Differences in the endometrial transcript profile during the receptive period between women who were refractory to implantation and those who achieved pregnancy.

BACKGROUND: Gene expression profiling of normal receptive endometrium has been characterized, but intrinsic defects in endometrial gene expression associated with implantation failure have not been reported. METHODS: Women who had previously participated as recipients in oocyte donation cycles and repeatedly exhibited implantation failure (Group A, study group) or had at least one successful cycle (Group B, control group) and spontaneously fertile women (Group C, normal fertility group) were recruited. All were treated with exogenous estradiol and progesterone to induce an endometrial cycle, and an endometrial biopsy was taken on the seventh day of progesterone administration. RNA from each sample was analysed by cDNA microarrays to identify differentially expressed genes between groups. RESULTS: 63 transcripts were differentially expressed (>or=2-fold) between Groups A and B, of which 16 were subjected to real time RT-PCR. Eleven of these were significantly decreased in Group A with regard to Groups B and C. Among the dysregulated genes were MMP-7, CXCR4, PAEP and C4BPA. CONCLUSIONS: Repeated implantation failure in some oocyte recipients is associated with an intrinsic defect in the expression of multiple genes in their endometrium. Significantly decreased levels of several transcripts in endometria without manifest abnormalities is demonstrated for the first time and shown to be associated with implantation failure.

WebaCGH: an interactive online tool for the analysis and display of array comparative genomic hybridisation data.

UNLABELLED: Gene copy number variations occur both in normal cells and in numerous pathologies including cancer and developmental diseases. Array comparative genomic hybridisation (aCGH) is an emerging technology that allows detection of chromosomal gains and losses in a high-resolution format. When aCGH is performed on cDNA and oligonucleotide microarrays, the impact of DNA copy number on gene transcription profiles may be directly compared. We have created an online software tool, WebaCGH, that functions to (i) upload aCGH and gene transcription results from multiple experiments; (ii) identify significant aberrant regions using a local Z-score threshold in user-selected chromosomal segments subjected to smoothing with moving averages; and (iii) display results in a graphical format with full genome and individual chromosome views. In the individual chromosome display, data can be zoomed in/out in both dimensions (i.e. ratio and physical location) and plotted features can have 'mouse over' linking to outside databases to identify loci of interest. Uploaded data can be stored indefinitely for subsequent retrieval and analysis. WebaCGH was created as a Java-based web application using the open-source database MySQL. AVAILABILITY: WebaCGH is freely accessible at http://129.43.22.27/WebaCGH/welcome.htm CONTACT: Xiaolin Wu (forestwu@mail.nih.gov) or Ulises Urzúa (uurzua@med.uchile.cl).

Transcriptomic analysis of an in vitro murine model of ovarian carcinoma: functional similarity to the human disease and identification of prospective tumoral markers and targets.

Ovarian cancer is an aggressive disease of poor prognostic when detected at advanced stage. It is widely accepted that the ovarian surface epithelium plays a central role in disease etiology, but little is known about disease progression at the molecular level. To identify genes involved in ovarian tumorigenesis, we carried out a genome-wide transcriptomic analysis of six spontaneously transformed mouse ovarian surface epithelial (MOSE) cell lines, an in vitro model for human ovarian carcinoma. Loess normalization followed by statistical analysis with control of multiple testing resulted in 509 differentially expressed genes using an adjusted P-value < or = 0.05 as cut-off. The top 20 differentially expressed genes included 10 genes (Spp1, Cyp1b1, Btg1, Cfh, Mt1, Mt2, Igfbp5, Gstm1, Gstm2, and Esr1) implicated in various aspects of ovarian carcinomas, and other 3 genes (Gsto1, Lcn7, and Alcam) associated to breast cancer. Upon functional analysis, the majority of alterations affected genes involved in glutathione metabolism and MAPK signaling pathways. Interestingly, over 20% of the aberrantly expressed genes were related to extracellular components, suggestive of potential markers of disease progression. In addition, we identified the genes Pura, Cnn3, Arpc1b, Map4k4, Tgfb1i4, and Crsp2 correlated to in vivo tumorigenic parameters previously reported for these cells. Taken together, our findings support the utility of MOSE cells in studying ovarian cancer biology and as a source of novel diagnostic and therapeutic targets.

A distinct and unique transcriptional program expressed by tumor-associated macrophages (defective NF-kappaB and enhanced IRF-3/STAT1 activation).

To identify the molecular basis underlying the functions of tumor-associated macrophages (TAMs), we characterized the gene expression profile of TAMs isolated from a murine fibrosarcoma in comparison with peritoneal macrophages (PECs) and myeloid suppressor cells (MSCs), using a cDNA microarray technology. Among the differentially expressed genes, 15 genes relevant to inflammation and immunity were validated by real-time polymerase chain reaction (PCR) and protein production. Resting TAMs showed a characteristic gene expression pattern with higher expression of genes coding for the immunosuppressive cytokine IL-10, phagocytosis-related receptors/molecules (Msr2 and C1q), and inflammatory chemokines (CCL2 and CCL5) as expected, as well as, unexpectedly, IFN-inducible chemokines (CXCL9, CXCL10, CXCL16). Immunohistology confirmed and extended the in vitro analysis by showing that TAMs express M2-associated molecules (eg, IL-10 and MGL1), as well as CCL2, CCL5, CXCL9, CXCL10, and CXCL16, but no appreciable NOS2. Lipopolysaccharide (LPS)-mediated activation of TAMs resulted in defective expression of several proinflammatory cytokines (eg, IL-1beta, IL-6, TNF-alpha) and chemokines (eg, CCL3), as opposed to a strong up-regulation of immunosuppressive cytokines (IL-10, TGFbeta) and IFN-inducible chemokines (CCL5, CXCL9, CXCL10, CXCL16). Thus, profiling of TAMs from a murine sarcoma revealed unexpected expression of IFN-inducible chemokines, associated with an M2 phenotype (IL-10high, IL-12low), and divergent regulation of the NF-kappaB versus the IRF-3/STAT1 pathway.

Gene expression profiles of serous, endometrioid, and clear cell subtypes of ovarian and endometrial cancer.

PURPOSE: The presence of similar histologic subtypes of epithelial ovarian and endometrial cancers has long been noted, although the relevance of this finding to pathogenesis and clinical management is unclear. Despite similar clinical characteristics, histologic subtypes of cancers of the ovary and endometrium are treated according to organ of origin. This study compares the gene expression profiles of analogous histologic subtypes of cancers of the ovary and endometrium using the same genomic platform to determine the similarities and differences between these tumors. EXPERIMENTAL DESIGN: Gene expression profiles of 75 cancers (endometrioid, serous, and clear cell) of the ovary and endometrium, five renal clear cell cancers, and seven normal epithelial brushings were determined using a 11,000-element cDNA array. All images were analyzed using BRB ArrayTools. Validation was done using real-time PCR on select genes and immunohistochemical staining. RESULTS: Comparison across endometrial and ovarian cancers and serous and endometrioid tumors showed expression patterns reflecting their organ of origin. Clear cell tumors, however, showed remarkably similar expression patterns regardless of their origin, even when compared with renal clear cell samples. A set of 43 genes was common to comparisons of each of the three histologic subtypes of ovarian cancer with normal ovarian surface epithelium. CONCLUSIONS: The comparison of the gene expression profiles of endometrioid and serous subtypes of ovarian and endometrial cancer are largely unique to the combination of a particular subtype in a specific organ. In contrast, clear cell cancers show a remarkable similarity in gene expression profiles across organs (including kidney) and could not be statistically distinguished.

Microarray comparative genomic hybridization profile of a murine model for epithelial ovarian cancer reveals genomic imbalances resembling human ovarian carcinomas.

Microarray comparative genomic hybridization (mCGH) is emerging as a high-resolution technology to detect gene dosage alterations in numerous pathologies, including cancer. We optimized cDNA microarrays to identify genome-wide imbalances in spontaneously transformed mouse ovarian surface epithelial cell lines, an in vitro murine model for ovarian cancer. Amplification of chromosome 19 and a more variable gain pattern of chromosomes 15 and 5 were detected and independently validated using conventional metaphase CGH. In addition, cryptic aberrations in segments of chromosomes 4, 7, 8, 9, 11, 17, and X, allowed identification of 2 related genomic variants among six cell lines studied. Mouse-human synteny revealed an overall early transformation stage with approximately 80% conservation relative to human ovarian malignancies of epithelial origin including low malignant potential tumors, serous carcinoma, and carcinoma cell lines. Importantly, three of the cells bear gained segments 13 and 41 Mbp length of chromosomes 5 and 15, respectively, which are syntenic to human 22q11-13, 8q24 and 12p11-q24, the two latter chromosomal regions thought to define one pathway of karyotypic changes in the development of human ovarian tumors. Our findings support the utility of mouse ovarian surface epithelial (MOSE) cells in studying initiation and progression of human ovarian cancer and as a suitable model to evaluate therapeutic approaches.

Three microarray platforms: an analysis of their concordance in profiling gene expression.

BACKGROUND: Microarrays for the analysis of gene expression are of three different types: short oligonucleotide (25-30 base), long oligonucleotide (50-80 base), and cDNA (highly variable in length). The short oligonucleotide and cDNA arrays have been the mainstay of expression analysis to date, but long oligonucleotide platforms are gaining in popularity and will probably replace cDNA arrays. As part of a validation study for the long oligonucleotide arrays, we compared and contrasted expression profiles from the three formats, testing RNA from six different cell lines against a universal reference standard. RESULTS: The three platforms had 6430 genes in common. In general, correlation of gene expression levels across the platforms was good when defined by concordance in the direction of expression difference (upregulation or downregulation), scatter plot analysis, principal component analysis, cell line correlation or quantitative RT-PCR. The overall correlations (r values) between platforms were in the range 0.7 to 0.8, as determined by analysis of scatter plots. When concordance was measured for expression ratios significant at p-values of <0.05 and at expression threshold levels of 1.5 and 2-fold, the agreement among the platforms was very high, ranging from 93% to 100%. CONCLUSION: Our results indicate that the long oligonucleotide platform is highly suitable for expression analysis and compares favorably with the cDNA and short oligonucleotide varieties. All three platforms can give similar and reproducible results if the criterion is the direction of change in gene expression and minimal emphasis is placed on the magnitude of change.

Inhibition of melanoma growth and metastasis by ATF2-derived peptides.

The resistance of melanoma to apoptosis, as well as its growth and metastasis capabilities, can be overcome by expression of a peptide derived from amino acid (aa) 51 to 100 of ATF2. Here we show that expression of ATF2((51-100)) in human melanoma cells reduced their growth in nude mice, which was additionally inhibited upon treatment with protein kinase inhibitors UCN-01 or SB203580. Injection of a fusion protein consisting of HIV-TAT and aa 51 to 100 of ATF2 into SW1 melanomas efficiently inhibits their growth and their metastasis up to complete regression. Additionally, expression of a 10aa peptide that corresponds to aa 51 to 60 of ATF2 sensitizes melanoma cells to spontaneous apoptosis, which coincides with activation of caspase 9 and poly(ADP-ribose) polymerase cleavage, and inhibit their growth in vivo. The 10aa peptide increases the association of c-Jun NH(2)-terminal kinase with c-Jun but not with ATF2, resulting in concomitant increase in TRE-mediated transcription. Our study points to mechanisms underlying the activities of the ATF2 peptide while highlighting its possible use in drug design.

Lsh, an epigenetic guardian of repetitive elements.

The genome is burdened with repetitive sequences that are generally embedded in silenced chromatin. We have previously demonstrated that Lsh (lymphoid-specific helicase) is crucial for the control of heterochromatin at pericentromeric regions consisting of satellite repeats. In this study, we searched for additional genomic targets of Lsh by examining the effects of Lsh deletion on repeat regions and single copy gene sequences. We found that the absence of Lsh resulted in an increased association of acetylated histones with repeat sequences and transcriptional reactivation of their silenced state. In contrast, selected single copy genes displayed no change in histone acetylation levels, and their transcriptional rate was indistinguishable compared to Lsh-deficient cells and wild-type controls. Microarray analysis of total RNA derived from brain and liver tissues revealed that <0.4% of the 15 247 examined loci were abnormally expressed in Lsh-/-embryos and almost two-thirds of these deregulated sequences contained repeats, mainly retroviral LTR (long terminal repeat) elements. Chromatin immunoprecipitation analysis demonstrated a direct interaction of Lsh with repetitive sites in the genome. These data suggest that the repetitive sites are direct targets of Lsh action and that Lsh plays an important role as 'epigenetic guardian' of the genome to protect against deregulation of parasitic retroviral elements.

An ATF2-derived peptide sensitizes melanomas to apoptosis and inhibits their growth and metastasis.

Melanomas are among the aggressive tumor types because of their notorious resistance to treatment and their high capacity to metastasize. ATF2 is among transcription factors implicated in the progression of melanoma and its resistance to treatment. Here we demonstrate that the expression of a peptide spanning amino acids 50-100 of ATF2 (ATF2(50-100)) reduces ATF2 transcriptional activities while increasing the expression and activity of c-Jun. Altering the balance of Jun/ATF2 transcriptional activities sensitized melanoma cells to apoptosis, an effect that could be attenuated by inhibiting c-Jun. Inhibition of ATF2 via RNA interference likewise increased c-Jun expression and primed melanoma cells to undergo apoptosis. Growth and metastasis of SW1 and B16F10 mouse melanomas were inhibited by ATF2(50-100) to varying degrees up to a complete regression, depending on the mode (inducible, constitutive, or adenoviral delivery) of its expression.

Initiating oncogenic event determines gene-expression patterns of human breast cancer models.

Molecular expression profiling of tumors initiated by transgenic overexpression of c-myc, c-neu, c-ha-ras, polyoma middle T antigen (PyMT) or simian virus 40 T/t antigen (T-ag) targeted to the mouse mammary gland have identified both common and oncogene-specific events associated with tumor formation and progression. The tumors shared great similarities in their gene-expression profiles as compared with the normal mammary gland with an induction of cell-cycle regulators, metabolic regulators, zinc finger proteins, and protein tyrosine phosphatases, along with the suppression of some protein tyrosine kinases. Selection and hierarchical clustering of the most variant genes, however, resulted in separating the mouse models into three groups with distinct oncogene-specific patterns of gene expression. Such an identification of targets specified by particular oncogenes may facilitate development of lesion-specific therapeutics and preclinical testing. Moreover, similarities in gene expression between human breast cancers and the mouse models have been identified, thus providing an important component for the validation of transgenic mammary cancer models.

The feasibility of using fine needle aspiration from primary breast cancers for cDNA microarray analyses.

PURPOSE: Our aims in this pilot study were to determine whether fine needle aspirates (FNAs) provide a sufficient quantity of mRNA for cDNA microarray analysis, produce a set of quality control criteria to accept individual arrays, and determine whether gene expression profiles obtained from FNAs were representative of the source tumor. EXPERIMENTAL DESIGN: Twenty-seven women with breast cancer for treatment with primary surgery had a FNA before and at the time of surgery, and a portion of excised tumor was taken for array analysis. Control experiments were performed using two Ewing's sarcoma xenograft models. mRNA was extracted from the samples and hybridized with the reference (MCF7 cell line) on cDNA microarrays. Statistical methods were applied to identify acceptability criteria for the arrays. RESULTS: Statistical analyses demonstrated that an adequate array could be identified by calculating the SD of the log of fluorescence intensities from the arrays. Using this criterion, only 4 of the 27 patients (15%) had FNA samples suitable for array analysis. Gene expression profiles from the FNAs closely resembled that of the corresponding source tumors and were clearly distinguished from FNAs derived from the xenografts. CONCLUSIONS: SD is a useful quality index for the clinical application of cDNA microarrays. This "proof of principle" study demonstrates that FNAs from primary breast cancers can be used for microarray analysis, although without amplification, it is feasible in only a small proportion of patients. For this to be clinically useful, validated amplification techniques for FNA samples are probably required.