Caenorhabditis elegans Heterochromatin protein 1 (HPL-2) links developmental plasticity, longevity and lipid metabolism

Background Heterochromatin protein 1 (HP1) family proteins have a well-characterized role in heterochromatin packaging and gene regulation. Their function in organismal development, however, is less well understood. Here we used genome-wide expression profiling to assess novel functions of the Caenorhabditis elegans HP1 homolog HPL-2 at specific developmental stages. Results We show that HPL-2 regulates the expression of germline genes, extracellular matrix components and genes involved in lipid metabolism. Comparison of our expression data with HPL-2 ChIP-on-chip profiles reveals that a significant number of genes up- and down-regulated in the absence of HPL-2 are bound by HPL-2. Germline genes are specifically up-regulated in hpl-2 mutants, consistent with the function of HPL-2 as a repressor of ectopic germ cell fate. In addition, microarray results and phenotypic analysis suggest that HPL-2 regulates the dauer developmental decision, a striking example of phenotypic plasticity in which environmental conditions determine developmental fate. HPL-2 acts in dauer at least partly through modulation of daf-2/IIS and TGF-β signaling pathways, major determinants of the dauer program. hpl-2 mutants also show increased longevity and altered lipid metabolism, hallmarks of the long-lived, stress resistant dauers. Conclusions Our results suggest that the worm HP1 homologue HPL-2 may coordinately regulate dauer diapause, longevity and lipid metabolism, three processes dependent on developmental input and environmental conditions. Our findings are of general interest as a paradigm of how chromatin factors can both stabilize development by buffering environmental variation, and guide the organism through remodeling events that require plasticity of cell fate regulation.

iTRAQ identification of candidate serum biomarkers associated with metastatic progression of human prostate cancer

A major challenge in the management of patients with prostate cancer is identifying those individuals at risk of developing metastatic disease, as in most cases the disease will remain indolent. We analyzed pooled serum samples from 4 groups of patients (n = 5 samples/group), collected prospectively and actively monitored for a minimum of 5 yrs. Patients groups were (i) histological diagnosis of benign prostatic hyperplasia with no evidence of cancer 'BPH', (ii) localised cancer with no evidence of progression, 'non-progressing' (iii) localised cancer with evidence of biochemical progression, 'progressing', and (iv) bone metastasis at presentation 'metastatic'. Pooled samples were immuno-depleted of the 14 most highly abundant proteins and analysed using a 4-plex iTRAQ approach. Overall 122 proteins were identified and relatively quantified. Comparisons of progressing versus non-progressing groups identified the significant differential expression of 25 proteins (p<0.001). Comparisons of metastatic versus progressing groups identified the significant differential expression of 23 proteins. Mapping the differentially expressed proteins onto the prostate cancer progression pathway revealed the dysregulated expression of individual proteins, pairs of proteins and 'panels' of proteins to be associated with particular stages of disease development and progression. The median immunostaining intensity of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), one of the candidates identified, was significantly higher in osteoblasts in close proximity to metastatic tumour cells compared with osteoblasts in control bone (p = 0.0353, Mann Whitney U). Our proteomic approach has identified leads for potentially useful serum biomarkers associated with the metastatic progression of prostate cancer. The panels identified, including eEF1A1 warrant further investigation and validation.

Inhibition of the AKT/mTOR and erbB pathways by gefitinib, perifosine and analogs of gonadotropin-releasing hormone I and II to overcome tamoxifen resistance in breast cancer cells

Endocrine resistance in breast cancer remains a major clinical problem and is caused by crosstalk mechanisms of growth factor receptor cascades, such as the erbB and PI3K/AKT pathways. The possibilities a single breast cancer cell has to achieve resistance are manifold. We developed a model of 4-hydroxy-tamoxifen (OHT)‑resistant human breast cancer cell lines and compared their different expression patterns, activation of growth factor receptor pathways and compared cells by genomic hybridization (CGH). We also tested a panel of selective inhibitors of the erbB and AKT/mTOR pathways to overcome OHT resistance. OHT‑resistant MCF-7-TR and T47D-TR cells showed increased expression of HER2 and activation of AKT. T47D-TR cells showed EGFR expression and activated MAPK (ERK-1/2), whereas in resistant MCF-7-TR cells activated AKT was due to loss of CTMP expression. CGH analyses revealed remarkable aberrations in resistant sublines, which were predominantly depletions. Gefitinib inhibited erbB signalling and restored OHT sensitivity in T47D-TR cells. The AKT inhibitor perifosine restored OHT sensitivity in MCF-7-TR cells. All cell lines showed expression of receptors for gonadotropin-releasing hormone (GnRH) I and II, and analogs of GnRH-I/II restored OHT sensitivity in both resistant cell lines by inhibition of erbB and AKT signalling. In conclusion, mechanisms to escape endocrine treatment in breast cancer share similarities in expression profiling but are based on substantially different genetic aberrations. Evaluation of activated mediators of growth factor receptor cascades is helpful to predict response to specific inhibitors. Expression of GnRH-I/II receptors provides multi-targeting treatment strategies.

Met-thodology

Met-regulated expression signature defines a subset of human hepatocellular carcinomas with poor prognosis and aggressive phenotype. Kaposi-Novak P, Lee JS, Gomez-Quiroz L, Coulouarn C, Factor VM, Thorgeirsson SS. Identification of specific gene expression signatures characteristic of oncogenic pathways is an important step toward molecular classification of human malignancies. Aberrant activation of the Met signaling pathway is frequently associated with tumour progression and metastasis. In this study, we defined the Met-dependent gene expression signature using global gene expression profiling of WT and Met-deficient primary mouse hepatocytes. Newly identified transcriptional targets of the Met pathway included genes involved in the regulation of oxidative stress responses as well as cell motility, cytoskeletal organization, and angiogenesis. To assess the importance of a Met-regulated gene expression signature, a comparative functional genomic approach was applied to 242 human hepatocellular carcinomas (HCCs) and 7 metastatic liver lesions. Cluster analysis revealed that a subset of human HCCs and all liver metastases shared the Met-induced expression signature. Furthermore, the presence of the Met signature showed significant correlation with increased vascular invasion rate and microvessel density as well as with decreased mean survival time of HCC patients. We conclude that the genetically defined gene expression signatures in combination with comparative functional genomics constitute an attractive paradigm for defining both the function of oncogenic pathways and the clinically relevant subgroups of human cancers. [Abstract reproduced by permission of J Clin Invest 2006;116:1582-1595].

The quantitative genetic basis of offspring solicitation and parental response in a passerine bird with biparental care

The coevolution of parental investment and offspring solicitation is driven by partly different evolutionary interests of genes expressed in parents and their offspring. In species with biparental care, the outcome of this conflict ma!: be influenced by the sexual conflict over parental investment, Models for the resolution of such family conflicts have made so far untested assumptions about genetic variation and covariation in the parental resource provisioning response and the level of offspring solicitation. Using a combination of cross-fostering and begging playback experiments, we show that, in the great tit (Parus major), (i) the begging call intensity of nestlings depends on their common origin, suggesting genetic variation for this begging display, (ii) only mothers respond to begging calls by increased food provisioning, and (iii! the size of the parental response is positively related to the begging call intensity of nestlings in the maternal but not paternal line. This study indicates that genetic covariation, its differential expression in the maternal and paternal lines and/or early environmental and parental effects need to be taken into account when predicting the phenotypic outcome of the conflict over investment between genes expressed in each parent and the offspring. [References: 36]

Establishment of murine cell lines by constitutive and conditional immortalization

Mouse cell lines were immortalized by introduction of specific immortalizing genes. Embryonic and adult animals and an embryonal stem cell line were used as a source of primary cells. The immortalizing genes were either introduced by DNA transfection or by ecotropic retrovirus transduction. Fibroblasts were obtained by expression of SV40 virus large T antigen (TAg). The properties of the resulting fibroblast cell lines were reproducible, independent of the donor mouse strains employed and the cells showed no transformed properties in vitro and did not form tumors in vivo. Endothelial cell lines were generated by Polyoma virus middle T antigen expression in primary embryonal cells. These cell lines consistently expressed relevant endothelial cell surface markers. Since the expression of the immortalizing genes was expected to strongly influence the cellular characteristics fibroblastoid cells were reversibly immortalized by using a vector that allows conditional expression of the TAg. Under inducing conditions, these cells exhibited properties that were highly similar to the properties of constitutively immortalized cells. In the absence of TAg expression, cell proliferation stops. Cell growth is resumed when TAg expression is restored. Gene expression profiling indicates that TAg influences the expression levels of more than 1000 genes that are involved in diverse cellular processes. The data show that conditionally immortalized cell lines have several advantageous properties over constitutively immortalized cells.

Gap junction channels and cardiac impulse propagation

The role of gap junction channels on cardiac impulse propagation is complex. This review focuses on the differential expression of connexins in the heart and the biophysical properties of gap junction channels under normal and disease conditions. Structural determinants of impulse propagation have been gained from biochemical and immunocytochemical studies performed on tissue extracts and intact cardiac tissue. These have defined the distinctive connexin coexpression patterns and relative levels in different cardiac tissues. Functional determinants of impulse propagation have emerged from electrophysiological experiments carried out on cell pairs. The static properties (channel number and conductance) limit the current flow between adjacent cardiomyocytes and thus set the basic conduction velocity. The dynamic properties (voltage-sensitive gating and kinetics of channels) are responsible for a modulation of the conduction velocity during propagated action potentials. The effect is moderate and depends on the type of Cx and channel. For homomeric-homotypic channels, the influence is small to medium; for homomeric-heterotypic channels, it is medium to strong. Since no data are currently available on heteromeric channels, their influence on impulse propagation is speculative. The modulation by gap junction channels is most prominent in tissues at the boundaries between cardiac tissues such as sinoatrial node-atrial muscle, atrioventricular node-His bundle, His bundle-bundle branch and Purkinje fibers-ventricular muscle. The data predict facilitation of orthodromic propagation.

The 14-bp deletion polymorphism in the HLA-G gene displays significant differences between ulcerative colitis and Crohn's disease and is associated with ileocecal resection in Crohn's disease

HLA-G is a non-classical MHC class Ib molecule predominantly expressed in cytotrophoblasts and under pathological conditions also in chronically inflamed and in malignant tissues. Recently an increased expression of HLA-G was found in ulcerative colitis (UC), but not in Crohn's disease (CD). The HLA-G gene is located in IBD3, a linkage region for inflammatory bowel disease (IBD). A 14-bp deletion polymorphism (Del+/Del-) within exon 8 of the HLA-G gene might influence transcription activity and is therefore of potential functional relevance. To investigate whether the 14-bp deletion polymorphism is associated with IBD, 371 patients with CD, 257 patients with UC and 739 controls were genotyped. The heterozygous genotype (P = 0.031) and the Del+ phenotype (P = 0.038) were significantly increased, whereas the homozygous Del- phenotype (P = 0.038) was significantly decreased in UC when compared with CD. Thus, the 14-bp deletion polymorphism within the HLA-G gene displayed significant differences between UC and CD. Moreover, a significant increase of the Del+ allele (P = 0.002) and the Del+/Del+ genotype (P = 0.013) and a consecutive decrease of the Del-/- genotype (P = 0.024) were observed in those CD cases positive for ileocecal resection. Thus, a potential effect of the HLA-G gene in IBD may affect both UC and CD. Other polymorphisms linked to the 14-bp deletion polymorphism might also contribute to immunopathogenesis. As there are several partly functional polymorphisms within the promoter region potentially influencing HLA-G expression, further studies in IBD are necessary in the context of differential expression of HLA-G between UC and CD.

Clinical significance of cell cycle- and apoptosis-related markers in biliary tract cancer: a tissue microarray-based approach revealing a distinctive immunophenotype for intrahepatic and extrahepatic cholangiocarcinomas

Cholangiocarcinoma is the second most common malignant tumor of the liver. We analyzed, immunohistochemically, the significance of cell cycle- and apoptosis-related markers in 128 cholangiocarcinomas (42 intrahepatic, 70 extrahepatic, and 16 gallbladder carcinomas) combined in a tissue microarray. Follow-up was available for 57 patients (44.5%). In comparison with normal tissue (29 specimens), cholangiocarcinomas expressed significantly more frequently p53, bcl-2, bax, and COX-2 (P.05 <). Intrahepatic tumors were significantly more frequently bcl-2+ and p16+, whereas extrahepatic tumors were more often p53+ (P < .05). Loss of p16 expression was associated with reduced survival of patients. Our data show that p53, bcl-2, bax, and COX-2 have an important role in the pathogenesis of cholangiocarcinomas. The differential expression of p16, bcl-2, and p53 between intrahepatic and extrahepatic tumors demonstrates that there are location-related differences in the phenotype and the genetic profiles of these tumors. Moreover, p16 was identified as an important prognostic marker in cholangiocarcinomas.

Reduction in rat phosphatidylethanolamine binding protein-1 (PEBP1) after chronic corticosterone treatment may be paralleled by cognitive impairment: a first study

Chronic stress is associated with hippocampal atrophy and cognitive dysfunction. This study investigates how long-lasting administration of corticosterone as a mimic of experimentally induced stress affects psychometric performance and the expression of the phosphatidylethanolamine binding protein (PEBP1) in the adult hippocampus of one-year-old male rats. Psychometric investigations were conducted in rats before and after corticosterone treatment using a holeboard test system. Rats were randomly attributed to 2 groups (n = 7) for daily subcutaneous injection of either 26.8 mg/kg body weight corticosterone or sesame oil (vehicle control). Treatment was continued for 60 days, followed by cognitive retesting in the holeboard system. For protein analysis, the hippocampal proteome was separated by 2D electrophoresis (2DE) followed by image processing, statistical analysis, protein identification via peptide mass fingerprinting and gel matching and subsequent functional network mapping and molecular pathway analysis. Differential expression of PEBP1 was additionally quantified by Western blot analysis. Results show that chronic corticosterone significantly decreased rat hippocampal PEBP1 expression and induced a working and reference memory dysfunction. From this, we derive the preliminary hypothesis that PEBP1 may be a novel molecular mediator influencing cognitive integrity during chronic corticosterone exposure in rat hippocampus.

Strategies to determine the biological function of microRNAs

MicroRNAs (miRNAs) are regulators of gene expression that control many biological processes in development, differentiation, growth and metabolism. Their expression levels, small size, abundance of repetitive copies in the genome and mode of action pose unique challenges in studies elucidating the function of miRNAs. New technologies for identification, expression profiling and target gene validation, as well as manipulation of miRNA expression in vivo, will facilitate the study of their contribution to biological processes and disease. Such information will be crucial to exploit the emerging knowledge of miRNAs for the development of new human therapeutic applications.

Integrative analysis of RUNX1 downstream pathways and target genes

BACKGROUND: The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia. RESULTS: Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFbeta, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFbeta. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes. CONCLUSION: This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications.

MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses

OBJECTIVE: MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue-specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA-140 (miR-140). METHODS: To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR-140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin-1beta (IL-1beta) on miR-140 expression. Double-stranded miR-140 (ds-miR-140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA. RESULTS: Microarray analysis showed that miR-140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR-140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR-140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL-1beta suppressed miR-140 expression. Transfection of chondrocytes with ds-miR-140 down-regulated IL-1beta-induced ADAMTS5 expression and rescued the IL-1beta-dependent repression of AGGRECAN gene expression. CONCLUSION: This study shows that miR-140 has a chondrocyte differentiation-related expression pattern. The reduction in miR-140 expression in OA cartilage and in response to IL-1beta may contribute to the abnormal gene expression pattern characteristic of OA.

Selecting control genes for RT-QPCR using public microarray data

BACKGROUND: Gene expression analysis has emerged as a major biological research area, with real-time quantitative reverse transcription PCR (RT-QPCR) being one of the most accurate and widely used techniques for expression profiling of selected genes. In order to obtain results that are comparable across assays, a stable normalization strategy is required. In general, the normalization of PCR measurements between different samples uses one to several control genes (e.g. housekeeping genes), from which a baseline reference level is constructed. Thus, the choice of the control genes is of utmost importance, yet there is not a generally accepted standard technique for screening a large number of candidates and identifying the best ones. RESULTS: We propose a novel approach for scoring and ranking candidate genes for their suitability as control genes. Our approach relies on publicly available microarray data and allows the combination of multiple data sets originating from different platforms and/or representing different pathologies. The use of microarray data allows the screening of tens of thousands of genes, producing very comprehensive lists of candidates. We also provide two lists of candidate control genes: one which is breast cancer-specific and one with more general applicability. Two genes from the breast cancer list which had not been previously used as control genes are identified and validated by RT-QPCR. Open source R functions are available at http://www.isrec.isb-sib.ch/~vpopovic/research/ CONCLUSION: We proposed a new method for identifying candidate control genes for RT-QPCR which was able to rank thousands of genes according to some predefined suitability criteria and we applied it to the case of breast cancer. We also empirically showed that translating the results from microarray to PCR platform was achievable.

Age-dependent suppression of SERCA2a mRNA in pediatric atrial myocardium

Differential expression of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) and phospholamban (PLB) has been shown in heart failure and atrial arrhythmias. We investigated the influence of volume overload and age on their expression in pediatric atrial myocardium. Right atrial specimens from 18 children with volume overloaded right atrium (VO) and 12 patients without overload were studied. Each group was further divided into patients less than and older than 12 months of age. Only in the younger patients SERCA2a was significantly reduced in the VO group. In younger patients PLB mRNA level tended to be lower in VO. The PLB:SERCA protein ratio was significantly reduced in the VO group. Age itself did not influence the SERCA2a and PLB expression, if the hemodynamic overload was not taken into account. This study is the first to show a combined influence of volume overload and age on atrial SERCA2a expression.