Understanding the impact of epigenetic mechanisms on tumorigenesis is essential, as epigenetic alterations are associated with tumor initiation and progression. Because epigenetic changes are reversible, they are potential targets for cancer therapy. Nucleosome Remodeling Factor (NURF) is a chromatin-remodeling complex that regulates gene expression by changing nucleosome positioning along the DNA sequence. Previous studies have shown a role for NURF in embryonic development as well as regulating genes involved in tumor progression. In this work we investigated the impact of eliminating NURF function in tumorigenesis in vivo. BALB/c mice challenged with syngeneic 67NR breast cancer cell lines, injected into the mammary fat pad, lacking NURF, due to knockdown of its essential subunits Bptf, showed reduction in tumor growth comparing to control tumors. The observed reduction in tumor growth was abrogated in immunodeficient mice lacking a functional immune system. Bptf KD and control 67NR cells grew at similar rates in vitro. Similar findings were observed in our lab using 66cl4 breast cancer cell lines. Using immunofluorescence staining, no significant difference in CD8+, CD4+, NK and MDSC cells infiltrations into the tumor microenvironment was observed in 66cl4 tumors. Preliminary results from 67NR tumors suggested more CD4+ and CD8+ cells. Gene expression profile of tumor tissues from BALB/c mice injected with 67NR and 66cl4 cell lines showed enrichment of genes associated with immune response. Our findings suggested a role of the immune system in targeting tumor cells lacking Bptf in vivo.

Telomerase, a ribonucleoprotein enzyme minimally composed of an RNA template (hTR) and a catalytically active protein subunit (hTERT), synthesizes telomeric repeats onto chromosome ends and is obligatory for continuous tumor cell proliferation, as well as malignant progression of breast cancer cells. Telomerase is an attractive anticancer therapeutic target because its activity is present in over 90% of human cancers, including more than 95% of breast carcinomas, but undetectable in most somatic cells. Traditions chemo- and radio-therapies lack the ability to effectively control and cure breast cancer, in part because residual cells are or become resistant to DNA damaging modalities.While various telomerase inhibition strategies cause cancer cells to undergo apoptosis car senescence, there is often a lag period between administration and biologic effect (Corey, 2002). Our goal in this study was to compare the efficacy of different telomerase inhibition strategies in concert with standard chemotherapeutic agents at triggering senescence and/or apoptosis in cultures of breast cancer cells. We hypothesized that telomerase inhibition strategies will sensitize breast cancer cells to traditional chemotherapies, potentially reducing the lag phase, allowing for more potent anti-tumor effects at lower doses, and therefore ultimately imparting less toxicity to the patient.We blocked telomerase by targeting hTR and hTERT, individually and collectively utilizing synthetic short interfering RNA (siRNA), short hairpin RNA (siRNA), and a dominant negative form of hTERT (DN-hTERT) in MCF-7 breast cancer cells. We analyzed the efficiency of telomerase inhibition for each strategy alone and then treated the cells with two mainstay chemotherapeutic agents, Adriamycin (AdR) and Taxol. The most effective telomerase inhibition strategies were synthetic siRNA and DN-hTERT, individually. After treatment with various concentrations of AdR or Taxol, breast cancer cells with inhibited telomerase grew significantly slower and exhibited widespread senescence or apoptosis within a much shorter time period and at a dose that is insufficient to trigger cytostasis. In addition, we provide evidence that cells in which telomerase was inhibited were more sensitive to anti-cancer agents, whether the drug inhibited topoisomerase II resulting in DNA damage (AdR) or blocked mitosis via protracted microtubule stabilization (Taxol). Collectively, our data indicate that alone, anti-telomerase inhibition strategies differ in their efficacy. However, when used in the adjuvant setting with diverse acting chemotherapeutic agents, there is a potent synergy resulting in chemotherapeutic sensitization characterized in part by widespread senescence and/or apoptosis.

Most organisms experience progressive declines in physiological function as they age. A number of studies in a variety of species support a strong link between oxidative damage, age-related functional declines and life span determination. Here, manipulating the expression levels of superoxide dismutase (SOD) isoenzymes SOD1 and SOD2, resulted in altered functional senescence and survival characteristics in Drosophila. Overexpression of cytosolic Sod1 using the yeast GAL4/UAS system conferred a 30-34% increase in mean life span and resulted in an attenuated senescence of odor avoidance behavior in aging flies. Tissue-specific Sod1 overexpression selectively in the nervous system or muscle failed to reproduce these delayed aging phenotypes suggesting that Sod1 overexpression in these tissues alone was not primarily responsible for the aging effects observed. Graded reduction of mitochondrially localized Sod2 expression in a series of Sod2 mutants led to progressive reductions in life span, accelerated age-related functional declines, mitochondrial oxidative damage and neuronal cell death. Tissue-specific Sod2 knock-down using RNA interference revealed that muscle is a key tissue underlying the accelerated age-related functional decline and mortality observed upon loss of SOD2. Sod2 knock-down in the musculature caused a degenerative phenotype consisting of a dramatic reduction in muscle mitochondrial content and ATP levels, elevated cell death and progressive locomotor dysfunction which culminated in early-onset mortality. Collectively, these studies highlight the important role of SOD enzymes in protecting against the impact of oxidative damage on senescence and survival. These findings also lend further support to the oxidative damage hypothesis of aging.

A great need exists for an abundant, easily accessible source of patient-specific cells that will function for use in regenerative medicine. One promising source is the adult stem cell derived from adipose tissue (ASCs). Isolated from waste lipoaspiration, these cells could serve as a readily available source for the regeneration of damaged tissues. To further define the biology of ASCs, we have isolated multiple cell strains from different adipose tissue sources, indicating wide-spread distribution in the body. We find that a widely used set of cell surface markers fail to distinguish ASCs from normal fibroblasts. However, our ASC isolations are multipotent while fibroblasts show no differentiation potential. In further contrast to fibroblasts, these cells also show expression of genes associated with pluripotent cells, Oct-4, SOX2, and NANOG. Together, our data suggest that while the cell surface profile of ASCs do not distinguish them from normal fibroblasts and their lack of telomerase shows their limited proliferation capacity, the expression of genes closely linked to pluripotency and their differentiation capacity clearly define ASCs as multipotent stem cells. iPS cells are another promising cell type for tissue regeneration, due to their expression of hTERT and their capacity to differentiate into all three germ layers. Interestingly, telomerase is activated during the induction process, accomplished by the exogenous expression of four genes in normal, non-hTERT-expressing fibroblasts. To elucidate the mechanisms behind this activation, we examined the overexpression of these four factors in BJ fibroblasts and ASCs, which resulted in undetectable hTERT expression. We then demonstrated a lack of an acetylated histone H3K9 with the opposing di-methylation, indicative of a closed chromatin state at the hTERT promoter. Subsequent treatment of cells with TSA alone showed an upregulation of hTERT mRNA without telomerase activity. However, telomerase activity was found when ASCs, but not BJs were treated with TSA and all four factors, indicating differential regulation of hTERT in cells of similar mesenchymal origins. Our data suggest that while hTERT’s expression is universally dependent on the presence of a relaxed chromatin state and sufficient transactivating factors, other cell to cell differences can prevent its expression.

Krüppel like factors (KLFs) are a family of 17 proteins whose main function is gene regulation by binding to DNA elements in the promoters of various genes. KLF transcription factors recognize CACCC-elements and act as activators or repressors of the gene expression. Among the 17 family members, KLF1, KLF2, and KLF4 share high homology to each other. KLF1 is the founding member of the family and is an erythroid-specific protein. KLF2 is expressed in erythroid, endothelial, and other cells. KLF4 is expressed in endothelial, smooth muscle, and other cells. In this thesis, the functions of these KLFs were reviewed in the context of subjects related to erythropoiesis and cardiovascular development. A mouse model lacking KLF1, KLF2, and KLF4 was used to investigate whether these genes have overlapping functions in regulating the embryonic β-globin genes during early embryogenesis. Quantitative RT-PCR assays were used to measure the expression level of Ey- and βh1- globin mRNA at embryonic day 9.5 (E9.5). It was found that KLF1-/-KLF2-/- and KLF1-/-KLF2-/-KLF4-/- embryos express significantly decreased amounts of Ey- and βh1-globin genes when compared to WT and KLF4-/- embryos. There were no significant changes in the levels of Ey- and βh1-globin mRNA between KLF1-/-KLF2-/- and KLF1-/-KLF2-/-KLF4-/- embryos. It was demonstrated here that KLF1 does not regulate KLF2 in mouse erythroid cells at E10.5.

Melanoma differentiation associated gene-7 (mda-7) was discovered in the Fisher laboratory by subtraction hybridization of temporally spaced subtracted cDNA libraries prepared from terminally differentiated human melanoma cells treated with human fibroblast interferon (IFN-β) and the protein kinase C activator mezerein (MEZ), an approach called ‘differentiation induction subtraction hybridization’ (DISH). mda-7 is located in human chromosome 1q32–33 and based on sequence homology, chromosomal localization, and its functional properties, the mda-7 gene is now classified as a member of the IL-10 family of cytokines and named IL-24. The mda-7/IL-24 cDNA encodes a protein of 206-amino acids with a predicted size of ~24-kDa, which contains an interleukin (IL)-10 signature motif at amino acids 101–121 (SDAESCYLVHTLLEFYLKTVF) shared by other members of the IL-10 family of cytokines. Sequence analysis revealed the presence of a 49-amino acid signal peptide suggesting that the molecule could be cleaved and secreted. Expression of MDA-7/IL-24 protein was detected in cells of the immune system (mainly by expression in tissues associated with the immune system, such as spleen, thymus and PBMC) and normal human melanocytes. Of interest, a progressive loss of MDA-7/IL-24 expression during melanoma progression suggests an inverse relationship between MDA-7/IL-24 expression and the evolution of melanocytes to various stages of melanoma. mda-7/IL-24 induces growth suppression in human melanoma and other cancer cells, without affecting normal cells. Subsequent studies provided consistent evidence that ectopic expression of mda-7/IL-24 employing a replication incompetent adenovirus (Ad.mda-7) resulted in apoptosis induction and cell death in a wide variety of solid tumors including melanoma, malignant glioma, carcinomas of the breast, kidney, cervix, colorectum , liver, lung, ovary and prostate sparing normal cellular counterparts, i.e., such as normal melanocytes, astrocytes, fibroblasts, and mesothelial and epithelial cells. The in vitro antitumor activity of mda-7/IL-24 readily translated into the in vivo situation in animal models containing human breast, prostate, lung and colorectal carcinomas and in malignant glioma xenografts. Moreover, the ability of mda-7/IL-24 to induce a potent “bystander cancer-specific killing effect” provides an unprecedented opportunity to use this molecule to target for destruction not only primary tumors, but also metastases. Based on its profound cancer-selective tropism, substantiated by in vivo human xenograft studies in nude mice, mda-7/IL-24 (administered as Ad.mda-7) was evaluated in a Phase I clinical trial in patients with melanomas and solid cancers. These studies document that mda-7/IL-24 is well tolerated and demonstrates evidence of significant (44%) clinical activity. This review focuses on the recent enhancements in our understanding of the mode of action of mda-7/IL-24 and its potential applications as a unique and promising effective cytokine-based gene therapy for human cancers. The first chapter explored the efficacy of a tropism-modified Ad-based cancer gene therapy approach for eradicating low CAR colorectal cancer cells. We show that in low CAR human colorectal cancer cells (RKO), a recombinant Ad.5/3 virus delivering mda-7/IL-24 (Ad.5/3-mda-7) is more efficient than Ad.5 delivering mda-7 (Ad.5-mda-7) in expressing MDA-7/IL-24 protein, inducing cancer-specific apoptosis and inhibiting in vivo tumor growth in a nude mouse xenograft model. Additionally, our in vitro and in vivo data confirms that BI-97C1 (Sabutoclax) profoundly sensitizes mda-7/IL-24 mediated toxicity in colorectal cancer. Thus, Ad.5/3-mda-7, alone and/or in combination with BI-97C1 (Sabutoclax), might represent an improved and more effective therapeutic approach for colorectal and other cancers. In view of the essential roles of anti-apoptotic Bcl-2 family proteins in tumorigenesis and chemoresistance, efforts are focused on developing small molecule inhibitors of Bcl-2 family proteins as potential therapeutics for cancer. Unfortunately, due to the unique structure of Mcl-1 as compared with Bcl-2 and Bcl-xL, currently employed inhibitors, such as ABT-737 or its clinical counterpart, ABT-263, display limited affinity for Mcl-1. Using nuclear magnetic resonance (NMR) binding assays and computational docking studies, we have recently identified a series of new Apogossypol derivatives, compound 3 (BI-79D10) and compound 11 (BI-97C1), with pan-Bcl-2- inhibitory potency. BI-79D10 binds to Bcl- xL, Bcl-2, and Mcl-1 with IC50 values of 190, 360, and 520 nmol/L, respectively. BI-97C1 (Sabutoclax) is an optically pure individual Apogossypol derivative that retains all the properties of BI-79D10 along with superior in vitro and in vivo efficacy. Because Mcl-1 is over-expressed in the majority of PCs, we hypothesized that suppressing Mcl-1 by treating human PC cells with BI-97C1 (Sabutoclax) would sensitize them to mda-7/IL-24-mediated cytotoxicity. The second chapter study highlights the noteworthy potential of a combinatorial approach involving mda-7/IL-24, a broad-acting anticancer gene, and BI-97C1 (Sabutoclax), which targets Mcl-1, to sensitize PC to mda-7/IL-24-mediated cytotoxicity, thereby enhancing therapeutic efficacy. Our data suggests that treatment with the combination regimen of mda-7/IL-24 and BI-97C1 (Sabutoclax) induces autophagy that facilitates apoptosis in association with up regulation of NOXA, accumulation of Bim, and activation of Bax and Bak. Treatment with mda-7/IL-24 and BI-97C1 (Sabutoclax) inhibited the growth of PC xenografts and suppressed PC development in an immunocompetent transgenic mouse model of PC. The third chapter study explored the efficacy of a tropism-modified CRCA cancer gene therapy approach for eradicating low CAR prostate cancer cells. We showed that in low CAR PC3 cells Ad.5/3-CTV is more efficient than Ad.5-CTV in delivering transgene (mda-7/IL-24), infecting tumor cells, expressing MDA-7/IL-24 protein, inducing cancer-specific apoptosis, inhibiting in vivo tumor growth and exerting an antitumor ‘bystander’ effect in a nude mouse human prostate cancer xenograft and suppressed PC development in an immunocompetent transgenic mouse model of PC model.

Cyclin Dependent Kinase-2 Associated Protein-1 (CDK2AP1) plays an important role in cell cycle regulation, by inhibiting CDK2 and by targeting it for proteolysis. It is also known to bind the DNA polymerase alpha-primase complex and regulate the initiation step of DNA synthesis. Its overexpression has been shown to inhibit growth, reduce invasion and increase apoptosis in a number of cancer cell lines. In studies in which mouse embryonic stem cells (mESCs) with targeted deletion of the Cdk2ap1 gene were used, Cdk2ap1 was shown to be required for epigenetic silencing of Oct4 during differentiation. The goal of this thesis was to examine the role of CDK2AP1 in somatic cells (primary human dermal fibroblasts (HDFs)) and human embryonic stem cells (hESCs) and specifically assess its impact on proliferation, self-renewal and differentiation. In the first part of this study, using a short-hairpin RNA (shRNA) approach, we investigated the effect of CDK2AP1 downregulation in HDFs. Outcomes indicated: (a) reduced proliferation, (b) premature senescence, (c) cell cycle alterations, (d) DNA damage, and (e) an increase in p53, p21, and the p53-responsive apoptotic genes BAX and PUMA. Simultaneous downregulation of p53 and CDK2AP1 in HDFs confirmed that observed phenotype was p53 dependent. In the second part of this study, using a shRNA approach, we investigated the role of CDK2AP1 on hESC fate associated with self-renewal and differentiation. We found that CDK2AP1 knockdown in hESCs resulted in: (a) reduced self-renewal (b) enhanced differentiation (c) cell cycle alterations and (d) increase in p53 expression. Results indicate that the knockdown of CDK2AP1 in hESCs enhances differentiation and favors it over a self-renewal fate. Thus, this study has successfully identified novel functions for CDK2AP1, as its knockdown has a significant impact on self-renewal, differentiation and senescence. Results obtained from this study could contribute to development of directed differentiation strategies for generating uniform populations of differentiated phenotypes from hESCs for clinical applications.

While age-related sex chromosomal aneuploidy is a well-characterized phenomenon, the relationship between autosomal loss and age remains unclear. The emergence of the specific and highly sensitive fluorescence in situ hybridization (FISH) technology has enabled investigators to study interphase cells, thereby overcoming problems inherent with the study of metaphase spreads for acquired aneuploidy assessment. Despite all the advantages of this technique, there are some limitations that could be misleading when scoring interphase autosomal aneuploidy. In this study we show that sex chromosomal hypoploidy is correlated with age. By using a twin study design, we evaluated Y chromosome hypoploidy frequencies and found that loss of the Y chromosome is likely to be a multifactorial phenotype, being influenced by both genetic and environmental factors. An analysis of acquired aneuploidy frequencies for 13 autosomes in men showed that only one autosome, chromosome 3, had an age-related increase in acquired aberrations levels. Using a multi-probe study design, we determined that an apparent loss of fluorescent signal(s) could result from the coincident positioning (overlaying) of the repeat sequences targeted by the probes (due to either somatic homolog pairing or aggregation of the heterochromatic regions). Therefore, caution should be taken when performing autosomal FISH analysis to avoid overestimation of autosomal aneuploidy in uncultured leukocytes.

It has been shown that the key components of the hsp90 chaperone complex, including hsp90, p23, hsp70, hsp40, and HOP (p60), associate with telomerase; however, their specific roles in telomerase function and tumor progression have not yet been defined. HSF1, the primary mammalian heat shock protein transcription factor, may affect telomerase activity and transformation by regulating the expression of several hsp90 chaperone complex proteins in response to stress as well as regulating the transcription of hTERT, the protein subunit of telomerase.In our in vitro model of prostate cancer progression, as cells progress from immortal but non-tumorigenic (P69) to tumorigenic (M2182) and eventually metastatic (M12) capabilities, both telomerase activity and global chaperone protein levels increase. Our hypothesis is that HSF1 affects telomerase activity directly at the level of transcription and indirectly at the protein level via its regulation of proteins of the hsp90 chaperone complex. Furthermore, upregulation of HSF1 and/or members of the hsp90 chaperone complex directly contribute to prostate cell transformation and that introduction of chaperone-related genes will convert non-tumorigenic prostate cells to a tumorigenic state.We have shown that ectopic overexpression of HSF1 induces increased expression of endogenous hsp90 in P69 cells. Furthermore, telomerase activity in the overexpressing HSF1 cell lines is increased as well and is the end result of two disparate, yet ultimately cooperating pathways. However, the increased telomerase activity does not correlate with increased tumorigenicity.In conjunction with this study, we have overexpressed hTERT in the P69 cell lines and found that telomerase activity is markedly increased in the absence of chaperone upregulation. We propose that the demand for increased folding and stability of the exogenous hTERT leads to a recruitment of telomerase associated chaperone proteins, which can be measured by increased activity after immunoprecipitations and nuclear translocation of hsp90 chaperone complex proteins.Taken together, these projects indicate a significant role for HSF1 and the hsp90 chaperone complex proteins on telomerase activity, and provide evidence that each may be a viable target for therapeutic intervention.

The research efforts refer to tracking homologus loci in the chromosomes of a pair of a species. The purpose is to infer the extent of maximum syntenic correlation when an exhaustive set of orthologs of the species are searched. Relevant bioinformatic analyses use comparative mapping of conserved synteny via Oxford grid. In medical diagnostic efforts, deducing such synteny correlation can help screening chromosomal aberration in genetic disorder pathology. Objectively, the present study addresses: (i) Cytogenetic framework of syntenic correlation and, (ii) applying information-theoretics to determine entropy-dictated synteny across an exhaustive set of orthologs of the test pairs of species. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection