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

∆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.

Does the endometrial gene expression of fertile women vary within and between cycles?

STUDY QUESTION: Does gene expression of putative endometrial implantation markers vary in expression between menstrual cycles? SUMMARY ANSWER: In fertile women the expression of certain genes exhibits a pattern of stable regulation.which is not affected even when sampled twice in one cycle. WHAT IS KNOWN ALREADY: Successful implantation occurs in a minority of IVF embryo transfers. In contrast to knowledge regarding the ovulatory process, there is a sparse understanding of endometrial genes critical to implantation. This lack of knowledge hinders progress in this field. STUDY DESIGN, SIZE, DURATION: Endometrial pipelle samples were collected based on blood endocrinological markers at 2 and 7 days post initial LH surge. Five samples were collected over four cycles where the interval between collections ranged from sequential months to three years. PARTICIPANTS/MATERIALS, SETTING, METHODS: Six fertile women attending an IVF clinic for male factor infertility, had samples collected. Global gene expression profiles were obtained from laser-microdissected, endometrial glands and stroma. Nineteen potential proliferation, cytokine and adhesion markers based on previous validated reports were studied. MAIN RESULTS AND THE ROLE OF CHANCE: There was a significant modification between LH+2 and LH+7 of expression for 23 genes-11 in 8 in glands and stroma, 4 in stroma only and 3 in glands only suggesting stable, controlled regulation. Nevertheless, genes exhibited individual characteristics, e.g MKI67 exhibited lower expression at LH+7 than LH+2 and CCL4 higher, whereas TRO expressed limited difference in both cell types. Stability between cycles was demonstrated for gene expression at both LH+2-more than 60% of genes had <25% variation and at LH+7-60% had <30% variation. Further, effects of prior collection of an LH+2 sample on gene expression at LH+7 were not detected. The range of mRNA expression suggested that a clinical/diagnostic sample at LH+2 and LH+7 is likely to be a better index of endometrial function than a single sample. The possibility of redundancy suggests a panel would be more informative than a single marker. LARGE SCALE DATA: Raw and normalized microarray data have been deposited with the EMBL's European Genome-Phenome Archive for collaborative analysis, reference ega-box-815 (Lappalainen I, Almeida-King J, Kumanduri V, Senf A, Spalding JD, Ur-Rehman S, Saunders G, Kandasamy J, Caccamo M, Leinonen R et al. The European Genome-phenome Archive of human data consented for biomedical research. Nat Genet 2015;47:692-695.) [https://www.ebi.ac.uk/ega/home]. LIMITATIONS, REASONS FOR CAUTION: This type of research has difficulties of recruitment of fertile women for multiple blood testing and repeat endometrial biopsies. Therefore, these data had decreased statistical power due to the overall participant numbers. However, the inclusion of four cycles for each participant permitted the aim of obtaining information on intercycle and intracycle variability to be achieved. WIDER IMPLICATIONS OF THE FINDINGS: Our results support the feasibility of a clinical means of identification of a functional receptive endometrium. The robustness of data from individual women suggests that samples from one cycle can generally be applied to subsequent cycles. STUDY FUNDING/COMPETING INTEREST(S): Funding was granted from the Tertiary Education Commission of New Zealand, Contract I.D.:UOOX06007. There are no competing interests.

Characterization of the cyanobacteria and associated bacterial community from an ephemeral wetland in New Zealand.

New Zealand ephemeral wetlands are ecologically important, containing up to 12% of threatened native plant species and frequently exhibiting conspicuous cyanobacterial growth. In such environments, cyanobacteria and associated heterotrophs can influence primary production and nutrient cycling. Wetland communities, including bacteria, can be altered by increased nitrate and phosphate due to agricultural practices. We have characterized cyanobacteria from the Wairepo Kettleholes Conservation Area and their associated bacteria. Use of 16S rRNA amplicon sequencing identified several operational taxonomic units (OTUs) representing filamentous heterocystous and non-heterocystous cyanobacterial taxa. One Nostoc OTU that formed macroscopic colonies dominated the cyanobacterial community. A diverse bacterial community was associated with the Nostoc colonies, including a core microbiome of 39 OTUs. Identity of the core microbiome associated with macroscopic Nostoc colonies was not changed by the addition of nutrients. One OTU was highly represented in all Nostoc colonies (27.6%-42.6% of reads) and phylogenetic analyses identified this OTU as belonging to the genus Sphingomonas. Scanning electron microscopy showed the absence of heterotrophic bacteria within the Nostoc colony but revealed a diverse community associated with the colonies on the external surface.

Altered transcription of murine genes induced in the small bowel by administration of probiotic strain Lactobacillus rhamnosus HN001.

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.

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.

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.

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 ß-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-ß signaling pathway, in activated dendritic cells. CONCLUSIONS: These results implicate Smad2 as an Idd21.1 candidate and Smad2 and the TGF-ß 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.

Heterogeneous gene expression changes in colorectal cancer cells share the WNT pathway in response to growth suppression by APHS-mediated COX-2 inhibition.

Cyclooxygenase-2 (COX-2), the prostaglandin (PG)-synthesizing enzyme overexpressed in colorectal cancer (CRC), has pleiotropic, cancer-promoting effects. COX-2 inhibitors (CIBs) interfere with many cancer-associated processes and show promising antineoplastic activity, however, a common mechanism of CIB action has not yet been established. We therefore investigated by microarray the global response towards the CIB APHS at a dose significantly inhibiting the growth of three COX-2-positive CRC but not of two COX-2-negative cell lines. None of the genes significantly (p = 0.005) affected by APHS were common to all three cell lines and 83% of the altered pathways were cell line-specific. Quantitative polymerase chain reaction (QPCR) on selected pathways confirmed cell line-specific expression alterations induced by APHS. A low stringency data analysis approach using BRB array tools coupled with QPCR, however, identified small expression changes shared by all COX-2-positive cell lines in genes related to the WNT pathway, the key driver of colonic carcinogenesis. Our data indicates a substantial cell line-specificity of APHS-induced expression alterations in CRC cells and helps to explain the divergent effects reported for CIBs. Further, the shared inhibition of the WNT pathway by APHS suggests one potential common mechanism behind the antineoplastic effects of COX-2 inhibition.

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 ( approximately 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.