The Gadd45 family of proteins is known to play a central role as cellular stress sensors that modulate the response of mammalian cells to different stressors, including oncogenic stress. Gadd45a expression is regulated during myeloid cell differentiation, and is also induced in response to acute stimulation with cytokines, myeloablation and inflammation. The proto-oncogene C-myc plays a pivotal role in growth control, differentiation and apoptosis in hematopoietic cells. Deregulated Myc in hematopoietic cells blocks the differentiation program and prevents normal homeostatic cellular apoptosis, which alters the balance of cell populations, often participating in leukemogenesis. The status of Gadd45a expression has been shown to impact on different cancers, including breast cancer and leukemia. How the stress response gene Gadd45a modulates oncogenic stress imparted by deregulated c-Myc in myeloid cells has not been investigated. We hypothesized that Gadd45a and its interacting partner proteins can modulate specific pro-survival or pro-apoptotic signaling pathways, altering the cellular response to oncogenic myc in myeloid cells. Gadd45a may play different roles in proliferating and differentiating cells, and myeloid cells in vivo are at all stages of myeloid development. Therefore, to understand how Gadd45a status impacts on proliferating and differentiating myeloid cells, we decided to study the effect of loss of Gadd45a in myc-expressing cells that are either proliferating or stimulated to undergo differentiation. Therefore, to address this issue we utilized bone marrow from wild-type (wt) and Gadd45a null mice, and retrovirally infected these cells to express constitutive Myc or empty vector control. Using these cells we have shown that bone marrow deficient in Gadd45a and expressing constitutive Myc, display decreased apoptosis under proliferating conditions, yet increased apoptosis in media containing the differentiation inducing cytokine GM-CSF. We show that in expansion media loss of Gadd45a in the presence of Myc elicits its function through the activation of p38, with evidence supporting a role for PU.1 and Mcl-1 expression, which are downstream of p-p38. In contrast, deregulated C-Myc and loss of Gadd45a does not signal through p-38 in GM-CSF, but surprisingly there is a decrease in cytokine receptor expression. This data demonstrates that Gadd45a may be required for optimal cytokine receptor expression in myeloid cells, which can impact on survival of the cells. Although in primary bone marrow Gadd45a status had no effect on differentiation of Myc expressing cells, the loss of Gadd45a in Hoxb8 generated cell lines shifted differentiation towards increased neutrophils. Determining the role of Gadd45a on the biological outcome of myeloid cells in response to deregulated c-Myc will provide vital information in understanding the function of Gadd45a in the development and progression of Myc expressing myeloid leukemia. / Molecular Biology and Genetics

Skeletal muscle atrophy is a common and often debilitating complication of diverse stresses including muscle disuse, fasting, aging, critical illness and many chronic illnesses. However, the pathogenesis of muscle atrophy is still poorly understood. The thesis herein describes my studies investigating the molecular mechanisms of skeletal muscle atrophy. Using mouse skeletal muscle and cultured skeletal myotubes as experimental systems, I discovered a novel stress-induced pathway in skeletal muscle that causes muscle atrophy. The pathway begins with stress-induced expression of ATF4, a basic leucine zipper (bZIP) transcription factor with an evolutionarily ancient role in cellular stress responses. I found that diverse stresses including fasting and muscle disuse increase expression of ATF4 in skeletal muscle. ATF4 then activates the growth arrest and DNA damage-inducible 45a (Gadd45a) gene, leading to increased expression of Gadd45a protein, an essential and inducible subunit of DNA demethylase complexes. Gadd45a localizes to skeletal myonuclei where it interacts with and stimulates demethylation of a specific region in the promoter of the cyclin dependent kinase inhibitor 1a (Cdkn1a) gene. By demethylating the Cdkn1a promoter, Gadd45a activates the Cdkn1a gene, leading to increased expression of Cdkn1a protein, also known as p21WAF1/CIP1. Cdkn1a stimulates protein breakdown (a critical pro-atrophy process) and inhibits anabolic signaling, protein synthesis and PGC-1α expression (processes that maintain healthy skeletal muscle and protect against atrophy). As a result, Cdkn1a causes skeletal muscle fibers to undergo atrophy. Importantly, interventions that reduce any one component of this pathway (ATF4, Gadd45a or Cdkn1a) reduce skeletal muscle atrophy during fasting, muscle disuse, and perhaps other skeletal muscle stresses such as illness and aging. Conversely, forced expression of any one component of this pathway is sufficient to cause skeletal muscle fiber atrophy in the absence of upstream stress. These data suggest the ATF4/Gadd45a/Cdkn1a pathway as a potential therapeutic target. Collectively, my studies demonstrate that the sequential, stress-induced expression of ATF4, Gadd45a and Cdkn1a is a critical process in the pathogenesis of skeletal muscle atrophy. This significantly advances our understanding of how muscle atrophy occurs and it opens up new avenues of investigation into the causes and treatment of muscle atrophy.

Skeletal muscle atrophy is a debilitating condition that commonly occurs as a secondary consequence of many acute and chronic medical conditions, including muscle disuse, heart and renal failure, starvation, cancer, HIV/AIDS, and aging. Though it leads to weakness, falls, and fractures, and reduces independence and quality of life for millions of Americans annually, no effective pharmacologic therapies for muscle atrophy exist. This is largely due to a poor understanding of the pathogenesis of skeletal muscle atrophy at a molecular level. In this thesis, I describe my studies into the molecular pathogenesis of skeletal muscle atrophy. Using mouse models, I showed that the gene encoding the pro-atrophy nuclear protein Gadd45a is regulated by distinct pathways after muscle denervation and fasting, and also identified a novel protein regulating skeletal muscle fiber size. First, we demonstrated that denervation-induced muscle atrophy, unlike atrophy mediated by fasting, does not require the bZIP transcription factor ATF4. However, the lysine deacetylase HDAC4 is sufficient to induce Gadd45a mRNA and necessary for Gadd45a mRNA induction after denervation, but not after fasting. Taken together, these data show that Gadd45a is a central convergence point for muscle atrophy caused by several stimuli, and also demonstrate that distinct pathways mediate Gadd45a induction in different models of skeletal muscle atrophy. Second, we identified spermine oxidase as a critical regulator of muscle fiber size. We observed that spermine oxidase mRNA and spermine oxidase protein were reduced by several distinct causes of muscle atrophy (i.e. immobilization, denervation, fasting, and aging). Furthermore, spermine oxidase overexpression increased muscle fiber size, while spermine oxidase knockdown caused muscle fiber atrophy. Restoring spermine oxidase expression significantly attenuated muscle atrophy after limb immobilization, denervation, and fasting. Finally, we identified p21 as a key upstream regulator of spermine oxidase expression, and spermine oxidase as a required mediator of p21-mediated skeletal muscle fiber atrophy. Collectively, these findings greatly advance our understanding of the molecular pathogenesis of skeletal muscle atrophy. These data demonstrate that Gadd45a is a convergence point for multiple pro-atrophy pathways and identify spermine oxidase as a novel therapeutic target for the treatment of skeletal muscle atrophy. These discoveries suggest several important new areas for future research, and further our understanding of this common, debilitating condition.

Recent evaluations of the regulatory in vitro genotoxicity tests have revealed that the mammalian cell tests are sensitive for the prediction of rodent carcinogenicity, but not very specific; they produce positive results for many non-carcinogens. Studies to investigate the potential causes of this poor specificity have focused on the high test concentrations of chemicals and the often associated high cytotoxicity. Others have sought to develop alternative in vitro assays that retain high sensitivity but also have higher specificity. One such test is the GADD45a-GFP “GreenScreen HC” genotoxicity assay, which uses elements of the cellular DNA damage response in a green fluorescent protein (GFP) reporter system to detect genotoxic hazard. Published validation studies for this assay have shown both high sensitivity and high specificity. Suggestions that the high specificity of the assay is attributable to the method of cytotoxicity estimation, relative cell density (RCD), and further that this method leads to overestimation of cytotoxicity and hence lower maximum test concentrations, are investigated in this study.In order to investigate whether high specificity of the GADD45a-GFP assay is a result of inaccurate estimation of cytotoxicity, RCD was compared with other cytotoxicity methods used in screening assays, as well as the cytotoxicity methods used in the regulatory in vitro mammalian genotoxicity tests. Results showed that RCD actually underestimates cytotoxicity in the GADD45-GFP assay and as a consequence, test data are collected up to and beyond the cytotoxicity-limited test concentrations reached in the regulatory tests. The use of RCD was found not to contribute to the high specificity of the GADD45a-GFP assay and indeed, its high specificity was retained regardless of the method used to estimate cytotoxicity.A largely overlooked element in the consideration of the poor specificity of in vitro mammalian genotoxicity assays is the supplementation of assays with exogenous metabolism systems, the most common of which is ‘S9’, a liver-derived preparation. S9 is used to enable the detection of compounds (pro-genotoxins) that undergo metabolic transformation in vivo resulting in the formation of genotoxic carcinogens. However, both the preparation and usage of S9 create a bias towards phase I metabolism, specifically the cytochrome P450 monooxygenases that can catalyse the formation of DNA-reactive species. The neglect of phase II metabolism (detoxification/conjugation) could potentially contribute to the poor specificity of the in vitro mammalian tests and this was explored in the study presented.A review of published data revealed that the prevalence of positive results amongst in vitro mammalian genotoxicity data generated in the presence of S9 was similar in collections of both rodent carcinogens and non-carcinogens. Thus, any increased sensitivity from using S9 is offset by decreased specificity for these tests. However, as inclusion of S9 affected sensitivity and specificity in equal measure it was concluded that S9 is not a confounding factor on the accuracy of the in vitro mammalian genotoxicity tests.

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