Les McNoe

Wilms tumour histology is determined by distinct types of precursor lesions and not epigenetic changes.

Current models of Wilms tumour development propose that histological features of the tumours are programmed by the underlying molecular aberrations. For example, tumours associated with WT1 mutations arise from intralobar nephrogenic rests (ILNR), concur with CTNNB1 mutations and have distinct histology, whereas tumours with IGF2 loss of imprinting (LOI) often arise from perilobar nephrogenic rests (PLNR). Intriguingly, ILNR and PLNR are found simultaneously in Wilms tumours in children with overgrowth who have constitutional IGF2 LOI. We therefore examined whether the precursor lesions or early epigenetic changes are the primary determinant of Wilms tumour histology. We examined the histological features and gene expression profiles of IGF2 LOI tumours and WT1‐mutant tumours which are associated with PLNR and/or ILNR. Two distinct types of IGF2 LOI tumours were identified: the first type had a blastemal‐predominant histology associated with PLNR, while the second subtype had a myogenic histology, increased expression of mesenchymal lineage genes and an association with ILNR, similar to WT1‐mutant tumours. These ILNR‐associated IGF2 LOI tumours also showed signatures of activation of the WNT signalling pathway: differential expression of β‐catenin targets (MMP2, RARG, DKK1) and WNT antagonist genes (DKK1, WIF1, SFRP4). Unexpectedly, the majority of these tumours had CTNNB1 mutations, which are normally only seen in WT1‐mutant tumours. The absence of WT1 mutations in tumours with IGF2 LOI indicated that CTNNB1 mutations occur predominantly in tumours arising from ILNR independent of the presence or absence of WT1 mutations. Thus, even though these two classes of tumours with IGF2 LOI have the same underlying predisposing epigenetic error, the tumour histology and the gene expression profiles are determined by the nature of the precursor cells within the nephrogenic rests and subsequent CTNNB1 mutations. Copyright

Gene and protein expression signature of endometrial glandular and stromal compartments during the window of implantation

OBJECTIVE To map the changes in messenger RNA (mRNA) and protein abundance during the window of implantation in specifically endometrial stromal and glandular epithelial cells obtained using laser microdissection microscopy (LDM). DESIGN Endometrial samples were collected from two menstrual cycles at 2 and 7 days after first significant rise in blood LH, and separate cell populations were obtained using LDM. A new generation linear polymerase chain reaction (PCR) amplified the mRNA, which were hybridized to both Affymetrix U133 Plus2 and Agilent 4x44K microarrays followed by gene set analysis. Immunohistochemistry assessed protein expression between the two collection times. SETTING In vitro fertilization clinic. PATIENT(S) Nine Caucasian, fertile, cycling women. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Cycle dating using blood markers; microarrays on laser microdissected glands and stroma; dual platform microarray confirmation; immunohistochemical analysis of cell cycle proteins. RESULT(S) The two microarray platforms showed concordance. During the window of implantation, a statistically significant network of 22 mRNA associated with the cell cycle was down-regulated. Immunohistochemistry identified altered localization in stroma. CONCLUSION(S) Microarrays demonstrated glands and stroma have distinct mRNA signatures, each dependent on the day of the cycle. We characterized two compartments of the receptive endometrium with a transcriptomic signature identifying regulation of only the cell cycle. Immunohistochemical analysis of cell cycle proteins identified a signature staining pattern of nuclear relocalization of a group of cyclins of stromal cells, which may be clinically applicable.

Slow proliferation as a biological feature of colorectal cancer metastasis.

Background:We have recently reported an inverse relationship between colon cancer progression and tumour proliferative activity. Here, we extend our findings by evaluating the proliferative activity of liver metastatic lesions and primary colorectal cancers (CRC) that differ in their metastatic potential.Methods:Using an earlier established multi-gene proliferation signature (GPS), proliferative levels were analysed in 73 primary CRCs and 27 liver metastases.Results:Compared with primary CRCs, we observed a significantly lower expression of the GPS in liver metastases and confirmed their lower proliferative levels by quantitative RT–PCR and Ki-67 immunostaining. No difference could be detected in apoptotic indices as assessed by M30 immunostaining, indicating that the net growth rate is lower in metastases relative to primary tumours. Notably, relapsed primaries or those with established metastases had significantly lower proliferative activity than CRCs that were non-metastatic and did not relapse.Conclusion:Our results suggest that slow proliferation is a biological characteristic of both liver metastases and those primary tumours with the ability to metastasise. The delineation of the mechanisms underlying the inverse association between proliferation and CRC aggressiveness may be important for the development of new therapeutic strategies.

The chromosome 9p21.3 coronary heart disease risk allele is associated with altered gene expression in normal heart and vascular tissues

Genome-wide association studies have identified a coronary artery disease (CAD) risk locus in a non-coding region at 9p21.3, the nearest genes being CDKN2A and CDKN2B. To understand the pathways by which this locus might influence CAD susceptibility, we investigated associations between the 9p21.3 risk genotype and global gene expression in heart tissue from donors with no diagnosed heart disease (n = 108, predominant cause of death, cerebral vascular accident) and in carotid plaque (n = 106), aorta (n = 104) and mammary artery (n = 88) tissues from heart valve and carotid endarterectomy patients. Genotyping was performed with Taqman assays and Illumina arrays, and gene expression profiles generated with Affymetrix microarrays. Associations were analyzed with an additive genetic model. In heart tissue, 46 genes were putatively altered in association with the 9p21.3 risk allele (70% down-regulated, fold-change >1.1 per allele, p<0.05 adjusted for age, gender, ethnicity, cause of death). These genes were enriched for biomarkers of myocardial infarction (p = 1.53×10−9), response to wounding (p = 2.65×10−10) and inflammatory processes (p<1.97×10−7). Among the top 10 most down-regulated genes, 7 genes shared a set of transcription factor binding sites within conserved promoter regions (p<1.14×10−5), suggesting they may be co-regulated. Canonical pathway modelling of the most differentially expressed transcripts across all tissues (154 genes, 60% down-regulated, fold-change >1.1 per allele, p<0.01) showed that 75% of the genes could be transcriptionally regulated through the cell cycle G1 phase progression pathway (p<1.08×10−258), in which CDKN2A and CDKN2B play a regulatory role. These data suggest that the cell cycle G1 phase progression pathway is activated in individuals with the 9p21.3 risk allele. This may contribute to a proliferative phenotype that promotes adverse cardiac hypertrophy and vascular remodeling, leading to an increased CAD risk.

Evaluation of Global RNA Amplification and Its Use for High-Throughput Transcript Analysis of Laser-Microdissected Endosperm

Laser microdissection (LM) provides a useful method for isolating specific cells or tissues from biological samples. Here, we adapted microdissection protocols to allow high-resolution transcript analysis of different tissues from developing Arabidopsis seed. Sufficient RNA (∼50 ng) was extracted from endosperm tissue for RT-PCR. However, to obtain enough RNA for microarray analyses, it was necessary to amplify the RNA. PCR- and IVT-based amplification methods were investigated and several important technical aspects of amplification were identified (such as target truncation and alterations in signal intensity). We found that when starting from only 50 ng of RNA, amplification methods based on PCR and IVT produced sufficient product for reliable microarray hybridizations, with two-round IVT giving the best results. Microarray analyses, using endosperm-derived RNA amplified by two-round IVT, reproducibly identified endosperm enriched marker genes. Thus, when combined with RNA-amplification protocols, LM is a robust and reliable technique for high-throughput tissue-specific gene expression analysis.

Altered Transcription of Murine Genes Induced in the Small Bowel by Administration of Probiotic Strain Lactobacillus rhamnosus HN001

ABSTRACT Lactobacillus rhamnosus HN001 is a probiotic strain reported to increase resistance to epithelium-adherent and -invasive intestinal pathogens in experimental animals. To increase understanding of the relationship between strain HN001 and the bowel, transcription of selected genes in the mucosa of the murine small bowel was measured. Mice previously naive to lactobacilli (Lactobacillus-free mice) were examined after daily exposure to HN001 in drinking water. Comparisons were made to results from matched Lactobacillus-free mice. Infant and adult mice were investigated to provide a temporal view of gene expression in response to exposure to HN001. Genes sgk1, angptl4, and hspa1b, associated with the apoptosis pathway, were selected for investigation by reverse transcription-quantitative PCR on the basis of a preliminary duodenal DNA microarray screen. Normalized to gapdh gene transcription, these three genes were upregulated after 6 to 10 days exposure of adult mice to HN001. Angptl4 was shown by immunofluorescence to be upregulated in duodenal epithelial cells of mucosal samples. Epithelial cell migration was faster in HN001-exposed mice than in the Lactobacillus-free controls. Transcriptional responses in infant mice differed according to bowel region and age. For example, sgk1 was upregulated in duodenal, jejunal, and ileal mucosa of mice less than 25 days old, whereas angptl4 and hspa1b were upregulated at 10 days in the duodenum but downregulated in the jejunal mucosa until mice were 25 days old. Overall, the results provide links between a probiotic strain, mucosal gene expression, and host phenotype, which may be useful in delineating mechanisms of probiotic action.

Smad2: A Candidate Gene for the Murine Autoimmune Diabetes Locus Idd21.1

CONTEXT Congenic NOD.ABH(D18Mit8-D18Mit214) mice, which contain greater than 12.8 Mb of DNA encompassing Idd21.1 from diabetes-resistant Biozzi/ABH mice, have a lower frequency of diabetes compared with the parental nonobese diabetic (NOD) strain, possibly due to reduced pathogenicity of β-islet-infiltrating immune cells. OBJECTIVE The objective of the study was to identify an Idd21.1 candidate gene. METHODS The methods used in the study were adoptive transfer into scid mice lacking an adaptive immune system; dendritic cell phenotyping and gene expression analysis; and fine-mapping Idd21.1 by congenic mapping. RESULTS Diabetes incidences of NOD.scid.ABH(D18Mit8-D18Mit214) mice receiving splenocytes from NOD and NOD.ABH(D18Mit8-D18Mit214) were similar to that previously observed in NOD.scid recipients, suggesting that the diabetes resistance in NOD.ABH(D18Mit8-D18Mit214) is primarily mediated by the adaptive immune system, findings supported by adoptive transfer of CD4(+) T cells. In activated dendritic cells, there were no conclusive differences in cytokine profiles and activation marker expression. However, microarray analysis comparing gene expression between activated dendritic cells from NOD and NOD.ABH (D18Mit8-D18Mit214) revealed that Smad2, in a maximal 6.5-Mb region to which Idd21.1 was further resolved by congenic mapping, was differentially expressed (increased in NOD). Quantitative real-time PCR confirmed the differential expression of Smad2, and other genes in the TGF-β signaling pathway, in activated dendritic cells. CONCLUSIONS These results implicate Smad2 as an Idd21.1 candidate and Smad2 and the TGF-β signaling pathway in activated dendritic cells in diabetogenesis. With suggestive evidence from human genome-wide association studies supporting a role for SMAD7 in human type 1 diabetes, a comprehensive genetic investigation of the SMAD genes in type 1 diabetes is warranted.

Oligonucleotide array outperforms SNP array on formalin-fixed paraffin-embedded clinical samples

Compromised quality of formalin-fixed paraffin-embedded (FFPE)-derived DNA has compounded the use of archival specimens for array-based genomic studies. Recent technological advances have led to first successes in this field; however, there is currently no general agreement on the most suitable platform for the array-based analysis of FFPE DNA. In this study, FFPE and matched fresh-frozen (FF) specimens were separately analyzed with Affymetrix single nucleotide polymorphism (SNP) 6.0 and Agilent 4x44K oligonucleotide arrays to compare the genomic profiles from the two tissue sources and to assess the relative performance of the two platforms on FFPE material. Genomic DNA was extracted from matched FFPE-FF pairs of normal intestinal epithelium from four patients and were applied to the SNP and oligonucleotide platforms according to the manufacturer-recommended protocols. On the Affymetrix platform, a substantial increase in apparent copy number alterations was observed in all FFPE tissues relative to their matched FF counterparts. In contrast, FFPE and matched FF genomic profiles obtained via the Agilent platform were very similar. Both the SNP and the oligonucleotide platform performed comparably on FF material. This study demonstrates that Agilent oligonucleotide array comparative genomic hybridization generates reliable results from FFPE extracted DNA, whereas the Affymetrix SNP-based array seems less suitable for the analysis of FFPE material.

∆133p53 isoform promotes tumour invasion and metastasis via interleukin-6 activation of JAK-STAT and RhoA-ROCK signalling.

Abstract∆122p53 mice (a model of ∆133p53 isoform) are tumour-prone, have extensive inflammation and elevated serum IL-6. To investigate the role of IL-6 we crossed ∆122p53 mice with IL-6 null mice. Here we show that loss of IL-6 reduced JAK-STAT signalling, tumour incidence and metastasis. We also show that ∆122p53 activates RhoA-ROCK signalling leading to tumour cell invasion, which is IL-6-dependent and can be reduced by inhibition of JAK-STAT and RhoA-ROCK pathways. Similarly, we show that Δ133p53 activates these pathways, resulting in invasive and migratory phenotypes in colorectal cancer cells. Gene expression analysis of colorectal tumours showed enrichment of GPCR signalling associated with ∆133TP53 mRNA. Patients with elevated ∆133TP53 mRNA levels had a shorter disease-free survival. Our results suggest that ∆133p53 promotes tumour invasion by activation of the JAK-STAT and RhoA-ROCK pathways, and that patients whose tumours have high ∆133TP53 may benefit from therapies targeting these pathways.Aberrant expression of the Δ133p53 isoform is linked to many cancers. Here, the authors utilise a model of the Δ133p53 isoform that is prone to tumours and inflammation, showing that Δ133p53 promotes tumour cell invasion by activation of the JAK-STAT and RhoA-ROCK pathways in an IL-6 dependent manner.

Heterologous hybridisation to a Pinus microarray: profiling of gene expression in Pinus radiata saplings exposed to ethephon

BackgroundThe design, construction and application of a Pinus microarray platform are described. The oligonucleotide microarray was developed using publicly available Pinus cDNA sequences mostly derived from Pinus taeda to test whether heterologous hybridisation of microarray probes will generate useful data when hybridised with cRNA constructed from the dominant New Zealand forestry species Pinus radiata.MethodsA comprehensive consensus sequence collection of Pinus cDNA sequences was collated into a non-redundant database used for automated design of 60-mer oligonucleotide microarray probes. The microarray slides, manufactured by Agilent Technologies (Palo Alto, California), were used to monitor gene expression in an induction experiment using 2-chloroethylphosphonic acid, common name ethephon and the active ingredient of the plant growth regulator Ethrel® (Bayer Crop Science). The transcriptomes from tissues of 2-year old Pinus radiata saplings +/− ethephon treatment were compared by hybridisation onto the Pinus microarray slides.ResultsStatistically significant differentially expressed genes identified by heterologous hybridisation to the Pinus microarray following ethephon induction included the up-regulation of genes in the xylem that were related to the metabolism of phenylpropanoids and flavonoids, and also defence responses, specifically against fungal/insect attack and oxidative stress. Bark, mucilaginous xylem and xylem generated largely mutually exclusive cohorts of genes and Gene Ontology (GO) classes. The results are also interpreted in reference to gross and microscopic morphological changes. Samples of gene responses were validated by quantitative RT-PCR.ConclusionThese results confirm the successful development of a Pinus microarray and demonstrate the utility of the microarray for transcriptomic research in Pinus radiata through heterologous hybridisation.