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A number of amino acid deprivations have been tested in our laboratory, for the selective eradication of tumour cells in vitro. Withdrawal of the essential amino acid, L-arginine, produced the greatest differential effect on cell proliferation. Normal cells ceased to proliferate and remained viable in G, of the cell cycle, while tumour cells attempted to proliferate in conditions unfavourable for growth, resulting in rapid cell death. Of the six tumour cell lines studied here, four displayed the latter response, while the other two responded in an apparently similar manner to normal cells. Most tumour cells cannot arrest in G1 and are therefore defective in G1 cell cycle control (particularly at the R-point). Analysis of a normal cell line, and the two tumour cell lines that survived arginine deprivation, revealed that cdk4 was downregulated, and the cells were found to possess functional p53. The other four tumour cell lines had dysfunctional p53 and did not downregulate cdk4 upon arginine withdrawal, or relied upon cdk6 for pRb phosphorylation. Arginine is required for histone synthesis during S phase. Histone synthesis in the absence of arginine was compared in a normal and a tumour cell line. Normal fibroblasts synthesise histones to support previously initiated DNA synthesis for the first 24 h of arginine deprivation until the cells reach the R-point. However, HeLa cells cannot synthesise adequate amounts of histone proteins, despite continued DNA synthesis and this is to their detriment. A novel cancer therapy has been developed which exploits the differential response of normal and tumour cells to arginine deprivation. Extracorporeal dialysis was used to reduce blood arginine levels in normal and tumour-bearing dogs. Arginine was successfully reduced to <10 M within the first 12 h of dialysis and this low level was maintained for up to 5 days, but arginine was not reduced for a long enough period of time to see significant tumour regression.

Thesis advisor: Anthony T. Annunziato / The histone acetyltransferase Hat1 is an enzyme that specifically acetylates newly synthesized histone H4 at positions K5 and K12 (or their homologous positions) in all eukaryotes. In Schizosaccharomyces pombe, the deletion of hat1 presents a mutant phenotype. The telomeres in a hat1-del strain become permissive for transcription, as analyzed by a telomeric ura4 marker gene. In this study, we evaluate the efficacy of high copy suppression of this hat1 deletion. Due to high-frequency recombination events in the telomere, it became necessary to create a hat1-rad52 double deletion strain that also contains a telomeric ura4 reporter. High copy suppressor screens for recovery of telomeric silencing yielded several promising transformants. Multiple rounds of testing were performed to assess the recovery of transcriptional repression at the telomere. It was found that despite the anti-recombination effect of deleting rad52, the ura4 reporter was still lost from the telomere through recombination. Additional observation of the hat1-del rad52-del ura4-tel strain revealed a significant synthetic slow-growth phenotype. The double mutant displays a greatly decreased growth rate compared to hat1-del, as well as increased cellular length. Further study showed unique phenotypes on various media, and gene expression studies showed unique patterns of regulation in this double mutant when compared to both a wild- type and its single mutant counterparts (hat1-del, rad52-del). In summary, the telomeric ura4 marker in a hat1-del strain of S. pombe is not stable and is lost by recombination at a high frequency. This has led to the discovery of a double mutant (hat1-del rad52-del) that displays a severe synthetically sick phenotype. / Thesis (MS) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Anthony T. Annunziato / Thesis advisor: Charles Hoffman / The Hat1 complex was characterized in <italic>S. pombe</italic>. Through tandem affinity purification and mass spectrometry, it was determined that Hat1 is associated with Mis16 (an orthologue of HAT2). Unlike HAT2 in <italic>S. cerevisiae</italic>, we confirm <italic>mis16</italic> to be an essential gene in <italic>S. pombe</italic>. As expected, the <italic>S. pombe</italic> Hat1 complex was found to acetylate lysines 5 and 12 of histone H4. In contrast to budding yeast, deletion of <italic>hat1</italic> alone resulted in the loss of telomeric silencing without concomitant mutations of the H3 N-terminal domain. Deletion of <italic>hat1</italic> caused an increase of H4 acetylation at telomeres. Additionally, the hyperacetylation of histones also results in the loss of telomeric silencing. Loss of Hat1 did not affect silencing at the inner most repeat (imr) or outer repeat (otr) regions of the centromere, but did appear to increase silencing at the central core region (cnt) of the centromere. The experiments described herein demonstrate Hat1 to be essential for the establishment of proper telomeric silencing in fission yeast, and suggest that the timely acetylation of H4 during chromatin assembly is a unique factor in generating the correct epigenetic state at telomeres in <italic>S. pombe</italic>. Additionally, Hat1 and its acetylation of new H4 may have entirely different roles during telomeric silencing than during silencing at the centromeric central core. Our studies in HeLa cells demonstrated that transcription is involved in the exchange of H2A/H2B in acetylated chromatin regions. The finding that cytosolic H2A can be acetylated at lysine 5 is the first demonstration that cytosolic H2A can be specifically modified <italic>in vivo</italic>. Our results support a model in which H2A/H2B exchange during transcription is mediated by the NAP1 chaperone. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

As a centromere-specific protein complex in direct contact with the DNA, CenH3-containing nucleosomes are generally thought to act as the distinguishing epigenetic mark of active centromere location. Confusingly, seemingly disparate models have been proposed for the structure of CenH3 nucleosomes. The most widely supported model is an octameric structure that, like histone H3 nucleosomes, contains two subunits of each histone. Another more contentious, yet persistent model is the hemisome model proposed for fly and human CenH3 nucleosomes. In this case it is suggested that CenH3 nucleosomes contain only single subunit of each histone. One reason for this lack of consensus is that seemingly contradicting models are often proposed, even with material from the same organism, with little overlap in experimental approaches. For example, the proposed hemisome model for fly and human CenH3 nucleosomes is predominantly based on atomic force microscopy (AFM) imaging where the height of nucleosomes on a surface is measured. These AFM measurements are the main data used by protagonists for the hemisome model. However, data supporting an octameric model for human, and other, CenH3 nucleosomes is largely based on biochemical analysis of nucleosomes prepared in vitro, with little cross-over in the methodology used to generate data to support either model. In order to reach a consensus the same analyses needs to be applied to CenH3 nucleosomes assembled in vitro or extracted from cells. Here, recombinant Schizosaccharomyces pombe CENP-ACnp1 and H3 histones expressed and purified from E. coli have been assembled into nucleosomes. To our knowledge this is the first time that recombinant S. pombe nucleosomes have been produced, allowing the stoichiometry and composition of these nucleosomes to be examined in detail by a variety of biochemical and biophysical assays. The application of AFM has enabled the height of these recombinant nucleosomes to be measured and tests the ability of AFM to infer stoichiometry using defined material. The intriguing conclusion is that octameric CenH3 nucleosomes uniquely behave as tetrameric “hemisomes” as defined by AFM. In recent years the contribution of DNA sequence to directing H3 nucleosome location has received a great deal of interest. Since CENP-ACnp1 nucleosomes wrap DNA differently to H3 nucleosomes their preference for sequences that produce a stable nucleosome is expected to be altered. The development of protocols to assemble recombinant CENP-ACnp1 nucleosomes in vitro has also been used here to assess the contribution of primary DNA sequence to CENP-ACnp1 nucleosome positioning. CENP-ACnp1 and H3 nucleosomes were reconstituted on genomic DNA at low density and the resulting nucleosomal DNA from CENP-ACnp1 and H3 particles compared by Illumina sequencing. The stability of CENP-ACnp1 and H3 nucleosomes on specific ‘H3’ and ‘CENP-ACnp1’ sequences was cross-checked. Comparing these data with in vivo CENP-ACnp1 nucleosome positions has allowed the contribution of primary DNA sequence to CENP-ACnp1 nucleosome positioning to be explored.

We have previously shown that dietary fish oil and fiber, particularly the highly-fermentable pectin, are protective against colon cancer in a rat model of carcinogenesis. Therefore, based upon the current body of literature and our previous experimental findings, we hypothesized that one mechanism by which dietary fish oil+pectin suppress the promotion stage of colon cancer is through butyrate, the fermentation product of fiber, targeting (in particular) the p21Waf1/Cip1 gene and, via targeted histone hyperacetylation, inducing its expression. We found that dietary butyrate supplementation increased the concentration of fecal butyrate (mole %) in the distal colon, and that this increase corresponded to an increase in histone H4 acetylation. Similarly, diets supplemented with butyrate increased p21Waf1/Cip1 expression despite azoxymethane (AOM) treatment, which was not seen in non-butyrate supplemented diets. Furthermore, fish oil+butyrate diets resulted in the highest levels of apoptosis and the lowest levels of ACF, while corn oil+butyrate diets resulted in the lowest levels of apoptosis and the highest levels of ACF. Thus, it appears that the protective effect of fish oil+butyrate is due to the unique properties of fish oil, providing an environment in which butyrate’s enhancement of histone acetylation and p21 expression are pro-apoptotic, thereby diminishing pre-neoplastic ACF development.

Breast cancer is the most common malignancy and a major cause of cancer-related death among Canadian women. Although treatment of primary breast tumours is highly successful through surgery, metastatic breast cancer is difficult to treat. Cancer progression and metastasis require the accumulation of numerous genetic and epigenetic alterations. Normal cells that acquire such alterations can transform into cancer cells, resulting in primary tumour formation. Primary tumours are a heterogeneous population, containing cells of various metastatic potentials. Cells that acquire a high potential for metastasis can spread to secondary locations. Our model system consists of two subpopulations, with different metastatic potential, derived from the same rat mammary adenocarcinoma. Using this model, a differentially expressed novel gene, termed MS-1, was discovered. Due to significant expression of this gene in the poorly metastatic subpopulation and lack of expression in the highly metastatic subpopulation, MS-1 may have involvement in metastasis suppression. Several breast cancer metastasis suppressor genes have been identified on the basis that they are down-regulated during the progression of metastasis. Epigenetic mechanisms, such as DNA methylation, account for loss of expression in several of these genes. Hypermethylation of CpG islands within gene promoters results in deacetylation of histone proteins and produces a compact chromatin structure that is unfavourable for transcription. A CpG island spans the 5 untranslated region, exon 1 and part of intron 1 of the MS-1 gene. Our data reveals aberrant methylation patterns of this CpG island in our model. Also, MS-1 expression appears to be partially induced by both DNA methylation and histone deacetylation inhibitors. Following a screen of several cancer cell lines of various metastatic potential, it appears that the presence of DNA methylation in the CpG island of MS-1 correlates with the lack of MS-1 expression. Therefore, these results suggest that MS-1 may be silenced in cells of high metastatic potential through epigenetic mechanisms.

Histone Deacetylase Inhibitors (HDACi) are a new class of chemotherapeutics which have shown promise in pre-clinical and clinical settings. HDACi have been shown to act by re-programming gene expression, with the transcription of some genes such as p21WAF1 being activated, while others like SRC and BCL2L1 are repressed. The mechanism behind HDACi gene expression changes remains unknown; although it has been shown to involve a direct interaction with gene promoters. Using a quantitative qRT-PCR approach, the effect of various HDACi on the transcription of p21WAF1, SRC and BCL2L1 was examined. TSA and apicidin led to an up regulation of p21WAF1 mRNA levels while c-Src and Bcl-xL mRNA levels were downregulated. Short c-Src mRNA transcripts were unaffected following TSA and apicidin treatments, despite the full length transcripts being repressed. Repression of full length c-Src and Bcl-xL mRNA transcripts was not seen following treatment with MS-275 and MGCD0103, although p21WAF1 mRNA expression was induced. ChIP experiments revealed that following HDACi treatment, histone acetylation levels and RNA Polymerase II occupancy increased in the promoter regions of both the SRC and BCL2L1 genes. RNA Polymerase II occupancy lasted less than 15 minutes in the 3’ regions of the gene following treatment with apicidin and TSA, but was more long-term following MS-275 and MGCD0103 treatment. The protein phosphatase inhibitor Calyculin A completely blocked HDACi mediated repression of c-Src and Bcl-xL mRNA, suggesting a role for protein phosphatases in the mechanism behind HDACi. It is therefore hypothesized that HDACi work through at least two different mechanisms. Whether or not an HDACi leads to gene repression depends on its ability to disrupt an HDAC/protein phosphatase complex and not on their HDAC specificities. The disruption of the complex leads to the release of an active protein phosphatase. The released phosphatase can then presumably act on various factors changing a gene from an active to paused state, possibly through promoter proximal pausing. HDACi unable to disrupt this complex are unable to induce gene repression. Collectively, these studies highlight not only the complexity of HDACi mediated effects within the cell, but also present a new explanation behind HDACi mediated gene repression.

Breast cancer is the most common malignancy and a major cause of cancer-related death among Canadian women. Although treatment of primary breast tumours is highly successful through surgery, metastatic breast cancer is difficult to treat. Cancer progression and metastasis require the accumulation of numerous genetic and epigenetic alterations. Normal cells that acquire such alterations can transform into cancer cells, resulting in primary tumour formation. Primary tumours are a heterogeneous population, containing cells of various metastatic potentials. Cells that acquire a high potential for metastasis can spread to secondary locations. Our model system consists of two subpopulations, with different metastatic potential, derived from the same rat mammary adenocarcinoma. Using this model, a differentially expressed novel gene, termed MS-1, was discovered. Due to significant expression of this gene in the poorly metastatic subpopulation and lack of expression in the highly metastatic subpopulation, MS-1 may have involvement in metastasis suppression. Several breast cancer metastasis suppressor genes have been identified on the basis that they are down-regulated during the progression of metastasis. Epigenetic mechanisms, such as DNA methylation, account for loss of expression in several of these genes. Hypermethylation of CpG islands within gene promoters results in deacetylation of histone proteins and produces a compact chromatin structure that is unfavourable for transcription. A CpG island spans the 5 untranslated region, exon 1 and part of intron 1 of the MS-1 gene. Our data reveals aberrant methylation patterns of this CpG island in our model. Also, MS-1 expression appears to be partially induced by both DNA methylation and histone deacetylation inhibitors. Following a screen of several cancer cell lines of various metastatic potential, it appears that the presence of DNA methylation in the CpG island of MS-1 correlates with the lack of MS-1 expression. Therefore, these results suggest that MS-1 may be silenced in cells of high metastatic potential through epigenetic mechanisms.

We have previously shown that dietary fish oil and fiber, particularly the highly-fermentable pectin, are protective against colon cancer in a rat model of carcinogenesis. Therefore, based upon the current body of literature and our previous experimental findings, we hypothesized that one mechanism by which dietary fish oil+pectin suppress the promotion stage of colon cancer is through butyrate, the fermentation product of fiber, targeting (in particular) the p21Waf1/Cip1 gene and, via targeted histone hyperacetylation, inducing its expression. We found that dietary butyrate supplementation increased the concentration of fecal butyrate (mole %) in the distal colon, and that this increase corresponded to an increase in histone H4 acetylation. Similarly, diets supplemented with butyrate increased p21Waf1/Cip1 expression despite azoxymethane (AOM) treatment, which was not seen in non-butyrate supplemented diets. Furthermore, fish oil+butyrate diets resulted in the highest levels of apoptosis and the lowest levels of ACF, while corn oil+butyrate diets resulted in the lowest levels of apoptosis and the highest levels of ACF. Thus, it appears that the protective effect of fish oil+butyrate is due to the unique properties of fish oil, providing an environment in which butyrate’s enhancement of histone acetylation and p21 expression are pro-apoptotic, thereby diminishing pre-neoplastic ACF development.