TP53-binding protein variants and breast cancer risk: a case-control study

INTRODUCTION: The TP53-binding protein (53BP1) has been shown to influence TP53-mediated transcriptional activation, thus playing a pivotal role in DNA damage signalling. Genetic aberrations in TP53 and in ATM and CHEK2 predispose to cancer. We have therefore examined the effects of 53BP1 single nucleotide polymorphisms (D353E, G412S, and K1136Q) and the novel 53BP1 6bp deletion (1347_1352delTATCCC) on breast cancer risk. METHODS: Allelic discrimination was performed to investigate the frequencies of 53BP1 D353E, G412S, and K1136Q and of 1347_1352delTATCCC in 353 patients with breast cancer and 960 control individuals. RESULTS: No significant association of 53BP1 D353E, G412S, or K1136Q with breast cancer risk was detected. 53BP1 1347_1352delTATCCC, leading to the loss of an isoleucine and a proline residue, showed a nonsignificant inverse association with breast cancer risk (odds ratio = 0.61, 95% confidence interval = 0.22 to 1.68, P = 0.34). CONCLUSION: The lack of association casts doubt on the putative effects of D353E, G412S, and K1136Q on breast cancer risk. Investigating a larger study cohort might elucidate the influence of the 6bp deletion 1347_1352delTATCCC. Studying the functional effect and the impact of this variant on the risk of other cancers may be revealing

Mediator of DNA Damage Checkpoint 1 (MDC1) Contributes to High NaCl-Induced Activation of the Osmoprotective Transcription Factor TonEBP/OREBP

Background: Hypertonicity, such as induced by high NaCl, increases the activity of the transcription factor TonEBP/OREBP whose target genes increase osmoprotective organic osmolytes and heat shock proteins. Methodology: We used mass spectrometry to analyze proteins that coimmunoprecipitate with TonEBP/OREBP in order to identify ones that might contribute to its high NaCl-induced activation. Principal Findings: We identified 20 unique peptides from Mediator of DNA Damage Checkpoint 1 (MDC1) with high probability. The identification was confirmed by Western analysis. We used small interfering RNA knockdown of MDC1 to characterize its osmotic function. Knocking down MDC1 reduces high NaCl-induced increases in TonEBP/OREBP transcriptional and transactivating activity, but has no significant effect on its nuclear localization. We confirm six previously known phosphorylation sites in MDC1, but do not find evidence that high NaCl increases phosphorylation of MDC1. It is suggestive that MDC1 acts as a DNA damage response protein since hypertonicity reversibly increases DNA breaks, and other DNA damage response proteins, like ATM, also associate with TonEBP/OREBP and contribute to its activation by hypertonicity. Conclusions/Significance: MDC1 associates with TonEBP/OREBP and contributes to high NaCl-induced increase of tha

Proteomic characterization of HIV-modulated membrane receptors, kinases and signaling proteins involved in novel angiogenic pathways

<p>Abstract</p> <p>Background</p> <p>Kaposi's sarcoma (KS), hemangioma, and other angioproliferative diseases are highly prevalent in HIV-infected individuals. While KS is etiologically linked to the human herpesvirus-8 (HHV8) infection, HIV-patients without HHV-8 and those infected with unrelated viruses also develop angiopathies. Further, HIV-Tat can activate protein-tyrosine-kinase (PTK-activity) of the vascular endothelial growth factor receptor involved in stimulating angiogenic processes. However, Tat by itself or HHV8-genes alone cannot induce angiogenesis <it>in vivo </it>unless specific proteins/enzymes are produced synchronously by different cell-types. We therefore tested a hypothesis that <it>chronic </it>HIV-<it>replication in non-endothelial cells </it>may produce novel factors that provoke angiogenic pathways.</p> <p>Methods</p> <p>Genome-wide proteins from HIV-infected and uninfected T-lymphocytes were tested by subtractive proteomics analyses at various stages of virus and cell growth <it>in vitro </it>over a period of two years. Several thousand differentially regulated proteins were identified by mass spectrometry (MS) and >200 proteins were confirmed in multiple gels. Each protein was scrutinized extensively by protein-interaction-pathways, bioinformatics, and statistical analyses.</p> <p>Results</p> <p>By functional categorization, 31 proteins were identified to be associated with various signaling events involved in angiogenesis. 88% proteins were located in the plasma membrane or extracellular matrix and >90% were found to be essential for regeneration, neovascularization and angiogenic processes during embryonic development.</p> <p>Conclusion</p> <p>Chronic HIV-infection of T-cells produces membrane receptor-PTKs, serine-threonine kinases, growth factors, adhesion molecules and many diffusible signaling proteins that have not been previously reported in HIV-infected cells. Each protein has been associated with endothelial cell-growth, morphogenesis, sprouting, microvessel-formation and other biological processes involved in angiogenesis (p = 10^-4to 10^-12). Bioinformatics analyses suggest that overproduction of PTKs and other kinases in HIV-infected cells has <it>suppressed </it>VEGF/VEGFR-PTK expression and promoted <it>VEGFR-independent </it>pathways. This unique mechanism is similar to that observed in neovascularization and angiogenesis during embryogenesis. Validation of clinically relevant proteins by gene-silencing and translational studies <it>in vivo </it>would identify specific targets that can be used for early diagnosis of angiogenic disorders and future development of inhibitors of angiopathies. This is the first comprehensive study to demonstrate that HIV-infection alone, without any co-infection or treatment, can induce numerous "embryonic" proteins and kinases capable of generating novel <it>VEGF-independent </it>angiogenic pathways.</p

TopBP1 functions with 53BP1 in the G1 DNA damage checkpoint

The replication stress protein TopBP1 co-localizes with 53BP1 at sites of DNA damage and mediates its G1 cell cycle checkpoint function

The cohesin complex is required for the DNA damage-induced G2/M checkpoint in mammalian cells

Cohesin complexes mediate sister chromatid cohesion. Cohesin also becomes enriched at DNA double-strand break sites and facilitates recombinational DNA repair. Here, we report that cohesin is essential for the DNA damage-induced G2/M checkpoint. In contrast to cohesin's role in DNA repair, the checkpoint function of cohesin is independent of its ability to mediate cohesion. After RNAi-mediated depletion of cohesin, cells fail to properly activate the checkpoint kinase Chk2 and have defects in recruiting the mediator protein 53BP1 to DNA damage sites. Earlier work has shown that phosphorylation of the cohesin subunits Smc1 and Smc3 is required for the intra-S checkpoint, but Smc1/Smc3 are also subunits of a distinct recombination complex, RC-1. It was, therefore, unknown whether Smc1/Smc3 function in the intra-S checkpoint as part of cohesin. We show that Smc1/Smc3 are phosphorylated as part of cohesin and that cohesin is required for the intra-S checkpoint. We propose that accumulation of cohesin at DNA break sites is not only needed to mediate DNA repair, but also facilitates the recruitment of checkpoint proteins, which activate the intra-S and G2/M checkpoints

Alteration of p53-binding protein 1 expression during skin carcinogenesis: association with genomic instability.

Epidermal cells are the first cells to be exposed to environmental genotoxic agents such as ultraviolet (UV) and ionizing radiations (IR), which induce DNA double strand breaks (DSBs) and activate DNA damage response (DDR) to maintain genomic integrity. Defective DDR can result in genomic instability (GIN) which is considered to be a central aspect of any carcinogenic process. P53-binding protein 1 (53BP1) belongs to a family of evolutionarily conserved DDR proteins. Because 53BP1 molecules localize at the sites of DSBs and rapidly form nuclear foci, the presence of 53BP1 nuclear foci can be considered as a cytological marker for endogenous DSBs reflecting GIN. The levels of GIN were analyzed by immunofluorescence studies of 53BP1 in 56 skin tumors that included 20 seborrheic keratosis (SK), 8 actinic keratosis (AK), 9 Bowen’s disease (BD), 9 squamous cell carcinoma (SCC), and 10 basal cell carcinoma (BCC). This study demonstrated a number of nuclear 53BP1 foci in human skin tumorigenesis, suggesting a constitutive activation of DDR in skin cancer cells. Because AK showed a high DDR type of 53BP1 immunoreactivity, GIN seems to be induced at the precancerous stage. Furthermore, invasive cancers exhibited a high level of intense, abnormal 53BP1 nuclear staining with nuclear accumulation of p53, suggesting a disruption of DDR leading to a high level of GIN in cancer cells. The results of this study suggest that GIN has a crucial role in the progression of skin carcinogenesis. The detection of 53BP1 expression by immunofluorescence can be a useful histological marker to estimate the malignant potential of human skin tumors