Thesis (Ph. D.)--Ohio State University, 2008.

Thesis (M.S.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains viii, 140 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 119-140).

Thesis (M.S.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains x, 101 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 81-101).

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Histone deacetylases (HDACs) are an ancient class of enzymes that have been conserved throughout evolution and are found in diverse organisms such as animals, plants, fungi, eubacteria and archaebacteria. In C. elegans, twelve HDACs have been identified so far. These HDACs have been grouped into four different classes (Class I, II, III and IV) based on their cofactor requirements and sequence homologies. hda-1 is one of the three Class I HDACs in C. elegans and plays a role in the morphogenesis of several organs including the vulva. This thesis focuses on the role of hda-1 in vulval morphogenesis. The hermaphrodite vulva has twenty-two cells which can be further divided into seven different cell types: VulAs, VulBls, VulB2s, VulCs and VulDs (secondary great granddaughters), YulEs and VulFs (primary great granddaughters). The analysis of expression pattern of hda-1 revealed that hda-1 is expressed in the progeny of both the primary and secondary vulval precursor cells (VPCs). To examine hda-1 mutant phenotype in detail, I examined the expression pattern of five different vulval cell-type specific markers (cdh-3, zmp-1, ceh-2, egl-17 and daf-6) in hda-1 animals. The results revealed that hda-1 is necessary for proper differentiation of multiple vulval cell types. To study the evolutionary conservation of hda-1 function, I examined the role of hda-1 ortholog in C. briggsae. C. briggsae is a close relative of C. elegans and has almost identical vulval morphology. Knocking down Cbr-hda-1 in C. briggsae animals resulted in defective vulval phenotype. Consistent with this result, the expression of two cell- fate specific markers (C. briggsae orthologs of zmp-1 and egl-17) was found to be altered in Cbr-hda-1 RNAi treated animals. Thus, hda-1 function in the vulva appears to be conserved in these two species. To identify the hda-1 targets in vulval morphogenesis in C. elegans, microarray approach was taken. Two genes fos-1 and lin-29 were identified as putative targets and were examined in some detail. Among the targets identified (these still need to be validated), I focused on fos-1 and lin-29 for detailed investigation. The RNAi-mediated knockdown of hda-1 caused alterations in the expression pattern ofthefos-1 transcript,fos-1b. To examine interaction between fos-1 and lin-29, I used double RNAi approach and examinedfos-1 (RNAi), lin- 29 (RNAi), hda-1 (cw2) animals. It was found that fewer animals exhibit defects in vulval morphology in these animals as compared to fos-1 (RNAi), hda-1 (cw2) animals. While this suggests a possible interaction between lin-29 and hda-1 in the vulva, these results need to be validated by doing additional experiments. In summary, the work described in this thesis demonstrates that hda-1 plays an important role in vulval morphogenesis and regulates the expression of several important genes. Also, the function of hda-1 in C. elegans and C. briggsae is evolutionarily conserved. / Thesis / Master of Science (MSc)

The first part of this work describes the synthesis of a new histone deacetylase (HDAC) inhibitor (HDI). HDAC enzymes modify core histones, influence nucleosome structure and change gene transcription by removing the acetyl groups from lysine residues on proteins. HDIs are showing exciting potential as a new class of drugs for cancer and a variety of other diseases. A new HDAC inhibitor based on the hydroxamic acid motif has been synthesized. Two characteristic structural features were incorporated into the design of the novel inhibitor. A cyclic peptide mimetic of known structure was fused to a hydroxamic acid moiety through an aliphatic chain. The HDAC inhibitor provided significant inhibitory activity against HDACs with an IC50 value of 46 ± 15 nM, and against HDAC8 with an IC50 value of 208 ± 20 nM. The potent HDAC inhibitory activity of the HDAC inhibitor demonstrates the importance of the rim recognition region in the design of HDIs. The hydrophobic cyclic turn mimic allows the formation of a tight complex between HDI and HDAC enzymes. The second part of this work is to synthesize secondary structure mimics and incorporate them into the helix-turn-helix (HTH) motif. One of the important methods to study the conformation of the biologically active peptides is to incorporate the rigid peptidomimetics into the relevant peptides. Important information can be obtained from the study of conformationally constrained peptides. HTH proteins are well characterized and found in many organisms from prokaryotes to eukaryotes. The relatively small size, simple structure, and significance in stabilizing tertiary structures make the HTH peptide an attractive target to mimic. Both a Gly HTH turn mimic and a Ser HTH turn mimic were synthesized using stereoselective hydrogenation and macrocyclization starting from unnatural amino acids in a yield of 33% and 14%, respectively. The synthesis of Fmoc protected HTH turn mimics allowed incorporation into HTH peptides using Fmoc chemistry on solid phase. The incorporation of the HTH turn mimics into the peptides proved to be challenging, either by sequential elongation or by segment condensation. Alternative peptide synthesis strategies were employed in attempts to solve the problems. / Ph. D.

The increasing knowledge of molecular drivers of tumorigenesis has fueled targeted cancer therapies based on specific inhibitors. Beyond “classic” oncogene inhibitors, epigenetic therapy is an emerging field. Epigenetic alterations can occur at any time during cancer progression, altering the structure of the chromatin, the accessibility for transcription factors and thus the transcription of genes. They rely on post-translational histone modifications, particularly the acetylation of histone lysine residues, and are determined by the inverse action of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Importantly, HDACs are often aberrantly overexpressed, predominantly leading to the transcriptional repression of tumor suppressor genes. Thus, histone deacetylase inhibitors (HDACis) are powerful drugs, with some already approved for certain hematological cancers. Albeit HDACis show activity in solid tumors as well, further refinement and the development of novel drugs are needed. This review describes the capability of HDACis to influence various pathways and, based on this knowledge, gives a comprehensive overview of various preclinical and clinical studies on solid tumors. A particular focus is placed on strategies for achieving higher efficacy by combination therapies, including phosphoinositide 3-kinase (PI3K)-EGFR inhibitors and hormone- or immunotherapy. This also includes new bifunctional inhibitors as well as novel approaches for HDAC degradation via PROteolysis-TArgeting Chimeras (PROTACs).

BACKGROUND: Prostate cancer (PCa) is the most commonly diagnosed solid tumor and the second leading cancer death in the United States, and also one of the major cancer-related deaths in Chinese. Androgen deprivation therapy (ADT) is the first line treatment for metastatic PCa. PCa ultimately relapses with subsequent ADT treatment failure and becomes castrate-resistant (CR). It is important to develop effective therapies with a surrogate marker towards CR PCa. METHOD: Histone deacetylase (HDAC) inhibitors were examined to determine their effects in androgen receptor (AR)/ cellular prostatic acid phosphatase (cPAcP)-positive PCa cells, including LNCaP C-33, C-81, C4-2 and C4-2B and MDA PCa2b androgen-sensitive and androgen-independent cells, and AR/cPAcP-negative PCa cells, including PC-3 and DU 145 cells. Cell growth was determined by cell number counting. Western blot analyses were carried out to determine AR, cPAcP and PSA protein levels. RESULTS: cPAcP protein level was increased by HDAC inhibitor treatment. Valproic acid, a HDAC inhibitor, suppressed the growth of AR/cPAcP-positive PCa cells by over 50% in steroid-reduced conditions, higher than on AR/cPAcP-negative PCa cells. Further, HDAC inhibitor pretreatments increased androgen responsiveness as demonstrated by PSA protein level quantitation. CONCLUSION: Our results clearly demonstrate that HDAC inhibitors can induce cPAcP protein level, increase androgen responsiveness, and exhibit higher inhibitory activities on AR/cPAcP-positive PCa cells than on AR/cPAcP-negative PCa cells. Upon HDAC inhibitor pretreatment, PSA level was greatly elevated by androgens. This data indicates the potential clinical importance of cPAcP serving as a useful biomarker in the identification of PCa patient sub-population suitable for HDAC inhibitor treatment.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Perturbations of the complex intrinsic and extrinsic factors that contribute to cellular differentiation can have many consequences ranging from dedifferentiation to cell death. The overall objective of my research is to investigate the factors that contribute to the maintenance of mature midbrain neurons and glia. In order to address this objective, I first carried out a detailed immunocytochemical analysis to demonstrate that histone deacetylase inhibitor (HDACI) treatment of differentiated midbrain neurons in culture results in an overall destabilization of neuronal phenotype, which leads to caspase-independent cell death. GFAP positive astrocytes are refractory to the effects of HDACI treatment, suggesting that inhibition of HDACs has differential effects on neurons and glia. HDACI treatment alone was not sufficient to induce neuronal dedifferentiation as evidenced by RT-PCR analysis of stem/progenitor markers, and recovery experiments. Finally, I demonstrate that cortical neurons do not undergo cell death in response to HDACI treatment, suggesting that there may be microenvironmental factors that promote the susceptibility of midbrain neurons to the neurotoxic effects of HDACI. In the second part of this thesis I determined the molecular mechanism that was at least partly responsible for the effects of HDACI treatment on midbrain neurons. Gene expression profiling of HDACI treated midbrain cultures revealed a strong down-regulation of immune related factors. This observation is supported by the loss of microglia in HDACI treated midbrain cultures. I also provide evidence that Toll-like receptor (TLR) signaling, likely through the activation of Interleukin-6 (IL-6) expression, mediates HDAC-dependent neuronal survival. These data provide new evidence that the neuroimmune system is an extrinsic regulator for the homeostasis and survival of neurons.