidence of 1:6000 and is the second most common autosomal recessive hereditary disease in early childhood. The disease is characterized by the degeneration of spinal motor neurons with weakness of the proximal limb. This is caused by the deficiency of the SMN (survival of motor neuron) protein. Therefore therapeutical strategies aim to increase the SMN protein level. It has been shown that histone deacetylase inhibitors could increase SMN protein level in neuronal control cells and non-neuronal cells of SMA patients. The aim of the presented study was to investigate whether the HDAC inhibitors valproic acid, SAHA and FK228 had an effect on the SMN protein level in cortical neural progenitor cells or primary embryonic fibroblasts of a SMA mice model. The second question was if morphological pathologies in primary cultured embryonic motor neurons of this SMA mouse model could be altered. There was a significant increase in SMN protein level by the use of valproic acid and FK228 in cortical neuronal precursor cells. However, there was no effect on the SMN protein level in primary embryonic fibroblasts. In cortical neuronal precursor cells and primary cultured embryonic motor neurons, the HDAC inhibitors valproic acid and FK228 displayed and concentration-dependent toxic effect on the survival, axonal length and the size of the growth cone. No obvious influence on the morphological changes in the mice model could be seen. In conclusion a positive effect on the SMN protein level by the use of HDAC inhibitors could be detected, but with a toxic effect on neuronal cells at the same time.

Targeting the epigenome, including the use of histone deacetylase (HDAC) inhibitors, is a novel strategy for cancer chemoprevention. Sulforaphane (SFN), a compound found at high levels in broccoli and broccoli sprouts, is a potent inducer of Phase 2 detoxification enzymes and inhibits tumorigenesis in animal models. SFN also has a marked effect on cell cycle checkpoint controls and cell survival/apoptosis in various cancer cells, through mechanisms that are poorly understood. Based on the structure of known histone deacetylase inhibitors, it was hypothesized that SFN may possess HDAC inhibitory properties. Initial studies confirmed that, indeed, at physiologically-relevant concentrations, SFN inhibited HDAC activity in human colorectal cancer cells, with a concomitant increase in acetylated histones H3 and H4, induction of p21 expression, and increased acetylated histone H4 associated with the P21 promoter. A metabolite of SFN, SFN-Cysteine, was found to be the active HDAC inhibitor. Furthermore, in BPH-1, LnCaP, and PC-3 human prostate epithelial cells, SFN inhibited HDAC activity and increased acetylation of histones. SFN also induced p21 expression, with an increase in acetylated histone H4 associated with the P21 promoter in BPH-1 cells. The downstream effects of HDAC inhibition by SFN included induction of pro-apoptotic proteins and repression of anti-apoptotic proteins, and an increase in multi-caspase activity. Dietary SFN suppressed the growth of human prostate cancer PC-3 xenografts and inhibited HDAC activity in the xenografts, peripheral blood mononuclear cells (PBMC), and prostates. In time-course studies, a single oral dose of SFN induced histone acetylation at 6 and 24 h in mouse colonic mucosa, and long-term dietary SFN treatment increased histone acetylation in the ileum, colon, PBMC, and prostates. Moreover, dietary SFN suppressed intestinal tumorigenesis significantly in Apc[superscrip min] mice, with an increase in acetylated histones detected in the normal-looking ileum and polyps and polyps from the colon. Overall, the data presented in this thesis support a novel mechanism for chemoprevention by SFN in vivo, through inhibition of histone deacetylase. The findings also imply that SFN will offer significant protection against at least two of the major cancer killers in the US, namely colon and prostate cancer. / Graduation date: 2005

Histone Deacetylase (HDAC) plays a vital role in cellular processes, for example gene expression, cell growth, and apoptosis. Finding drug candidates to inhibit the over activity of HDACs in cancer is a growing area of interest. Inhibitors, thus far, have three important motifs to be studied: the zinc binding group, a hydrophobic linker, and a cap group. By altering these groups on the inhibitor, not only can activity be increased but also selectivity within the classes of HDACs. We present the design of two novel sets of molecules that contain either a 1,2,3-triazole or 1,2,4-triazole. The 1,2,3-triazoles were synthesized using “click chemistry” with a novel pyridyl triazine catalyst. The 1,2,4-triazoles were synthesized utilizing substitution chemistry. This set of molecules was designed after suberoylanilide hydroxamic acid (SAHA) but replaced the hydroxamate with the triazole as the zinc binding group. The activity of these inhibitors against HDAC 1, HDAC 6, and SIRT 1 were tested using the Biomol Fluor de Lys in vitro kits. Though none of the synthesized compounds were strong activators or inhibitors of any of the classes of HDACs, trends were observed that could lead to the design of more potent inhibitors.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 115-136).

Friedreich’s ataxia (FRDA) is an inherited recessive disorder characterized by progressive neurological disability and heart disease. It is caused by a pathological intronic hyperexpansion of a GAA repeat in the FXN gene, encoding the essential mitochondrial protein frataxin. At the homozygous state, the GAA expansion induces a heterochromatin state with decreased histone acetylation and increased methylation, resulting in a partial deficiency of frataxin expression. This was established in cells from FRDA patients. We showed that the same chromatin changes exist in a GAA based mouse model, KIKI, generated in our laboratory. Furthermore, treatment of KIKI mice with a novel Histone Deacetylase Inhibitor (HDACi), 106, a pimelic diphenylamide that increases frataxin levels in FRDA cell culture, restored frataxin levels in the nervous system and heart of KIKI mice and induced histone hyperacetylation near the GAA repeat. As shown by microarrays, most of the differentially expressed genes in KIKI were corrected towards wild type. In an effort to improve the pharmacological profile of compound 106, we synthesized more compounds based on its structure and specificity. We characterized two of these compounds in FRDA patients’ peripheral blood lymphocytes and in the KIKI mouse model. We observed a sustained frataxin upregulation in both systems, and, by following the time course of the events, we concluded that the effects of these compounds last longer than the time of direct exposure to HDACi. Our results support the pre-clinical development of a therapeutic approach based on pimelic diphenylamide HDACis for FRDA. Laboratory tools to follow disease progression and assess drug efficacy are needed in a slowly progressive neurodegenerative disease such as FRDA. We used microarrays to characterize the gene expression profile in peripheral lymphocytes from FRDA patients, carriers and controls. We identified gene expression changes in heterozygous, clinically unaffected GAA expansion carriers, suggesting that they present a biochemical phenotype, consistent with data from animal models of frataxin deficiency. We identified a subset of genes changing in patients as a result of pathological frataxin deficiency establishing robust gene expression changes in peripheral lymphocytes. These changes can be used as a biomarker to monitor disease progression and potentially assess drug efficacy. To this end, we used he same methodology to characterize the gene expression profiles in peripheral lymphocytes after treatment with pimelic diphenylamide HDACi. This treatment had relevant effects on gene expression on peripheral patients’ blood lymphocytes. It increased frataxin levels in a dose-dependent manner, and partially rescued the gene expression phenotype associated with frataxin deficiency in the tested cell model, thus providing the first application of a biomarker gene set in FRDA.

Understanding the specific mechanisms regulating chemotherapeutic drug anti-cancer activities will uncover novel strategies to enhance the efficacy of these drugs in clinical settings. Activating Transcription Factor 3 (ATF3) is a stress inducible gene whose expression has been associated with survival outcomes in cancer models. This study characterizes the chemotherapeutic drugs, cisplatin and Histone Deacetylase Inhibitor (HDACi), M344 as novel inducers of ATF3 expression. Cisplatin is a DNA damaging agent widely used in various tumour types including lung, head and neck, and ovarian carcinomas. The HDAC inhibitor, SAHA, has recently been approved as a single agent in the treatment of subcutaneous T-cell lymphoma and HDACis themselves show potential for synergistic anti-cancer effects when used in combination with established chemotherapeutic drugs, including cisplatin. This study evaluates the mechanisms by which cisplatin and HDACi induce ATF3, as well as the role ATF3 plays as a mediator of cisplatin-induced cytotoxicity and the enhanced cytotoxicity between HDACi and cisplatin in combination. In this study, we demonstrate that cytotoxic doses of cisplatin and carboplatin consistently induced ATF3 expression in a panel of human tumour derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response (ISR) independent mechanism, all previously implicated in stress mediated ATF3 induction. Analysis of MAPKinase pathway involvement in ATF3 induction by cisplatin revealed a MAPKinase dependent mechanism. Cisplatin treatment, in combination with specific inhibitors to each MAPKinase pathway (JNK, ERK and p38) resulted in decreased ATF3 induction at the protein level. MAPKinase pathway inhibition led to decreased ATF3 mRNA expression and a reduction in the cytotoxic effects of cisplatin as measured by MTT cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific shRNAs also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3 -/- MEFs were shown to be less sensitive to cisplatin induced cytotoxicity as compared with ATF3+/+ MEFs. Taken together, we identified cisplatin as a MAPKinase pathway dependent inducer of ATF3 whose expression regulates in part cisplatin’s cytotoxic effects. Furthermore, we demonstrated that the HDAC inhibitor M344 was also an inducer of ATF3 expression at the protein and mRNA level in the same human derived cancer cell lines. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatment was also shown to enhance the cytotoxic effects of cisplatin in these cancer cell lines. Unlike cisplatin, the mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways. Utilizing ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) M334 induction of ATF3 was shown to depend on the presence of ATF4, a known regulator of ATF3 expression as part of the ISR pathway. HDACi treatment did not affect the level of histone acetylation associated with the ATF3 promoter as determined through Chromatin immunoprecipitation (ChIP) analysis, suggesting that ATF3 induction was not a direct effect of HDACi mediated histone acetylation. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.

<p>The peritrophic matrix (PM) lines the insect midgut and is composed of chitin and protein. It is required for organization of digestion and for protection of epithelial cells from mechanical damage, pathogens, and toxins. The PM of <i>Mamestra configurata</i> (Lepidoptera: Noctuidae), bertha armyworm, a serious pest of cruciferous oilseed rape, was studied. The multilayered PM is delaminated from the anterior midgut epithelium during molting Phase II by periodic pulses and degraded during the molting Phase I stage. These events are controlled by chitin synthase-B, and chitinolytic enzymes, such as chitinase and β-<i>N</i>-acetylglucosaminidase. Eighty-two PM proteins were identified and classified as: i) peritrophins, ii) enzymes and iii) other proteins. Peritrophins were further classified as simple, binary, complex and repetitive according to their structural organization and phylogenetic analysis of peritrophin A domains. The expression of most genes encoding PM proteins was specific to the midgut and independent of larval feeding status, developmental stage, or PM formation.</p> <p>This study includes the first report of chitin deacetylase (CDA) activity in the insect midgut suggesting that the PM may contain chitosan. Digestive enzymes, such as insect intestinal lipases (IILs) and serine proteases were also associated with the PM. The IIL genes differed in their expression during larval development; however, serine protease genes were expressed continuously and serine protease activity was present in the midgut of feeding and nonfeeding stages. <i>M. configurata</i> IIM4, a complex peritrophin, was susceptible to degradation by Mamestra configurata nucleopolyhedrovirus-A challenge, as the first evidence of IIM degradation by an alphabaculovirus enhancin. <i>M. configurata</i> IIM2, a binary peritrophin, was unaffected by baculoviral challenge and such resistance of an IIM has not been reported previously. The current study is also the first demonstration of silencing by RNA interference (RNAi) of any gene encoding a PM protein, in this case <i>M. configurata</i> CDA1 (McCDA1) and McPM1. In addition, both <i>in vitro</i> and <i>per os</i> feeding experiments revealed <i>McCDA1</i> silencing starting at 24 or 36 hours posttreatment, as one of the most successful demonstrations of RNAi in a lepidopteran.</p>

Histone deacetylase inhibitors (HDIs) are a group of chemo-preventive and chemo-therapeutic agents that have generated significant attention in clinical trials, given their ability to selectively induce cell cycle arrest, differentiation and/or apoptosis of tumor cells. Presently, these agents are proposed to function by altering gene expression levels, primarily by promoting histone hyperacetylation and gene transcription. However, in this thesis, HDIs are reported to control the expression of genes from the c-Src kinase family and p21WAF1 by means other than transcriptional activation. <p>Overexpression and activation of c-Src, a 60kDa non-receptor tyrosine kinase, has been implicated in the development, growth, progression, and metastasis of several human cancers, especially those of the colon. Butyrate and the more specific histone deacetylase inhibitor trichostatin A (TSA) were both found to effectively inhibit the expression of c-Src mRNA and protein in a number of tumor cell lines, including those of the colon, liver and breast. Expression of the SRC oncogene is alternatively regulated by the SRC1A and SRC1 promoters. HDIs were shown to repress c-Src expression by inhibiting transcription of both of these promoters, independent of any new protein synthesis. Furthermore, butyrate and TSA similarly regulated the expression of the c-Src family kinase (SFK) members Yes, Fyn, Lyn and Lck in human colon cancer cell lines. In addition, TATA binding protein (TBP) associated factor 1 (TAF1) was shown to be necessary for basal transcription of the SRC1A, YES and LYN promoters, but was not required for HDI mediated repression. <p>Induction of the potent cyclin dependent kinase inhibitor p21WAF1 has been identified to be a key feature of HDI mediated cell cycle arrest. The level of p21WAF1 expression has been extensively reported to be directly upregulated by HDIs in a p53 independent manner that requires Sp family binding sites in the p21WAF1 proximal promoter to induce transcription. However, HDIs were shown to be capable of inducing p21WAF1 gene expression, dependent on new protein synthesis, by increasing mRNA stability. To date, p21WAF1 mRNA stability has been extensively studied and a number of cis-acting elements in the 3 untranslated region (UTR) of the p21WAF1 mRNA have been implicated in the regulation of mRNA stability, such as AU rich elements (AREs) and a 42 nucleotide HuD/Elav binding element. Similarly, in this work, two novel cis-acting elements were identified in the 3 UTR of p21WAF1 and were shown to facilitate basal and HDI induced post-transcriptional regulation of p21WAF1 mRNA stability in HepG2 cells. Collectively, these studies highlight the intricacy of HDI mediated effects and challenge the preconceptions regarding the molecular mechanism of these anti-tumor agents.

Understanding the specific mechanisms regulating chemotherapeutic drug anti-cancer activities will uncover novel strategies to enhance the efficacy of these drugs in clinical settings. Activating Transcription Factor 3 (ATF3) is a stress inducible gene whose expression has been associated with survival outcomes in cancer models. This study characterizes the chemotherapeutic drugs, cisplatin and Histone Deacetylase Inhibitor (HDACi), M344 as novel inducers of ATF3 expression. Cisplatin is a DNA damaging agent widely used in various tumour types including lung, head and neck, and ovarian carcinomas. The HDAC inhibitor, SAHA, has recently been approved as a single agent in the treatment of subcutaneous T-cell lymphoma and HDACis themselves show potential for synergistic anti-cancer effects when used in combination with established chemotherapeutic drugs, including cisplatin. This study evaluates the mechanisms by which cisplatin and HDACi induce ATF3, as well as the role ATF3 plays as a mediator of cisplatin-induced cytotoxicity and the enhanced cytotoxicity between HDACi and cisplatin in combination. In this study, we demonstrate that cytotoxic doses of cisplatin and carboplatin consistently induced ATF3 expression in a panel of human tumour derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response (ISR) independent mechanism, all previously implicated in stress mediated ATF3 induction. Analysis of MAPKinase pathway involvement in ATF3 induction by cisplatin revealed a MAPKinase dependent mechanism. Cisplatin treatment, in combination with specific inhibitors to each MAPKinase pathway (JNK, ERK and p38) resulted in decreased ATF3 induction at the protein level. MAPKinase pathway inhibition led to decreased ATF3 mRNA expression and a reduction in the cytotoxic effects of cisplatin as measured by MTT cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific shRNAs also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3 -/- MEFs were shown to be less sensitive to cisplatin induced cytotoxicity as compared with ATF3+/+ MEFs. Taken together, we identified cisplatin as a MAPKinase pathway dependent inducer of ATF3 whose expression regulates in part cisplatin’s cytotoxic effects. Furthermore, we demonstrated that the HDAC inhibitor M344 was also an inducer of ATF3 expression at the protein and mRNA level in the same human derived cancer cell lines. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatment was also shown to enhance the cytotoxic effects of cisplatin in these cancer cell lines. Unlike cisplatin, the mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways. Utilizing ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) M334 induction of ATF3 was shown to depend on the presence of ATF4, a known regulator of ATF3 expression as part of the ISR pathway. HDACi treatment did not affect the level of histone acetylation associated with the ATF3 promoter as determined through Chromatin immunoprecipitation (ChIP) analysis, suggesting that ATF3 induction was not a direct effect of HDACi mediated histone acetylation. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.

<p>The peritrophic matrix (PM) lines the insect midgut and is composed of chitin and protein. It is required for organization of digestion and for protection of epithelial cells from mechanical damage, pathogens, and toxins. The PM of <i>Mamestra configurata</i> (Lepidoptera: Noctuidae), bertha armyworm, a serious pest of cruciferous oilseed rape, was studied. The multilayered PM is delaminated from the anterior midgut epithelium during molting Phase II by periodic pulses and degraded during the molting Phase I stage. These events are controlled by chitin synthase-B, and chitinolytic enzymes, such as chitinase and β-<i>N</i>-acetylglucosaminidase. Eighty-two PM proteins were identified and classified as: i) peritrophins, ii) enzymes and iii) other proteins. Peritrophins were further classified as simple, binary, complex and repetitive according to their structural organization and phylogenetic analysis of peritrophin A domains. The expression of most genes encoding PM proteins was specific to the midgut and independent of larval feeding status, developmental stage, or PM formation.</p> <p>This study includes the first report of chitin deacetylase (CDA) activity in the insect midgut suggesting that the PM may contain chitosan. Digestive enzymes, such as insect intestinal lipases (IILs) and serine proteases were also associated with the PM. The IIL genes differed in their expression during larval development; however, serine protease genes were expressed continuously and serine protease activity was present in the midgut of feeding and nonfeeding stages. <i>M. configurata</i> IIM4, a complex peritrophin, was susceptible to degradation by Mamestra configurata nucleopolyhedrovirus-A challenge, as the first evidence of IIM degradation by an alphabaculovirus enhancin. <i>M. configurata</i> IIM2, a binary peritrophin, was unaffected by baculoviral challenge and such resistance of an IIM has not been reported previously. The current study is also the first demonstration of silencing by RNA interference (RNAi) of any gene encoding a PM protein, in this case <i>M. configurata</i> CDA1 (McCDA1) and McPM1. In addition, both <i>in vitro</i> and <i>per os</i> feeding experiments revealed <i>McCDA1</i> silencing starting at 24 or 36 hours posttreatment, as one of the most successful demonstrations of RNAi in a lepidopteran.</p>