Distinct versus overlapping functions of MDC1 and 53BP1 in DNA damage response and tumorigenesis

The importance of the DNA damage response (DDR) pathway in development, genomic stability, and tumor suppression is well recognized. Although 53BP1 and MDC1 have been recently identified as critical upstream mediators in the cellular response to DNA double-strand breaks, their relative hierarchy in the ataxia telangiectasia mutated (ATM) signaling cascade remains controversial. To investigate the divergent and potentially overlapping functions of MDC1 and 53BP1 in the ATM response pathway, we generated mice deficient for both genes. Unexpectedly, the loss of both MDC1 and 53BP1 neither significantly increases the severity of defects in DDR nor increases tumor incidence compared with the loss of MDC1 alone. We additionally show that MDC1 regulates 53BP1 foci formation and phosphorylation in response to DNA damage. These results suggest that MDC1 functions as an upstream regulator of 53BP1 in the DDR pathway and in tumor suppression

A c-Myc–SIRT1 feedback loop regulates cell growth and transformation

The protein deacetylase SIRT1 has been implicated in a variety of cellular functions, including development, cellular stress responses, and metabolism. Increasing evidence suggests that similar to its counterpart, Sir2, in yeast, Caenorhabditis elegans, and Drosophila melanogaster, SIRT1 may function to regulate life span in mammals. However, SIRT1's role in cancer is unclear. During our investigation of SIRT1, we found that c-Myc binds to the SIRT1 promoter and induces SIRT1 expression. However, SIRT1 interacts with and deacetylates c-Myc, resulting in decreased c-Myc stability. As a consequence, c-Myc's transformational capability is compromised in the presence of SIRT1. Overall, our experiments identify a c-Myc–SIRT1 feedback loop in the regulation of c-Myc activity and cellular transformation, supporting/suggesting a role of SIRT1 in tumor suppression

Proliferation and Ploidy During Cardiac Differentiation of Human Induced Pluripotent Stem Cells

Cardiomyocytes are a highly specialized cell type whose primary function is uninterrupted, rhythmic contraction. Despite the vital nature of the heart, cardiomyocytes do not readily proliferate, and consequently the heart is unable to sufficiently regenerate in response to acute insult such as myocardial infarction, or in response to chronic stressors such as hypertension. Thus there is great interest in the technologies that could stimulate cardiac regeneration and similarly great effort being expended to develop myocardial replacement therapies. Central to both of these endeavors is the need for a detailed understanding of cardiomyocyte cell cycle regulation; those factors stimulating proliferation and those inhibiting proliferative potential Chapter I reviews the current understanding of our knowledge of cardiomyocyte cell cycle regulation, proliferation, ploidy and regenerative potential in animal models and in humans. Chapter II describes the materials and methods used throughout this thesis. Chapter III examines the emergence of cardiomyocytes during the in vitro cardiac differentiation of human induced pluripotent cells, their replicative capacity, cell cycle distribution and gene expression profiles relating to these traits and attempts to align this data with data from in vivo models of murine cardiogenesis. In Chapter IV we demonstrate a flow cytometry based method for the identification of mono- and bi-nucleated cardiomyocytes. In Chapter V we further investigate ploidy and cell cycle arrest in response to endogenous and exogenous stress. Chapter VI summarizes the results presented in this thesis and proposes further studies that would expand our knowledge of the roles and regulation of cardiomyocyte ploidy

(A and B) ATM, Chk1, and Chk2 activation in wild-type (WT), MDC1, and double knockout (DKO or MDC1/53BP1) MEFs

MEFs of the indicated genotypes were irradiated at the indicated doses and harvested 1 h later (A) or irradiated (2 Gy) and harvested at different time points (B). Cell extracts were then blotted with the indicated antibodies. (C) MEFs were left untreated or irradiated (2 Gy). 1 h later, cells were stained for antiphospho-H3 antibodies, and mitotic populations were determined by FACS. Error bars represent SEM.<p><b>Copyright information:</b></p><p>Taken from "Distinct versus overlapping functions of MDC1 and 53BP1 in DNA damage response and tumorigenesis"</p><p></p><p>The Journal of Cell Biology 2008;181(5):727-735.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2396806.</p><p></p

Polo-Like Kinase 1 Is Essential for Early Embryonic Development and Tumor Suppression▿ ‡

Polo-like kinases (Plks) are serine/threonine kinases that are highly conserved in organisms from yeasts to humans. Previous reports have shown that Plk1 is critical for all stages of mitosis and may play a role in DNA replication during S phase. While much work has focused on Plk1, little is known about the physiological function of Plk1 in vivo. To address this question, we generated Plk1 knockout mice. Plk1 homozygous null mice were embryonic lethal, and early Plk1−/− embryos failed to survive after the eight-cell stage. Immunocytochemistry studies revealed that Plk1-null embryos were arrested outside the mitotic phase, suggesting that Plk1 is important for proper cell cycle progression. It has been postulated that Plk1 is a potential oncogene, due to its overexpression in a variety of tumors and tumor cell lines. While the Plk1 heterozygotes were healthy at birth, the incidence of tumors in these animals was threefold greater than that in their wild-type counterparts, demonstrating that the loss of one Plk1 allele accelerates tumor formation. Collectively, our data support that Plk1 is important for early embryonic development and may function as a haploinsufficient tumor suppressor

DBC1 Functions as a Tumor Suppressor by Regulating p53 Stability

DBC1 (deleted in breast cancer 1), also known as CCAR2 or KIAA1967, is an important negative regulator of SIRT1 and cellular stress response. Although the Dbc1 gene localizes at a region that is homozygously deleted in breast cancer, its role in tumorigenesis remains unclear. It has been suggested to be either a tumor suppressor or an oncogene. Therefore, the function of DBC1 in cancer needs to be further explored. Here, we report that Dbc1 knockout mice are tumor prone, suggesting that DBC1 functions as a tumor suppressor in vivo. Our data suggest that the increased tumor incidence in Dbc1 knockout mice is independent of Sirt1. Instead, we found that DBC1 loss results in less p53 protein in vitro and in vivo. DBC1 directly binds p53 and stabilizes it through competition with MDM2. These studies reveal that DBC1 plays an important role in tumor suppression through p53 regulation

Deleted in breast cancer–1 regulates SIRT1 activity and contributes to high-fat diet–induced liver steatosis in mice

The enzyme sirtuin 1 (SIRT1) is a critical regulator of many cellular functions, including energy metabolism. However, the precise mechanisms that modulate SIRT1 activity remain unknown. As SIRT1 activity in vitro was recently found to be negatively regulated by interaction with the deleted in breast cancer–1 (DBC1) protein, we set out to investigate whether DBC1 regulates SIRT1 activity in vivo. We found that DBC1 and SIRT1 colocalized and interacted, and that DBC1 modulated SIRT1 activity, in multiple cell lines and tissues. In mouse liver, increased SIRT1 activity, concomitant with decreased DBC1-SIRT1 interaction, was detected after 24 hours of starvation, whereas decreased SIRT1 activity and increased interaction with DBC1 was observed with high-fat diet (HFD) feeding. Consistent with the hypothesis that DBC1 is crucial for HFD-induced inhibition of SIRT1 and for the development of experimental liver steatosis, genetic deletion of Dbc1 in mice led to increased SIRT1 activity in several tissues, including liver. Furthermore, DBC1-deficient mice were protected from HFD-induced liver steatosis and inflammation, despite the development of obesity. These observations define what we believe to be a new role for DBC1 as an in vivo regulator of SIRT1 activity and liver steatosis. We therefore propose that the DBC1-SIRT1 interaction may serve as a new target for therapies aimed at nonalcoholic liver steatosis

Additional file 1: Figure S1. of Establishing and characterizing patient-derived xenografts using pre-chemotherapy percutaneous biopsy and post-chemotherapy surgical samples from a prospective neoadjuvant breast cancer study

Characterization and utilization of PDX models generated from both pretreatment biopsies and surgical samples in the BEAUTY study. Figure S2. Representative immunohistochemistry shows the change of subtype from luminal B to triple negative. The histology is depicted using H&E staining and the expression of ER, PR, HER2, and Ki-67 is compared between the representative PDX (passage 2) and the corresponding human tumor (M06). Figure S3. Immunohistochemistry shows different subtypes for xenografts derived from the same original patient tumor. The representative PDX tumors at passage 2, and corresponding human tumor (M14) are shown. Figure S4. In vivo taxane response for the other six PDX models tested. Passage 4 tumors were used for the drug tests. (PDF 4901 kb