The process of reversible growth arrest, otherwise known as cellular quiescence or the G₀ phase denoted by withdrawal from the cell cycle, is a poorly characterized state. Subsets of growth arrest-specific (GAS) genes are upregulated during quiescence, however, these subsets are specific to/dependent on the limiting factor or circumstance inducing growth arrest. Here I characterize the expression and regulation of the lipid trafficking GAS gene Fatty Acid-Binding Protein 4 in the quiescence-inducing conditions of contact inhibition and oxygen limitation (hypoxia). Chicken Embryo Fibroblasts (CEF) were cultured to high density or subjected to hypoxia, in which oxygen is the limiting factor inducing growth arrest, or serum starvation, in which nutrients is the limiting factor inducing growth arrest. Contact inhibition and hypoxia induced FABP4 expression, whereas cycling control CEF and serum depleted CEF did not. At higher, though still hypoxic, oxygen levels that did not robustly induce FABP4, proliferation assays showed a slight reduction in CEF proliferation. The GAS gene p20k lipocalin has been shown to exhibit similar expression patterns to FABP4, with its regulation determined by the presence of the transcription factor C/EBP-β. CEF overexpressing C/EBP-β also showed strong FABP4 induction. Furthermore, chromatin immunoprecipitation (ChIP) assays revealed that C/EBP-β bound directly to the FABP4 promoter in both normoxic and hypoxic cells, although only the latter condition induced FABP4 protein expression. In summary, these results suggest that FABP4 is induced during growth arrest specifically when oxygen is the limiting factor, as induction was not seen during growth arrest mediated by starvation-induced endoplasmic reticulum (ER) stress, where nutrients was the limiting factor. The induction of these hypoxia-responsive genes suggests that oxygen availability regulates the expression of a sub-class of growth arrest specific genes. Additionally, FABP4 was shown to be associated with growth arrest and the promotion of cell survival and proliferation, as depicted by proliferation assays. Lastly, C/EBP-β not only strongly induced FABP4 expression, but directly bound to the FABP4 promoter. This suggests that C/EBP-β is a regulator of FABP4, although there may be other interacting factors acting as activators or repressors as this FABP4-C/EBP-β interaction was observed in conditions permissive and non-permissive to FABP4 expression. / Thesis / Master of Science (MSc) / The process of reversible growth arrest is a poorly characterized state. Subsets of growth arrest-specific (GAS) genes are upregulated during quiescence, however, these subsets are specific to the limiting factor or circumstance inducing growth arrest. Here we characterize the expression and regulation of the lipid trafficking GAS gene Fatty Acid-Binding Protein 4 in the quiescence-inducing conditions of contact inhibition (CI) and hypoxia. Chicken Embryo Fibroblasts (CEF) were cultured to high density or subjected to hypoxia, in which oxygen is the limiting factor inducing growth arrest, or serum starvation, in which nutrients availability is the limiting factor. CI and hypoxia induced FABP4 expression, whereas control and serum depleted CEF did not. At higher, though still hypoxic, oxygen levels that did not robustly induce FABP4, proliferation assays showed a slight reduction in CEF proliferation. When overexpressing C/EBP-β, CEF showed strong FABP4 induction. Additionally, a direct interaction with the FABP4 promoter was observed in both normoxic and hypoxic cells, although only the latter condition induced expression. In summary, the induction of this hypoxia-responsive gene suggests that oxygen availability regulates the expression of a sub-class of growth arrest specific genes and that this induction may be regulated by C/EBP-β.

Transcription elongation regulator 1 (TCERG1) has previously been demonstrated to be an inhibitor of the transactivation and growth arrest activities of CCAAT/Enhancer binding protein alpha (C/EBPα). Furthermore, TCERG1 had been demonstrated to become relocalized from nuclear speckles to the pericentromeric regions where C/EBPα resides when both proteins are co-expressed in the cell. This thesis demonstrates that the deletion of a unique, imperfect series of 38 glutamine-alanine (QA) repeats near the amino terminus of TCERG1 is able to abrogate the ability of TCERG1 to inhibit C/EBPα-mediated growth arrest, the physical interaction between TCERG1 and C/EBPα, and the relocalization of TCERG1 from nuclear speckles when C/EBPα is co-expressed in the cell. The deletion of the QA domain demonstrated that there was a threshold amount of QA repeats required in TCERG1 for the relocalization and growth arrest inhibitory activities between TCERG1 and C/EBPα. It was demonstrated that between 11 and 20 QA repeats were required in TCERG1 to produce the relocalization from nuclear speckles or to be able to inhibit C/EBPα-mediated growth arrest. The physical interaction of TCERG1 and C/EBPα as examined by co-immunoprecipitation was also found to be QA dependent, with a diminishing interaction observed as the number of QA repeats in TCERG1 were reduced. However, experiments examining the isolated QA domain indicated that it was insufficient to relocalize an mCherry fluorescent protein fusion to either the nucleus or to pericentromeric regions where C/EBPα is concentrated. This inability to produce relocalization suggests that the QA domain requires another domain or domains from TCERG1 to mediate the relocalization activity. When expressed with the WT TCERG1, ΔQA TCERG1 was able to act in a dominant negative manner, preventing the relocalization of the WT TCERG1 protein to pericentromeric domains. Interestingly, the transactivation inhibitory activities of TCERG1 on C/EBPα do not appear to require the QA domain, but rather are localized to the carboxy half of TCERG1, somewhere within amino acids 612-1098. The data obtained provides the first report of a role for this unique QA repeat domain.

Breast cancer is one of the most prevalent and deadly forms of cancer in women and is not restricted by race or ethnicity. Although a wealth of knowledge has been amassed on the biology of breast cancer, including its risk factors, diagnosis, prognosis, prevention, and treatment, it remains a serious health concern and active area of research. Initial response to standard chemotherapeutic and radiotherapeutic regimens is generally strong for many patients, yet breast tumors often recur, leading to more aggressive and resistant tumors. Because recurrence is such a clinical issue, more effective therapeutic approaches are needed to eliminate partial clinical responses and undesirable side effects. Molecular chaperones like the heat shock protein 90 (Hsp90) family are regarded as ubiquitous, highly conserved proteins that mainly respond upon induction of stress or disruption in cellular homeostasis. Chaperones are critically involved in controlling the conformation, stability, function, and degradation of many oncogenic client proteins by assisting in trafficking, remodeling of improperly folded client proteins, and suppression of protein aggregation. Hsp90-mediated folding events are an ATP-dependent process that involves the association with various co-chaperones and stimulators, including Hsp70, Hsp40, HOP, p23, and Aha1 for client stabilization and modification. Notably, Hsp90 seems to be particularly associated with cell signaling clientele, such as nuclear hormone receptors, protein kinases, and many other oncogenic proteins that directly influence the hallmarks of cancer. In order to define the role of chaperones in breast cancer progression, we assessed chaperone expression levels in normal and tumor lines. Based on our initial finding of increased expression of Hsp90 and p23 in immortal and cancer cell lines, it is possible that this phenomenon may be reflected in normal breast tissue as well as breast carcinoma specimens. Indeed, we observed high Hsp90 expression in invasive carcinomas, such that high nuclear expression correlates with a greater TNM stage, while high cytoplasmic Hsp90 correlates with ER negativity, suggesting that elevated Hsp90 may be an indicator or marker of advanced disease. p23 expression also increases appreciably in established breast cancer cell lines with invasive breast tissue specimens displaying an opposite trend. Although p23 does not appear to have any relationship with TNM stage, significant relationships with ER and PR emerged, with higher nuclear p23 correlating to ER positivity and higher total p23 being positively associated with PR presence. Due to the differential expression of Hsp90 in normal, DCIS, and invasive breast carcinomas, we determined the impact on Hsp90 function, finding that total Hsp90 in tumor cells is associated with an increase in both complexed and uncomplexed Hsp90, which is in contrast to a previously reported study. The intrinsic nature of heat shock proteins makes them especially relevant to a cell’s defense against cancer initiation. The preferential accumulation of Hsp90 in cancer cells also forms the basis for the unique sensitivity of tumor cells to Hsp90 inhibition. The Hsp90 specific inhibitor, radicicol, is more potent in terms of blocking ATPase activity than other Hsp90-related compounds like geldanamycin, which is much more toxic. All Hsp90 inhibitors prevent the association of the co-chaperone p23 with Hsp90, resulting in destabilization of the client protein. For these reasons, it may be possible that Hsp90 inhibition would sensitize breast cancer cells to be more responsive to standard chemotherapeutics. We determined that radicicol negatively alters cellular proliferation, and in combination with Adriamycin, elicits a more robust decline in growth and the expression of Hsp90 client proteins. This finding was associated with an increase in senescent cells without a detectable affect on apoptosis. Radicicol in combination with cisplatin or Taxol contributed to an increase in cell death (apoptosis) and differentially altered the expression of client proteins. Finally, ER negative breast cancer cells do not display altered p53 expression upon radicicol and Adriamycin treatment. Blockade of ER activity in ER positive cells with tamoxifen induced significant reductions in proliferation and decreased p53 expression without a corresponding decrease in p21 levels. In conclusion, these results point to the utility of Hsp90 inhibition as a valid form of targeted therapy for breast cancer, and the value of radicicol as a potential adjuvant treatment option in combination with standard chemotherapeutics.

<p>All-trans retinoic acid (ATRA), a biologically active metabolite of vitamin A, is a powerful inducer of terminal differentiation and growth arrest of several myeloid cell lines <i>in vitro</i>. Although the efficacy of ATRA as an anti-cancer drug has been demonstrated by the successful treatment of acute promyelocytic leukemia (APL), knowledge concerning the molecular mechanisms directing ATRA-induced differentiation and cell cycle arrest of myeloid cells is lacking. Our results show, for the first time, that the complex regulation of cell cycle proteins and myeloid-specific transcription factors induced by ATRA relies on functional Stat1. We found that Stat1 is activated by both tyrosine-701 and serine-727 phosphorylation upon ATRA-induced differentiation of the human monoblastic cell line U-937. Expression of phosphorylation deficient mutants of Stat1 (Stat1Y701F or Stat1S727A) inhibited both ATRA-induced differentiation and cell cycle arrest of U-937 cells, pointing to a requirement of active Stat1 in these processes. </p><p>Detailed analysis of the molecular mechanism of ATRA-induced cell cycle arrest and differentiation showed that the onset of cell cycle arrest was associated with a decrease in c-Myc and cyclin E levels and upregulation of p27^Kip1 and p21^WAF1/CIP1. This was followed by a rapid fall in cyclin A and B and a coordinate dephosphorylation of the retinoblastoma protein (pRb). The inhibition of ATRA-induced cell-cycle arrest by constitutive expression of Stat1Y701F or Stat1S727A was associated with impaired regulation of these cyclins and p27^Kip1, positioning Stat1 activation upstream of these events. To further understand the process of ATRA-induced differentiation, the regulation of myeloid-specific transcription factors was investigated during ATRA-treatment. Notably, ATRA-induced upregulation of Stat2, ICSBP and C/EBP-ε was selectively impaired in sublines expressing Stat1Y701F or Stat1S727A, suggesting an important function of these factors downstream Stat1. Taken together, the work in this thesis clearly demonstrates that Stat1 plays a key role in ATRA-induced terminal differentiation of myeloid cells, through regulation of cell cycle proteins and myeloid-specific transcription factors. </p>

All-trans retinoic acid (ATRA), a biologically active metabolite of vitamin A, is a powerful inducer of terminal differentiation and growth arrest of several myeloid cell lines in vitro. Although the efficacy of ATRA as an anti-cancer drug has been demonstrated by the successful treatment of acute promyelocytic leukemia (APL), knowledge concerning the molecular mechanisms directing ATRA-induced differentiation and cell cycle arrest of myeloid cells is lacking. Our results show, for the first time, that the complex regulation of cell cycle proteins and myeloid-specific transcription factors induced by ATRA relies on functional Stat1. We found that Stat1 is activated by both tyrosine-701 and serine-727 phosphorylation upon ATRA-induced differentiation of the human monoblastic cell line U-937. Expression of phosphorylation deficient mutants of Stat1 (Stat1Y701F or Stat1S727A) inhibited both ATRA-induced differentiation and cell cycle arrest of U-937 cells, pointing to a requirement of active Stat1 in these processes. Detailed analysis of the molecular mechanism of ATRA-induced cell cycle arrest and differentiation showed that the onset of cell cycle arrest was associated with a decrease in c-Myc and cyclin E levels and upregulation of p27Kip1 and p21WAF1/CIP1. This was followed by a rapid fall in cyclin A and B and a coordinate dephosphorylation of the retinoblastoma protein (pRb). The inhibition of ATRA-induced cell-cycle arrest by constitutive expression of Stat1Y701F or Stat1S727A was associated with impaired regulation of these cyclins and p27Kip1, positioning Stat1 activation upstream of these events. To further understand the process of ATRA-induced differentiation, the regulation of myeloid-specific transcription factors was investigated during ATRA-treatment. Notably, ATRA-induced upregulation of Stat2, ICSBP and C/EBP-ε was selectively impaired in sublines expressing Stat1Y701F or Stat1S727A, suggesting an important function of these factors downstream Stat1. Taken together, the work in this thesis clearly demonstrates that Stat1 plays a key role in ATRA-induced terminal differentiation of myeloid cells, through regulation of cell cycle proteins and myeloid-specific transcription factors.

Transcription elongation regulator 1 (TCERG1) is a nuclear protein involved in transcriptional elongation and splicing events, suggesting these two activities may be connected. Moreover, TCERG1 was recently identified as a novel interactor and co-repressor of CCAAT/Enhancer Binding Protein &alpha; (C/EBP&alpha;) transcriptional activity, suggesting TCERG1 has additional biological roles. Interestingly, TCERG1 also inhibits the growth arrest activity of C/EBP&alpha;. Additionally, the original clone found to interact with C/EBP&alpha; consisted of only the amino-terminal domain of TCERG1 and functional analysis of this clone indicated that it retained the ability to repress both C/EBP&alpha; mediated growth arrest and transcriptional activity. Furthermore, a TCERG1 mutant whose amino-terminal region was deleted was unable to interact with or repress the transcriptional and growth arrest activities of C/EBP&alpha;, suggesting the functional domain(s) lie elsewhere. In this study, domains of TCERG1 were examined for the ability to inhibit C/EBP&alpha;-mediated growth arrest and the mechanism whereby this effect occurs. By exploiting fluorescent properties of expressed proteins fused with green fluorescent protein, the extent to which each TCERG1 mutant was able to reverse C/EBP&alpha;-mediated growth arrest of cultured cells was assessed. Our analyses suggest that the inhibitory activity of TCERG1 lies within the amino-terminal region and may involve WWI and WWII domains within this region. Additionally, laser scanning confocal microscopy (LCSM) was used to visualize the subnuclear localization of fluorescent proteins fused to TCERG1 and C/EBP&alpha;. When expressed alone, TCERG1 localized to splicing factor-rich nuclear speckles while C/EBP&alpha; was found to reside in discrete punctate foci, both localization patterns being distinct and different from each other. Results from co-localization studies after co-expressing both proteins indicate an alteration in the subnuclear distribution of TCERG1. Furthermore, TCERG1 co-localizes with C/EBP&alpha;, suggesting a possible mechanism whereby TCERG1 inhibits the growth arrest and transcriptional activities mediated by C/EBP&alpha;.

Transcription elongation regulator 1 (TCERG1) is a nuclear protein involved in transcriptional elongation and splicing events, suggesting these two activities may be connected. Moreover, TCERG1 was recently identified as a novel interactor and co-repressor of CCAAT/Enhancer Binding Protein &alpha; (C/EBP&alpha;) transcriptional activity, suggesting TCERG1 has additional biological roles. Interestingly, TCERG1 also inhibits the growth arrest activity of C/EBP&alpha;. Additionally, the original clone found to interact with C/EBP&alpha; consisted of only the amino-terminal domain of TCERG1 and functional analysis of this clone indicated that it retained the ability to repress both C/EBP&alpha; mediated growth arrest and transcriptional activity. Furthermore, a TCERG1 mutant whose amino-terminal region was deleted was unable to interact with or repress the transcriptional and growth arrest activities of C/EBP&alpha;, suggesting the functional domain(s) lie elsewhere. In this study, domains of TCERG1 were examined for the ability to inhibit C/EBP&alpha;-mediated growth arrest and the mechanism whereby this effect occurs. By exploiting fluorescent properties of expressed proteins fused with green fluorescent protein, the extent to which each TCERG1 mutant was able to reverse C/EBP&alpha;-mediated growth arrest of cultured cells was assessed. Our analyses suggest that the inhibitory activity of TCERG1 lies within the amino-terminal region and may involve WWI and WWII domains within this region. Additionally, laser scanning confocal microscopy (LCSM) was used to visualize the subnuclear localization of fluorescent proteins fused to TCERG1 and C/EBP&alpha;. When expressed alone, TCERG1 localized to splicing factor-rich nuclear speckles while C/EBP&alpha; was found to reside in discrete punctate foci, both localization patterns being distinct and different from each other. Results from co-localization studies after co-expressing both proteins indicate an alteration in the subnuclear distribution of TCERG1. Furthermore, TCERG1 co-localizes with C/EBP&alpha;, suggesting a possible mechanism whereby TCERG1 inhibits the growth arrest and transcriptional activities mediated by C/EBP&alpha;.

Conditions that lead to reversible growth arrest (quiescence), promote the expression of a set of genes called growth arrest specific (GAS) genes. GAS genes play a crucial role in initiating and maintaining the entry into quiescence, while also activating stress responses to help the cell overcome the effects of the stressors. Gene profiling study examining the transcriptome has shown a vast number of genes that are upregulated during quiescence, among them is GEM1 (GTP binding protein overexpressed in skeletal muscle). GEM1 transcripts were elevated 18-fold in response to quiescence. GEM1 is a small monomeric GTPase from the Ras superfamily. It is involved in regulation of cytoskeleton reorganization, and inhibition of voltage gated calcium channels that ultimately prevents hormone secretion. A preliminary study determined that GEM1 is packaged into extracellular vesicles (EV). GEM1 is also reported to promote lipid accumulation and adipogenesis in goat pre-adipocytes. GEM1 is also reported to bind transcription factors that are involved in lipid homeostasis pathways. Thus, it is probable that GEM1 may play a major role in EV formation and/or release, and lipid homeostasis. This study examined the expression of GEM1 at the protein level and validates its candidacy as a GAS gene. We also created two GEM1-shRNA retroviral constructs capable of partially downregulating GEM1 expression which can serve as a molecular tool for further characterizing the function of GEM1 in quiescent CEF. / Thesis / Bachelor of Science (BSc) / GEM1 is a small monomeric GTPase, implicated in a variety of roles in eukaryotes. It plays a role in regulating adipogenesis, and hormone secretion. Most notably it regulates cytoskeleton reorganization in response to changes in calcium concentrations. Gene profiling done by Bédard Lab identified that GEM1 transcripts were highly elevated in reversible growth arrested chicken embryo fibroblasts (CEF). In this study we further explore and characterize the protein expression of GEM1 in quiescent CEF. We also design and test shRNAi retroviral constructs to downregulate GEM1 in quiescent CEF.

<p>A microarray analysis of RNA from contact inhibited CEF indicated a hypoxic signature in the contact inhibition program of gene expression (Ghosh <em>et al</em>., 2009). The purpose of this thesis was to investigate whether GAS genes known to be induced during contact inhibition are inducible by hypoxia. The gene p20K was selected as the model for this investigation because it is a growth arrest specific (GAS) gene with a well-characterized promoter (Mao <em>et al</em>., 1993). p20K expression was shown to be positively regulated in hypoxia. It was then determined by transient expression assay that this induction occurred at the promoter level. Interestingly by dissecting the promoter it was found that the quiescent responsive unit (QRU) was required for promoter induction during hypoxia. It has previously been shown that the QRU was required for contact inhibition induction of p20K in a C/EBPβ dependent manner (Mao <em>et al</em>., 1993; Kim <em>et al</em>., 1999).</p> <p>The mechanism behind hypoxic induction of the QRU was then investigated. The kinetics of HIF1α and p20K induction during hypoxia demonstrated that HIF1α was transiently expressed between 2-8 hrs of hypoxia while p20K was induced after 8 hrs of hypoxia. Co-Immuniprecipitation assay was also used to determine if a HIF1α-C/EBPβ interaction occurred, however, this molecular interaction could not be shown. These experiments suggests that HIF1α is not involved with the induction of the QRU. Over-expression of the dominant negative C/EBPβΔ184 repressed p20K induction, thus implicating C/EBPβ in activation in both contact inhibition and hypoxia. We also observed by western blot analysis that the C/EBP family member CHOP was repressed during hypoxia, causing a decrease in the amount of CHOP-C/EBPβ complexes in the cell. It was also found that over-expression of CHOP antagonized the induction of p20K by hypoxia. In conclusion hypoxia represses CHOP levels resulting in an increase of potent C/EBPβ homodimers at the expense of the inactive CHOP-C/EBPβ heterodimers.</p> / Master of Science (MSc)

<p>The study of growth arrest specific (GAS) genes is critical for our understanding of quiescence cell states. C/EBP-β is a transcriptional activator which is central to the expression of GAS genes in growth arrested cells. C/EBP-β is involved in the activation of numerous pathways, including mitogenesis, cytokine signaling, stress response, etc. Thus, it requires signaling cues which confer specificity in terms of gene expression.</p> <p>Here we used the p20K gene in chicken embryonic fibroblasts as a model system to study the control mechanisms of GAS genes. p20K is expressed in conditions such as contact inhibition mediated growth arrest and mild hypoxia. Here we explored the control mechanism mediated by ERK2 at the p20K promoter (QRU), as a mode of regulation which confers C/EBP-β binding specificity.</p> <p>In this study we demonstrate that ERK2 is recruited to the QRU in proliferative cells, i.e. where p20K is repressed. Using ChIP analysis we show that ERK2 binds directly to the QRU in proliferative cell states, but not in growth arrested cell conditions. Using a similar approach we demonstrate that ERK2 binding to the QRU is lost in states of hypoxia, where p20K is strongly induced. Furthermore, we show that this interaction is specific to ERK2 and is not observed with the related ERK1 kinase. Lastly, we employed transient expression assays to illustrate that ERK2 acts as a transcriptional repressor of the QRU. Through these experiments we have illustrated that ERK2 mediated transcriptional repression is a novel control mechanism at the QRU which skews C/EBP-β mediated signaling networks in proliferating cells.</p> / Master of Science (MSc)