Platinum-based chemotherapies are effective agents in the treatment of a wide variety of human cancers. However, patients with recurrent disease can become resistant to platinum-based chemotherapy, leading to low overall survival rates. Activating transcription factor 3 (ATF3) is a stress-inducible gene that is a regulator of cisplatin-induced cytotoxicity. ATF3 protein expression was upregulated after cytotoxic doses of cisplatin treatment in a panel of cell lines. A chromatin immunoprecipitation assay showed that upon treatment with cisplatin, ATF3 directly bound to the CHOP gene promoter and this correlated with an increase in CHOP protein expression. In a 1200 compound library screen performed on cancer cell lines, disulfiram, a dithiocarbamate drug, was identified as an enhancer of the cytotoxic effects of cisplatin. This increased cytotoxic action was likely due to disulfiram and cisplatin’s ability to induce ATF3 independently through two separate mechanisms, namely the MAPK and integrated stress pathways. Furthermore, ATF3 protein and mRNA levels were variable amongst human ovarian and lung cancer tissues, suggesting the potential for basal expression of ATF3 to be predictive of cisplatin treatment response. Thus, understanding ATF3’s role in cisplatin-induced cytotoxicity will lead to novel therapeutic approaches that could improve this drug’s efficacy.

Congenital heart defects due to the aberrant development of the atrioventricular (AV) valves and septum are among the most common developmental abnormality in newborns and cause significant neonatal morbidity and mortality. A key point in cardiac morphogenesis occurs when cells within the endocardial cushions (ECCs), the precursors for the AV valvoseptal complex, delaminate and undergo an epithelial-to-mesenchymal transformation (EMT). The mesenchymal cells then proliferate and the cushion area elongates to form the AV valves and portions of the AV septae. The signals that initiate region-specific EMT during heart development are unknown. Cilia, known for their role in establishing left-right (LR) asymmetry, function to receive and integrate extracellular signals, including fluid flow, in a range of other organ systems. We hypothesize that cilia could also have a direct role in heart development outside of their role in LR development. Using immunohistochemistry, we demonstrated the presence of cilia on the myocardium, epicardium, and ECCs of wild-type mouse hearts at embryonic day (e) 9.5 and e12.5. To characterize the potential role of these cilia, we compared mice with mutations affecting ciliary biogenesis, motility, and mechanosensation. Using bright field microscopy and in situ hybridization, we analyzed the embryonic heart structure and the expression pattern of Gata4, an EMT transcription factor. We showed that compared to mice with immotile but structurally normal cilia, the mice without cilia had hypocellular ECCs, a thinned compact myocardium (CM), and an up-regulated expression of Gata4. These observations suggest that a subset of cilia called cardiac cilia have a role in cardiogenesis outside of their role in LR development and affect Gata4 expression. One possible function of cardiac cilia is as mechanosensors, integrating fluid flow and influencing cardiac morphogenesis including EMT and development of the CM.

Thesis (M.S.)--Auburn University, 2008. / Abstract. Includes bibliographical references (p. 39-42).

Temperate maize is one of few model species that relies mainly on endogenous indicators of the plant’s developmental stage to cue the onset of reproductive development. The INDETERMINATE1 (ID1) transcription factor is a key regulator of the floral transition and id1 mutants are very late-flowering. ID1 is expressed and remains localized in developing leaves, while florigenic signals originate in mature, photosynthetically active leaves. Since very little is known about the molecular components of the floral transition in maize, and in autonomously flowering species at large, this work utilized id1 mutants to analyze the transcriptional and physiological alterations associated with the floral transition in maize. Analyses of functional categories of transcriptional change between developing leaves of id1 non-flowering mutants and normal flowering maize suggest a role for ID1 in energy metabolism and epigenetic regulation of leaf development. In addition, a novel family of -glucosidase genes were found to be expressed exclusively in immature leaves of normal flowering maize in a pattern similar to the ID1 gene suggesting that these genes may act in concert downstream of ID1. Further, profiling of transcript and metabolite alterations in mature leaves, which are likely the source of floral cues, suggest that coordination of resource storage in the form of transitory starch is an important signal for floral promotion in maize. Finally, analysis of the floral transition in Balsas teosinte, the progenitor of modern maize and an obligate short-day plant, suggests that ID1 may define a regulatory module unique to the autonomous floral regulation pathway in maize and related grass species.

The Distal-less (Dll) gene has an ancient evolutionary origin. Chordates have retained duplicated Dll genes; vertebrates have six distinct paralogues (Dlx1 through Dlx6 in mammals) arranged in three cis-linked pairs that are co-expressed. Dlx genes are expressed in a conserved nested pattern that defines a proximal-distal axis in the pharyngeal arch tissue of vertebrates. Dlx5-/- and Dlx6-/- mouse neonates have similar phenotypic variations in the lower jaw and inner ear bones, with the Dlx6-/- phenotype being a less perturbed version of the Dlx5-/- phenotype. Conversely, Dlx5/6-/- double mutants have a homeotic transformation of the lower jaw into a second set of maxillary structures. The combination of expression patterns and null phenotypes has led to the proposal of a “Dlx code” that patterns the craniofacial tissue. However, the nature of this code, whether individual Dlx transcription factors supply unique functions, or whether they make a quantitative contribution to a more generic and shared Dlx function, is not well understood. One prediction of a quantitative model for Dlx function in the pharyngeal arches is the functional equivalency of the proteins encoded by divergent cis-linked Dlx paralogues. To address this aspect of the model, three core functions of Dlx5 and Dlx6 were compared quantitatively: suppression of cell growth, transcription activity and DNA binding affinity. In most respects both proteins behaved very similarly.

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No description available.

Efficient muscle regeneration is essential in mammals in order to overcome daily stress such as wounds, exercise and pathologic processes. This regeneration relies on muscle stem cells, the satellite cells. After a lesion, satellite cells are activated, proliferate and differentiate in fonctionnal muscle fibers. Our laboratory has previously shown that the transcription factor GATA-6 is expressed in the satellite cells. The present thesis confirms the expression of this factor in this cell type. Also, it seems that GATA-6 could be implicated in the maintaining of quiescence of these cells. The GATA-6 heterozygous mouse muscle is characterized by an increase level of Myf5 and Pax7+ cells. Moreover, suppression of one copy of the GATA-6 gene in a muscular dystrophy model mouse, the mdx mice, alleviates its phenotype. Further experiments on a muscle-specific GATA-6 null mouse will allow a better understanding of the role of GATA-6 in muscle regeneration. / Keywords. GATA-6, muscle regeneration, mdx, satellite cells

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.

The inhibitor of apoptosis (IAP) proteins traditionally regulate programmed cell death by binding to and inhibiting caspases. Recent studies have uncovered a variety of alternate cellular roles for several IAP family members. The cellular inhibitor of apoptosis 1 (cIAP1) protein, for instance, regulates different axes of the NF-κB signalling pathway. Given the extensive functions of NF-κB signalling in muscle differentiation and regeneration, I asked if cIAP1 also plays critical roles in skeletal muscle myogenesis. In a primary myoblast cell-culture system, genetic and pharmacological approaches revealed that loss of cIAP1 dramatically increases the fusion of myoblasts into myotubes. NF-κB signalling occurs along a classical and an alternative pathway, both of which are highly active in cIAP1-/- myoblasts. Suppression of the alternative pathway attenuates myotube fusion in wildtype and cIAP1-/- myoblasts. Conversely, constitutive activation of the alternative pathway increases myoblast fusion in wildtype myoblasts. cIAP1-/- mice have greater muscle weight and size than wildtypes, as well as an increased number of muscle stem cells. These results identify cIAP1 as a regulator of myogenesis through its modulation of classical and alternative NF-κB signalling pathways. Loss of the structural protein dystrophin in the mdx mouse model of Duchenne muscular dystrophy leads to chronic degeneration of skeletal muscle. The muscle pathology is strongly influenced by NF-κB signaling. Given the roles demonstrated for cIAP1 in cell culture and in vivo, I asked whether loss of cIAP1 would influence muscle pathology in the mdx mouse. To address this question, double-mutant mice were bred lacking both cIAP1 and dystrophin (cIAP1-/-;mdx). Histological analyses revealed that double-mutant mice exhibited reduced indications of damage on several measures, as compared to single-mutant (cIAP1+/+;mdx) controls. Unexpectedly, these reductions were seen in the “slow-twitch” soleus muscle but not in the “fast-twitch” extensor digitorum longus (EDL) muscle. The improvements in pathology of double-mutant solei were associated with reductions in muscle infiltration by CD68-expressing macrophages. Finally, the double-mutant mice exhibited improved endurance and resistance to damage during treadmill-running exercise. Taken together, these results suggest that loss of cIAP1, through its multiple regulatory functions, acts to improve myogenesis and increase muscle resistance to damage.