Nucleosome reorganisation in breast cancer tissues.

BACKGROUND: Nucleosome repositioning in cancer is believed to cause many changes in genome organisation and gene expression. Understanding these changes is important to elucidate fundamental aspects of cancer. It is also important for medical diagnostics based on cell-free DNA (cfDNA), which originates from genomic DNA regions protected from digestion by nucleosomes. RESULTS: We have generated high-resolution nucleosome maps in paired tumour and normal tissues from the same breast cancer patients using MNase-assisted histone H3 ChIP-seq and compared them with the corresponding cfDNA from blood plasma. This analysis has detected single-nucleosome repositioning at key regulatory regions in a patient-specific manner and common cancer-specific patterns across patients. The nucleosomes gained in tumour versus normal tissue were particularly informative of cancer pathways, with ~ 20-fold enrichment at CpG islands, a large fraction of which marked promoters of genes encoding DNA-binding proteins. The tumour tissues were characterised by a 5-10 bp decrease in the average distance between nucleosomes (nucleosome repeat length, NRL), which is qualitatively similar to the differences between pluripotent and differentiated cells. This effect was correlated with gene activity, differential DNA methylation and changes in local occupancy of linker histone variants H1.4 and H1X. CONCLUSIONS: Our study offers a novel resource of high-resolution nucleosome maps in breast cancer patients and reports for the first time the effect of systematic decrease of NRL in paired tumour versus normal breast tissues from the same patient. Our findings provide a new mechanistic understanding of nucleosome repositioning in tumour tissues that can be valuable for patient diagnostics, stratification and monitoring.

An X-ray micro-computer tomography study of the Malpighian tubules of the Blue Bottle Blow Fly (Calliphora vomitoria) Diptera: Calliphoridae.

Malpighian tubules are the insect equivalent of mammalian kidneys and normally drain into the gut at the junction between the mid and hind gut. The Malpighian tubules of the fruit fly Drosophila melanogaster are increasingly being used as a model for studying human renal tract development, histology, nephrolithiasis and urolithiasis. In the present study we report when using X-ray micro-computer tomography techniques, the larval, intrapuparial and adult stages of the larger Calliphora vomitoria can contain large amounts of calcium-rich concretions which are tightly packed in the lumen of both anterior Malpighian tubules. We show that it is feasible to utilise these calcium-rich concretions as a form of marking agent to delineate the various developmental stages of the Malpighian tubules including the crucial phase when the Malpighian tubules reconnect with the hind gut. In the majority of cases during the intrapuparial period the ureters of the Malpighian tubules did not start to re-canalise and thus reconnect with the developing hind gut until the 7th day of the 10-11 day. Just prior to ecdysis, virtually all the radio-opaque concretions in the Malpighian tubules had emptied into the hind gut and had then been completely excreted by the time the imago emerged from its puparium. In contrast, we show that in flies developing from larvae previously stained by ingesting Rhodamine B, a known substrate for both the Multi Xenobiotic Resistance and Multi Drug Resistant membrane transport systems, the efficiency with which these calcium-rich concretions are excreted by the imago as it emerges from its intrapuparial period can be significantly impaired. Therefore, it might be useful to include C. vomitoria as a model when studying renal tract development and urolithiasis using X-ray micro-computer tomography.

Use of X-ray micro-computed tomography to study the moult cycle of the freshwater amphipod Gammarus pulex.

Stages of the moult cycle of the amphipod Gammarus pulex have been previously characterised based on the examination of either apolysis of the 3rd dactyl, or the whole body and eye appearance. In the current study the aim was to compare these two established moult staging techniques with a novel X-ray micro-computed tomography (micro-CT) scan method. The micro-CT provides information on the degree of calcification of the external integument and of the internal structures, such as the gastric mill. The degree of calcification is predicted to change during the moult cycle. Successful micro-CT scans were obtained from 80 G. pulex specimens and the radiological appearance of the 28 specimens immediately immersed in 4 % PFA were not different to the 52 specimens stored in 4 % PFA for at least 28 days prior to scanning. These specimens could be classified into moult stages A, B, C, early D or late D based on the degree of calcification. Good agreement was obtained between all three methods of moult stage classification if fresh specimens were used, but if specimens had been preserved in 4% Paraformaldehyde (PFA) for more than 24 hours the loss of colour from the whole body and eye meant these methods were not suitable. This is the first time that a micro-CT method has been used to study G. pulex and shows that this method of moult staging is accurate and reliable.

Engineering hemoglobin to enable homogenous PEGylation without modifying protein functionality.

In order to infuse hemoglobin into the vasculature as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the molecule to enhance vascular retention. This aim can be achieved by PEGylation. However, using non-specific conjugation methods creates heterogenous mixtures and alters protein function. Site-specific PEGylation at the naturally reactive thiol on human hemoglobin (ßCys93) alters hemoglobin oxygen binding affinity and increases its autooxidation rate. In order to avoid this issue, new reactive thiol residues were therefore engineered at sites distant to the heme group and the α/ß dimer/dimer interface. The two mutants were ßCys93Ala/αAla19Cys and ßCys93Ala/ßAla13Cys. Gel electrophoresis, size exclusion chromatography and mass spectrometry revealed efficient PEGylation at both αAla19Cys and ßAla13Cys, with over 80% of the thiols PEGylated in the case of αAla19Cys. For both mutants there was no significant effect on the oxygen affinity or the cooperativity of oxygen binding. PEGylation at αAla19Cys had the additional benefit of decreasing the rates of autoxidation and heme release, properties that have been considered contributory factors to the adverse clinical side effects exhibited by previous hemoglobin based oxygen carriers. PEGylation at αAla19Cys may therefore be a useful component of future clinical products.

Engineering tyrosine residues into hemoglobin enhances heme reduction, decreases oxidative stress and increases vascular retention of a hemoglobin based blood substitute.

Hemoglobin (Hb)-based oxygen carriers (HBOC) are modified extracellular proteins, designed to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects, in part linked to the intrinsic oxidative toxicity of Hb. Previously a redox-active tyrosine residue was engineered into the Hb ß subunit (ßF41Y) to facilitate electron transfer between endogenous antioxidants such as ascorbate and the oxidative ferryl heme species, converting the highly oxidizing ferryl species into the less reactive ferric (met) form. We inserted different single tyrosine mutations into the α and ß subunits of Hb to determine if this effect of ßF41Y was unique. Every mutation that was inserted within electron transfer range of the protein surface and the heme increased the rate of ferryl reduction. However, surprisingly, three of the mutations (ßT84Y, αL91Y and ßF85Y) also increased the rate of ascorbate reduction of ferric(met) Hb to ferrous(oxy) Hb. The rate enhancement was most evident at ascorbate concentrations equivalent to that found in plasma (< 100 µM), suggesting that it might be of benefit in decreasing oxidative stress in vivo. The most promising mutant (ßT84Y) was stable with no increase in autoxidation or heme loss. A decrease in membrane damage following Hb addition to HEK cells correlated with the ability of ßT84Y to maintain the protein in its oxygenated form. When PEGylated and injected into mice, ßT84Y was shown to have an increased vascular half time compared to wild type PEGylated Hb. ßT84Y represents a new class of mutations with the ability to enhance reduction of both ferryl and ferric Hb, and thus has potential to decrease adverse side effects as one component of a final HBOC product.

Poly(ADP-ribosyl)ation associated changes in CTCF-chromatin binding and gene expression in breast cells.

CTCF is an evolutionarily conserved and ubiquitously expressed architectural protein regulating a plethora of cellular functions via different molecular mechanisms. CTCF can undergo a number of post-translational modifications which change its properties and functions. One such modifications linked to cancer is poly(ADP-ribosyl)ation (PARylation). The highly PARylated CTCF form has an apparent molecular mass of 180 kDa (referred to as CTCF180), which can be distinguished from hypo- and non-PARylated CTCF with the apparent molecular mass of 130 kDa (referred to as CTCF130). The existing data accumulated so far have been mainly related to CTCF130. However, the properties of CTCF180 are not well understood despite its abundance in a number of primary tissues. In this study we performed ChIP-seq and RNA-seq analyses in human breast cells 226LDM, which display predominantly CTCF130 when proliferating, but CTCF180 upon cell cycle arrest. We observed that in the arrested cells the majority of sites lost CTCF, whereas fewer sites gained CTCF or remain bound (i.e. common sites). The classical CTCF binding motif was found in the lost and common, but not in the gained sites. The changes in CTCF occupancies in the lost and common sites were associated with increased chromatin densities and altered expression from the neighboring genes. Based on these results we propose a model integrating the CTCF130/180 transition with CTCF-DNA binding and gene expression changes. This study also issues an important cautionary note concerning the design and interpretation of any experiments using cells and tissues where CTCF180 may be present.

Comparison of the oxidative reactivity of recombinant fetal and adult human hemoglobin: implications for the design of hemoglobin-based oxygen carriers.

Hemoglobin (Hb)-based oxygen carriers (HBOCs) have been engineered to replace or augment the oxygen carrying capacity of erythrocytes. However, clinical results have generally been disappointing, in part due to the intrinsic oxidative toxicity of Hb. The most common HBOC starting material is adult human or bovine Hb. However, it has been suggested that fetal Hb may offer advantages due to decreased oxidative reactivity. Large-scale manufacturing of HBOC will likely and ultimately require recombinant sources of human proteins. We, therefore, directly compared the functional properties and oxidative reactivity of recombinant fetal (rHbF) and recombinant adult (rHbA) Hb. rHbA and rHbF produced similar yields of purified functional protein. No differences were seen in the two proteins in: autoxidation rate; the rate of hydrogen peroxide reaction; NO scavenging dioxygenase activity; and the NO producing nitrite reductase activity. The rHbF protein was: less damaged by low levels of hydrogen peroxide; less damaging when added to human umbilical vein endothelial cells (HUVEC) in the ferric form; and had a slower rate of intrinsic heme loss. The rHbA protein was: more readily reducible by plasma antioxidants such as ascorbate in both the reactive ferryl and ferric states; less readily damaged by lipid peroxides; and less damaging to phosphatidylcholine liposomes. In conclusion in terms of oxidative reactivity, there are advantages and disadvantages to the use of rHbA or rHbF as the basis for an effective HBOC.

A novel mechanism for CTCF in the epigenetic regulation of Bax in breast cancer cells.

We previously reported the association of elevated levels of the multifunctional transcription factor, CCCTC binding factor (CTCF), in breast cancer cells with the specific anti-apoptotic function of CTCF. To understand the molecular mechanisms of this phenomenon, we investigated regulation of the human Bax gene by CTCF in breast and non-breast cells. Two CTCF binding sites (CTSs) within the Bax promoter were identified. In all cells, breast and non-breast, active histone modifications were present at these CTSs, DNA harboring this region was unmethylated, and levels of Bax mRNA and protein were similar. Nevertheless, up-regulation of Bax mRNA and protein and apoptotic cell death were observed only in breast cancer cells depleted of CTCF. We proposed that increased CTCF binding to the Bax promoter in breast cancer cells, by comparison with non-breast cells, may be mechanistically linked to the specific apoptotic phenotype in CTCF-depleted breast cancer cells. In this study, we show that CTCF binding was enriched at the Bax CTSs in breast cancer cells and tumors; in contrast, binding of other transcription factors (SP1, WT1, EGR1, and c-Myc) was generally increased in non-breast cells and normal breast tissues. Our findings suggest a novel mechanism for CTCF in the epigenetic regulation of Bax in breast cancer cells, whereby elevated levels of CTCF support preferential binding of CTCF to the Bax CTSs. In this context, CTCF functions as a transcriptional repressor counteracting influences of positive regulatory factors; depletion of breast cancer cells from CTCF therefore results in the activation of Bax and apoptosis.

BioVyon Protein A, an alternative solid-phase affinity matrix for chromatin immunoprecipitation.

Chromatin immunoprecipitation (ChIP) is an important technique in the study of DNA/protein interactions. The ChIP procedure, however, has limitations in that it is lengthy, can be inconsistent, and is prone to nonspecific binding of DNA and proteins to the bead-based solid-phase matrices that are often used for the immunoprecipitation step. In this investigation, we examined the utility of a new matrix for ChIP assays, BioVyon Protein A, a solid support based on porous polyethylene. In ChIP experiments carried out using two antibodies and seven DNA loci, the performance of BioVyon Protein A was significantly better, with a greater percentage of DNA pull-down in all of the assays tested compared with bead-based matrices, Protein A Sepharose, and Dynabeads Protein A. Furthermore, the rigid porous disc format within a column made the BioVyon matrix much easier to use with fewer steps and less equipment requirements, resulting in a significant reduction in the time taken to process the ChIP samples. In summary, BioVyon Protein A provides a column-based assay method for ChIP and other immunoprecipitation-based procedures; the rigid porous structure of BioVyon enables a fast and robust protocol with higher ChIP enrichment ratios.

Decreased poly(ADP-ribosyl)ation of CTCF, a transcription factor, is associated with breast cancer phenotype and cell proliferation.

PURPOSE: There is compelling evidence of a relationship between poly(ADP-ribosyl)ation and tumorigenesis; however, much less is known about the role of specific targets of poly(ADP-ribosyl)ation in tumor development. Two forms of the multifunctional transcription factor, CTCF, were previously identified: a 130-kDa protein (CTCF-130), characteristic for cell lines, and a 180-kDa protein (CTCF-180), modified by poly(ADP-ribosyl)ation. This study was aimed to investigate differential poly(ADP-ribosyl)ation of CTCF in normal and tumor breast tissues. EXPERIMENTAL DESIGN: Western blot analysis, mass spectrometry, and immunohistochemical and immunofluorescent stainings were used to characterize CTCF-130 and CTCF-180 in breast cell lines, primary cultures, and normal and tumor breast tissues. The immunoreactivity score was used for CTCF-130 quantification in tissues. RESULTS: We discovered that only CTCF-180 is detected in the normal breast tissues, whereas both CTCF-130 and CTCF-180 are present in breast tumors. Using an antibody specific for CTCF-130, we observed that 87.7% of breast tumors were positive for CTCF-130. A negative correlation existed between the levels of CTCF-130, tumor stage, and tumor size. Significantly, a transition from CTCF-180 to CTCF-130 was discovered in primary cultures generated from normal breast tissues, indicating a link between CTCF-130 and proliferation. Conversely, the appearance of CTCF-180 was observed following growth arrest in breast cell lines. CONCLUSIONS: Collectively, our data suggest that the loss of CTCF poly(ADP-ribosyl)ation is associated with cell proliferation and breast tumor development. We propose the use of CTCF-130 as a marker for tumor breast cells and lower levels of CTCF-130 as an indicator of unfavorable prognosis.