Thrombospondin-4 is a putative tumour-suppressor gene in colorectal cancer that exhibits age-related methylation

<p>Abstract</p> <p>Background</p> <p><it>Thrombospondin-4 </it>(<it>THBS4</it>) is a member of the extracellular calcium-binding protein family and is involved in cell adhesion and migration. The aim of this study was to evaluate the potential role of deregulation of <it>THBS4 </it>expression in colorectal carcinogenesis. Of particular interest was the possible silencing of expression by methylation of the CpG island in the gene promoter.</p> <p>Methods</p> <p>Fifty-five sporadic colorectal tumours stratified for the CpG Island Methylator Phenotype (CIMP) were studied. Immunohistochemical staining of THBS4 protein was assessed in normal and tumour specimens. Relative levels of <it>THBS4 </it>transcript expression in matched tumours and normal mucosa were also determined by quantitative RT-PCR. Colony forming ability was examined in 8 cell lines made to overexpress THBS4. Aberrant promoter hypermethylation was investigated as a possible mechanism of gene disruption using MethyLight. Methylation was also assessed in the normal colonic tissue of 99 patients, with samples biopsied from four regions along the length of the colon.</p> <p>Results</p> <p><it>THBS4 </it>expression was significantly lower in tumour tissue than in matched normal tissue. Immunohistochemical examination demonstrated that THBS4 protein was generally absent from normal epithelial cells and tumours, but was occasionally expressed at low levels in the cytoplasm towards the luminal surface in vesicular structures. Forced THBS4 over-expression caused a 50-60% repression of tumour colony growth in all eight cell lines examined compared to control cell lines. Tumours exhibited significantly higher levels of methylation than matched normal mucosa, and <it>THBS4 </it>methylation correlated with the CpG island methylator phenotype. There was a trend towards decreased gene expression in tumours exhibiting high <it>THBS4 </it>methylation, but the correlation was not significant. <it>THBS4 </it>methylation was detectable in normal mucosal biopsies where it correlated with increasing patient age and negatively with the occurrence of adenomas elsewhere in the colon.</p> <p>Conclusions</p> <p><it>THBS4 </it>shows increased methylation in colorectal cancer, but this is not strongly associated with altered gene expression, either because methylation has not always reached a critical level or because other factors influence <it>THBS4 </it>expression. <it>THBS4 </it>may act as a tumour suppressor gene, demonstrated by its suppression of tumour colony formation <it>in vitro</it>. <it>THBS4 </it>methylation is detectable in normal colonic mucosa and its level may be a biomarker for the occurrence of adenomas and carcinoma.</p

DNA methylation within the normal colorectal mucosa is associated with pathway-specific predisposition to cancer

There are two major molecular pathways to sporadic colorectal cancer, the chromosomal instability (CIN) and the CpG island methylator phenotype (CIMP) pathways. This study recruited 166 patients undergoing colonoscopy. Biopsy samples were collected from the cecum, transverse colon, sigmoid colon and rectum. DNA methylation was quantified at ‘type A’ (ESR1, GATA5, HIC1, HPP1, SFRP1) and ‘type C’ markers (MGMT, MLH1, CDKN2A, MINT2, MINT31, IGF2, CACNA1G, NEUROG1, SOCS1, RUNX3), and LINE-1. ‘Type A’ genes are frequently methylated in normal and neoplastic tissues, proportional to tissue age. ‘Type C’ methylation is more specific for neoplasia. The last five ‘type C’ markers comprise a CIMP panel. The mean ‘type A’ and CIMP-panel methylation Z-scores were calculated. In all, 88 patients had adenomatous lesions, 32 had proximal serrated polyps (PSPs) and 50 were normal. Most ‘type A’ genes showed direct correlations between methylation and age (ESR1, ρ=0.66, P<0.0001), with higher methylation distally (ESR1, P<0.0001). On multivariate analysis, ‘type A’ methylation was inversely associated with colorectal adenomas (odds ratio=0.23, P<0.001), the precursor to CIN cancers. CIMP-panel methylation was significantly associated with advanced PSPs (odds ratio=5.1, P=0.009), the precursor to CIMP cancers. DNA methylation in normal mucosa varied with age and region and was associated with pathway-specific pathology. In the future, the colorectal field could yield important information and potentially inform clinical practice

Expansion of Multipotent Stem Cells from the Adult Human Brain

<div><p>The discovery of stem cells in the adult human brain has revealed new possible scenarios for treatment of the sick or injured brain. Both clinical use of and preclinical research on human adult neural stem cells have, however, been seriously hampered by the fact that it has been impossible to passage these cells more than a very few times and with little expansion of cell numbers. Having explored a number of alternative culturing conditions we here present an efficient method for the establishment and propagation of human brain stem cells from whatever brain tissue samples we have tried. We describe virtually unlimited expansion of an authentic stem cell phenotype. Pluripotency proteins Sox2 and Oct4 are expressed without artificial induction. For the first time multipotency of adult human brain-derived stem cells is demonstrated beyond tissue boundaries. We characterize these cells in detail <i>in vitro</i> including microarray and proteomic approaches. Whilst clarification of these cells’ behavior is ongoing, results so far portend well for the future repair of tissues by transplantation of an adult patient’s own-derived stem cells.</p></div

Are stem cell sources different based on microarray analysis.

<p>Relatedness of cultures from 3 humans/4 sources/1 platform. <b>A.</b> Two way table showing number of genes (mean; n = 3) differing by more than three fold. Total number of genes in this comparison: 34694. <b>B.</b> ‘Globaltest’ analysis revealing probability for the null hypothesis after testing differences between cell culture groups (n = 3). <b>C</b>. Histogram: the white bars show a test statistic for a paired <i>t</i>-test of HPC vs SVZ (individual cultures from each human have been paired). The blue line shows what one would expect if there were no real differences (just random, due to technical noise etc). The pink bars are what the method estimates as real differences calculated as the difference between the white bars and the blue line). The method estimates that around 6% of the genes are different between SVZ and HPC. <b>D.</b> The similarity between the expression profiles is determined by hierarchical cluster analysis and shown as a dendrogram. The length of the line connecting two samples indicates the similarity between the samples (short line: highly similar pattern). Dendrogram generated by Norwegian Microarray Consortium from quantile normalized data.</p

<i>Failsafe</i> protocol for brain biopsy stem cell culture.

<p><i>Failsafe</i> protocol for brain biopsy stem cell culture.</p

Proliferation and phenotype of brain stem cells.

<p><b>A.</b> Average doubling time was 6.5 days. 50 doublings is >10¹⁴. This is a yield of 10¹⁸ from a small biopsy. There are 10¹⁴ cells in a human. <b>B.</b> Morphology of brain-derived stem cells growing adherently. <b>C and D.</b> Cultures derived from Hippocampus appeared identical to those from Subventricular zone. (Bars: +SD, n = 3). Markers of: +ve control, Prol4OHase; dividing cells, Ki67; stem cells, Oct 4, Sox2, Intβ1, EGFr, Mus, Nestin; glia, GFAP, S100; neurons, βTub3, Map2, NF; oligodendrocytes, O4, GalC; dopaminergic cells, TH, DT. <b>E.</b> Cultures maintained phenotype through many passages. (Bars: +SD, n = 3). <b>F. </b><b>Stratification of cultures (Grey Matter p9).</b> Cultures could be arbitrarily divided into zones based on the appearance of the cells in these zones. Immunophenotype confirmed that the differences in shape also reflected differences in phenotype and suggest that this stratification in some way reflects the dynamics involved in tissue organization (See also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071334#pone.0071334.s001" target="_blank">Figure S1</a>. Phenotype of brain stem cell cultures</b>).</p

Constituents of the optimal growth medium.

<p>Constituents of the optimal growth medium.</p

Karyotype of human-derived brain stem cell cultures.

<p>Karyotype of human-derived brain stem cell cultures. Ratios are for number of normal cells (not taking into account loss of Y chromosome) out of total number of cells examined.</p

Brain stem cells appear to be multipotent in chick embryo chimeras.

<p>Cells were first labeled genetically with a lentivirus expressing GFP under a CMV promoter. Cells were transplanted into the primitive streak of 20 h chick embryos. Top: Descendant cells in embryos harvested at two days later visible under blue excitation as green cells in a variety of tissue locations (C is an overlay of the image in A and the image in B) (HB: hind brain; MB: mid brain; E: eye; H: heart, LB: limb bud). Antibodies against phenotypic markers of the relevant tissues were used to confirm appropriate differentiation of heart muscle (CTNI), striated muscle (sα-actin), and neuronal cells (NF200) (pink stain; G, L, Q). Regions that had shown GFP-positive green human-descended cells (E, J, O) were then also processed for anti GFP-immunochemistry (black stain, F, K, P). As well some sections from regions processed for phenotypic markers (pink) were subsequently co-stained for GFP antigen (black, H, M, R). Insets of high magnification of 100 micron squares depict close proximity of black and pink staining. The black anti-GFP is perinuclear in pattern thus making it highly likely that the two markers are expressed together in individual cells. Panels D, I and N show no primary antibody controls for immunochemistry. Magnification: bars in B, 1 mm; D to R, 100 µm.</p

A proteomic comparison was made between SVZ and HPC cultures.

<p>A proteomic comparison was made between SVZ and HPC cultures for differentially expressed proteins using ‘Silac’ technology that discriminates proteins synthesized in culture incorporating ‘heavy’ amino acids. Data were obtained for 840 proteins. Twelve of these were shown to be upregulated >1.4 fold in SVZ and 11 were shown to be upregulated >1.4 fold in HPC (p<<0.05, n = 3). Column 4 indicates whether this upregulation is reflected in the RNA microarray data (bolded ratios are those that are not).</p