PI 3 Kinase Related Kinases-Independent Proteolysis of BRCA1 Regulates Rad51 Recruitment during Genotoxic Stress in Human Cells

The function of BRCA1 in response to ionizing radiation, which directly generates DNA double strand breaks, has been extensively characterized. However previous investigations have produced conflicting data on mutagens that initially induce other classes of DNA adducts. Because of the fundamental and clinical importance of understanding BRCA1 function, we sought to rigorously evaluate the role of this tumor suppressor in response to diverse forms of genotoxic stress.We investigated BRCA1 stability and localization in various human cells treated with model mutagens that trigger different DNA damage signaling pathways. We established that, unlike ionizing radiation, either UVC or methylmethanesulfonate (MMS) (generating bulky DNA adducts or alkylated bases respectively) induces a transient downregulation of BRCA1 protein which is neither prevented nor enhanced by inhibition of PIKKs. Moreover, we found that the proteasome mediates early degradation of BRCA1, BARD1, BACH1, and Rad52 implying that critical components of the homologous recombination machinery need to be functionally abrogated as part of the early response to UV or MMS. Significantly, we found that inhibition of BRCA1/BARD1 downregulation is accompanied by the unscheduled recruitment of both proteins to chromatin along with Rad51. Consistently, treatment of cells with MMS engendered complete disassembly of Rad51 from pre-formed ionizing radiation-induced foci. Following the initial phase of BRCA1/BARD1 downregulation, we found that the recovery of these proteins in foci coincides with the formation of RPA and Rad51 foci. This indicates that homologous recombination is reactivated at later stage of the cellular response to MMS, most likely to repair DSBs generated by replication blocks.Taken together our results demonstrate that (i) the stabilities of BRCA1/BARD1 complexes are regulated in a mutagen-specific manner, and (ii) indicate the existence of mechanisms that may be required to prevent the simultaneous recruitment of conflicting signaling pathways to sites of DNA damage

Nicotinamide Inhibits Alkylating Agent-Induced Apoptotic Neurodegeneration in the Developing Rat Brain

BACKGROUND: Exposure to the chemotherapeutic alkylating agent thiotepa during brain development leads to neurological complications arising from neurodegeneration and irreversible damage to the developing central nerve system (CNS). Administration of single dose of thiotepa in 7-d postnatal (P7) rat triggers activation of apoptotic cascade and widespread neuronal death. The present study was aimed to elucidate whether nicotinamide may prevent thiotepa-induced neurodegeneration in the developing rat brain. METHODOLOGY/PRINCIPAL FINDINGS: Neuronal cell death induced by thiotepa was associated with the induction of Bax, release of cytochrome-c from mitochondria into the cytosol, activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP-1). Post-treatment of developing rats with nicotinamide suppressed thiotepa-induced upregulation of Bax, reduced cytochrome-c release into the cytosol and reduced expression of activated caspase-3 and cleavage of PARP-1. Cresyl violet staining showed numerous dead cells in the cortex hippocampus and thalamus; post-treatment with nicotinamide reduced the number of dead cells in these brain regions. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) and immunohistochemical analysis of caspase-3 show that thiotepa-induced cell death is apoptotic and that it is inhibited by nicotinamide treatment. CONCLUSION: Nicotinamide (Nic) treatment with thiotepa significantly improved neuronal survival and alleviated neuronal cell death in the developing rat. These data demonstrate that nicotinamide shows promise as a therapeutic and neuroprotective agent for the treatment of neurodegenerative disorders in newborns and infants

Increased Expression of PcG Protein YY1 Negatively Regulates B Cell Development while Allowing Accumulation of Myeloid Cells and LT-HSC Cells

Ying Yang 1 (YY1) is a multifunctional Polycomb Group (PcG) transcription factor that binds to multiple enhancer binding sites in the immunoglobulin (Ig) loci and plays vital roles in early B cell development. PcG proteins have important functions in hematopoietic stem cell renewal and YY1 is the only mammalian PcG protein with DNA binding specificity. Conditional knock-out of YY1 in the mouse B cell lineage results in arrest at the pro-B cell stage, and dosage effects have been observed at various YY1 expression levels. To investigate the impact of elevated YY1 expression on hematopoetic development, we utilized a mouse in vivo bone marrow reconstitution system. We found that mouse bone marrow cells expressing elevated levels of YY1 exhibited a selective disadvantage as they progressed from hematopoietic stem/progenitor cells to pro-B, pre-B, immature B and re-circulating B cell stages, but no disadvantage of YY1 over-expression was observed in myeloid lineage cells. Furthermore, mouse bone marrow cells expressing elevated levels of YY1 displayed enrichment for cells with surface markers characteristic of long-term hematopoietic stem cells (HSC). YY1 expression induced apoptosis in mouse B cell lines in vitro, and resulted in down-regulated expression of anti-apoptotic genes Bcl-xl and NFκB2, while no impact was observed in a mouse myeloid line. B cell apoptosis and LT-HSC enrichment induced by YY1 suggest that novel strategies to induce YY1 expression could have beneficial effects in the treatment of B lineage malignancies while preserving normal HSCs

Abrogation of DNA vector-based RNAi during apoptosis in mammalian cells due to caspase-mediated cleavage and inactivation of Dicer-1

RNA interference (RNAi) is used as a reverse-genetic tool to examine functions of a gene in different cellular processes including apoptosis. As key cellular proteins are inactivated during apoptosis, and as RNAi requires cooperation of many cellular proteins, we examined whether DNA vector-based RNAi would continue to function during apoptosis. The short hairpin RNA transcribed from the DNA vector is processed by Dicer-1 to form small interfering RNA that is incorporated in the RNA-induced silencing complex (RISC) to guide a sequence-specific silencing of the target mRNA. We report here that DNA vector-based RNAi of three different genes, namely poly(ADP-ribose) polymerase-1, p14(ARF) and lamin A/C are abrogated during apoptosis. The failure of DNA vector-based RNAi was not at the level of Ago-2 or RISC-mediated step of RNAi but due to catalytic inactivation of Dicer-1 on specific cleavage at the STTD1476 and CGVD(1538) sites within its RNase IIIa domain. Using multiple approaches, caspase-3 was identified as the major caspase responsible for the cleavage and inactivation of Dicer-1. As Dicer-1 is also the common endonuclease required for formation of microRNA (miRNA) in mammalian cells, we observed decreased levels of mature forms of miR-16, miR-21 and let-7a. Our results suggest a role for apoptotic cleavage and inactivation of Dicer-1 in controlling apoptotic events through altered availability of miRNA. Cell Death and Differentiation (2009) 16, 858-868; doi: 10.1038/cdd.2009.15; published online 20 February 200

Par-4 links dopamine signaling and depression

Prostate apoptosis response 4 (Par-4) is a leucine zipper containing protein that plays a role in apoptosis. Although Par-4 is expressed in neurons, its physiological role in the nervous system is unknown. Here we identify Par-4 as a regulatory component in dopamine signaling. Par-4 directly interacts with the dopamine D2 receptor (D2DR) via the calmodulin binding motif in the third cytoplasmic loop. Calmodulin can effectively compete with Par-4 binding in a Ca2+- dependent manner, providing a route for Ca2+-mediated downregulation of D2DR efficacy. To examine the importance of the Par-4/D2DR interaction in dopamine signaling in vivo, we used a mutant mouse lacking the D2DR interaction domain of Par-4, Par-4 Delta LZ Primary neurons from Par-4 Delta LZ embryos exhibit an enhanced dopamine-cAMP-CREB signaling pathway, indicating an impairment in dopamine signaling in these cells. Remarkably, Par-4 Delta LZ mice display significantly increased depression-like behaviors. Collectively, these results provide evidence that Par-4 constitutes a molecular link between impaired dopamine signaling and depression.X1194sciescopu

Consensus report of the 8 and 9th Weinman Symposia on Gene x Environment Interaction in carcinogenesis: novel opportunities for precision medicine

The relative contribution of intrinsic genetic factors and extrinsic environmental ones to cancer aetiology and natural history is a lengthy and debated issue. Gene-environment interactions (G x E) arise when the combined presence of both a germline genetic variant and a known environmental factor modulates the risk of disease more than either one alone. A panel of experts discussed our current understanding of cancer aetiology, known examples of G x E interactions in cancer, and the expanded concept of G x E interactions to include somatic cancer mutations and iatrogenic environmental factors such as anti-cancer treatment. Specific genetic polymorphisms and genetic mutations increase susceptibility to certain carcinogens and may be targeted in the near future for prevention and treatment of cancer patients with novel molecularly based therapies. There was general consensus that a better understanding of the complexity and numerosity of G x E interactions, supported by adequate technological, epidemiological, modelling and statistical resources, will further promote our understanding of cancer and lead to novel preventive and therapeutic approaches