Integrating proteomic and functional genomic technologies in discovery-driven translational breast cancer research

The application of state-of-the-art proteomics and functional genomics technologies to the study of cancer is rapidly shifting toward the analysis of clinically relevant samples derived from patients, as the ultimate aim of translational research is to bring basic discoveries closer to the bedside. Here we describe the essence of a long-term initiative undertaken by The Danish Centre for Translational Breast Cancer Research and currently underway for cancer biomarker discovery using fresh tissue biopsies and bio-fluids. The Centre is a virtual hub that brings together scientists working in various areas of basic cancer research such as cell cycle control, invasion and micro-environmental alterations, apoptosis, cell signaling, and immunology, with clinicians (oncologists, surgeons), pathologists, and epidemiologists, with the aim of understanding the molecular mechanisms underlying breast cancer progression and ultimately of improving patient survival and quality of life. The unifying concept behind our approach is the use of various experimental paradigms for the prospective analysis of clinically relevant samples obtained from the same patient, along with the systematic integration of the biological and clinical data.     

Dynamic assembly and sustained retention of 53BP1 at the sites of DNA damage are controlled by Mdc1/NFBD1

53BP1 is a key component of the genome surveillance network activated by DNA double strand breaks (DSBs). Despite its known accumulation at the DSB sites, the spatiotemporal aspects of 53BP1 interaction with DSBs and the role of other DSB regulators in this process remain unclear. Here, we used real-time microscopy to study the DSB-induced redistribution of 53BP1 in living cells. We show that within minutes after DNA damage, 53BP1 becomes progressively, yet transiently, immobilized around the DSB-flanking chromatin. Quantitative imaging of single cells revealed that the assembly of 53BP1 at DSBs significantly lagged behind Mdc1/NFBD1, another DSB-interacting checkpoint mediator. Furthermore, short interfering RNA-mediated ablation of Mdc1/NFBD1 drastically impaired 53BP1 redistribution to DSBs and triggered premature dissociation of 53BP1 from these regions. Collectively, these in vivo measurements identify Mdc1/NFBD1 as a key upstream determinant of 53BP1's interaction with DSBs from its dynamic assembly at the DSB sites through sustained retention within the DSB-flanking chromatin up to the recovery from the checkpoint.     

TRIP12 and UBR5 Suppress Spreading of Chromatin Ubiquitylation at Damaged Chromosomes

Histone ubiquitylation is a prominent response to DNA double-strand breaks (DSBs), but how these modifications are confined to DNA lesions is not understood. Here, we show that TRIP12 and UBR5, two HECT domain ubiquitin E3 ligases, control accumulation of RNF168, a rate-limiting component of?a pathway that ubiquitylates histones after DNA breakage. We find that RNF168 can be saturated by increasing amounts of DSBs. Depletion of TRIP12 and UBR5 allows accumulation of RNF168 to supraphysiological levels, followed by massive spreading of ubiquitin conjugates and hyperaccumulation of ubiquitin-regulated genome caretakers such as 53BP1 and BRCA1. Thus, regulatory and proteolytic ubiquitylations are wired in a self-limiting circuit that promotes histone ubiquitylation near the DNA lesions but at the same time counteracts its excessive spreading to undamaged chromosomes. We provide evidence that this mechanism is vital for the homeostasis of ubiquitin-controlled events after DNA breakage and can be subverted during tumorigenesis.     

RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells

The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role of RPA in homologous recombination in assembly of the RAD51 and RAD52 proteins. Furthermore, our data suggest that replacement of RPA with the RAD51 and RAD52 proteins is affected by checkpoint signalling.     

Adiponectin in patients with various stages of coronary heart disease - comparison of its concentration in coronary arteries and peripheral venous circulation

Adiponectin is an adipocytes-produced protein and showing a number of antiatherogenic effects. Adiponectin seems to be extensively deposited in the intersticium of venous lesions of persons with myocardial infarction. It may exhibit antiatherogenic and reparative effects. A decreased adiponectin concentration may be a risk factor of the origin and complications of atherosclerosis. AIM OF THE STUDY: 1) Do the adiponectin concentrations in venous blood of persons with acute coronary syndrome (ACS) differ from those in persons with stress angina pectoris? 2) In these persons do adiponectin concentrations in venous blood differ from those in main coronary arteries? 3) Do adiponectin levels differ in the infarction and non-infarction arteries in persons with STEMI (ST Elevation Myocardial Infarct) and delay within 4 hours after the onset? 4) In persons with ACS does any correlation exist between venous adiponectin and common risk factors of cardiovascular complications? Adiponectin concentration was determined in samples of blood collected from the peripheral vein and during coronarography in various localizations in 4 groups of examined persons (I. - no signs of CAD, II. - stable stress angina pectoris, III. - ACS over 48 hours without elevations of ST segment, IV. - STEMI during first 4 hours after its origin and proved occlusion of coronary artery at coronarography). Coronary angiography, risk factors and anamnestic data were analyzed. The software Medcalc was used to perform statistical analysis. We examined 73 probands with signs of myocardial ischemia (mean age of 61.5 years, 64 % males), who were subjected to coronarography and 21 healthy volunteers. A mean delay (delay from the origin of complaints to the performed coronarography) was 3.1 +/- 0.5 hours in individuals in the group IV. In patients with ACS we found lower adiponectin concentrations in venous blood compared to healthy individuals and persons with stress AP, but changes were not statistically significant (I.: -5.9 +/- 2.7 ng/l, II.: -4.9 +/- 1.2 ng/l, III.: -5.2 +/- 4.1 ng/l, IV.: -4.6 +/- 2.7 ng/l); no differences were found also with BMI. No significant difference was recorded between the samples of venous blood and those of coronary arteries, nor between the infarction and the non-infarction arteries in the group IV. (5.2 +/- 2.6 ng/l vs. 4.8 +/- -2.7 ng/l). Significant negative correlations were observed between adiponectin concentrations and BMI (correlation coefficient -0.29), triacylglycerols (correlation coefficient -0.4), AOPP (correlation coefficient -0.39), and positive correlations with HDL (correlation coefficient 0.32). No correlation was recorded between adiponectin and CRP. Adiponectin concentrations in persons with ACS are lower than in healthy persons or patients with stable angina pectoris, but differences are not statistically significant. The absence of adiponectin differences between the infarction/non-infarction artery may support the hypothesis of adiponectin uptake in the ischemic lesion with subsequent decrease in blood adiponectin. On the contrary, adiponectin decrease may be a risk factor independent of the origin and development of ACS.