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PubMed2006

High-throughput RNAi screening by time-lapse imaging of live human cells.

RNA interference (RNAi) is a powerful tool to study gene function in cultured cells. Transfected cell microarrays in principle allow high-throughput phenotypic analysis after gene knockdown by microscopy. But bottlenecks in imaging and data analysis have limited such high-content screens to endpoint assays in fixed cells and determination of global parameters such as viability. Here we have overcome these limitations and developed an automated platform for high-content RNAi screening by time-lapse fluorescence microscopy of live HeLa cells expressing histone-GFP to report on chromosome segregation and structure. We automated all steps, including printing transfection-ready small interfering RNA (siRNA) microarrays, fluorescence imaging and computational phenotyping of digital images, in a high-throughput workflow. We validated this method in a pilot screen assaying cell division and delivered a sensitive, time-resolved phenoprint for each of the 49 endogenous genes we suppressed. This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens.

Automation↗
PubMed2006

Gene expression profile analysis of an isogenic tumour metastasis model reveals a functional role for oncogene AF1Q in breast cancer metastasis.

To study the molecular mechanisms underlying breast cancer metastasis, gene expression profile analysis was performed on two well-established breast cancer cell lines with high and low metastatic potentials: MDA-MB-435HM and MDA-MB-435LM. The analysis was conducted using cDNA microarrays containing 8000 genes. Of 60 differentially expressed genes, ALL1-fused gene from chromosome 1q (AF1Q), a putative oncogene not described previously in breast cancer, was identified and found to be over-expressed in MDA-MB-435HM cells compared with MDA-MB-435LM cells. The results indicate that AF1Q may play an important role in breast cancer metastasis. To test this hypothesis, we generated an AF1Q high-expression cell line by stable transfection of AF1Q cDNA into MDA-MB-435LM cells. Results showed that over-expression of AF1Q led to a marked increase in the invasive and metastatic potential of MDA-MB-435LM cells in vitro and in vivo, accompanied by the up-regulation of matrix metalloproteinase-2 (MMP-2), MMP-9, transcription factor Ets-1, and RhoC expression in both mRNA and protein levels. Consistent with this observation, reduced AF1Q expression in MDA-MB-435HM cells by small interfering RNA (siRNA) resulted in a significant decrease in the invasive potential of MDA-MB-435HM cells in vitro and in the protein expression of MMP-2, MMP-9, Ets-1, and RhoC, compared with either parental or non-silencing control cells. These data provide functional evidence that oncogene AF1Q may be a novel mediator of metastasis promotion in human breast cancer through regulation of the MMP pathway and RhoC expression.

Blood Proteins↗
PubMed2005

Identification of candidate genes associated with salivary adenoid cystic carcinomas using combined comparative genomic hybridization and oligonucleotide microarray analyses.

Adenoid cystic carcinoma (ACC) of the salivary gland often has a variable clinical course with a poor prognosis. To investigate DNA copy number aberrations associated with ACCs, we compared comparative genome hybridization data from ACCs (n = 6) with other types of salivary gland tumors such as adenocarcinomas (n = 3) and pleomorphic adenomas (n = 6). While 15 gain loci (1q32, 6p25, 6q21-q24, 7q11.2, 7q31, 10q11.2, 11p12-q12, 12q13, 12q14, 13q24, 16p13.3-13.2, 18p11.3, 18q23, 19q13.4, and Xq28) were detected, no DNA loss locus was evident. To examine the expression status of genes on the ACC-associated loci, transcriptional measurements of approximately 38000 human genes then were monitored using Affymetrix U133 Plus 2.0 GeneChips. A total of 4431 genes were found differentially expressed by at least two-fold between ACCs and normal salivary glands. Of them, 3162 genes were up-regulated and 1269 genes were down-regulated in ACCs. After obtaining locus information about the RNA transcripts from the Affymetrix database, we found 262 ACC-associated genes with increased expression on ACC-associated loci. To investigate functional network and gene ontology, the 262 genes were analyzed using Ingenuity Pathway Analysis Tool. The function with the highest P value was a cancer-related function (P = 2.52e-4 to 4.71e-2). In addition, we identified pituitary tumor-transforming gene 1 and transformation related protein 63 genes that were up-regulated by increasing DNA copy number and modulated expression of oncogenes. These results suggested that the combination of copy number and gene expression profiling provides an improved strategy for gene identification in salivary gland ACCs.

Adenocarcinoma↗
PubMed2004

Microarray analysis of cell-free fetal DNA in amniotic fluid: a prenatal molecular karyotype.

Metaphase karyotype analysis of fetal cells obtained by amniocentesis or chorionic villus sampling is the current standard for prenatal cytogenetic diagnosis, particularly for the detection of trisomy 21. We previously demonstrated that large quantities of cell-free fetal DNA (cffDNA) are easily extracted from amniotic fluid (AF). In this study, we explored potential clinical applications of AF cffDNA by testing its ability to hybridize to DNA microarrays for comparative genomic hybridization (CGH) analysis. cffDNA isolated from 11 male fetuses showed significantly increased hybridization signals on SRY and decreased signals on X-chromosome markers, compared with female reference DNA. cffDNA isolated from six female fetuses showed the reverse when compared with male reference DNA. cffDNA from three fetuses with trisomy 21 had increased hybridization signals on the majority of the chromosome 21 markers, and cffDNA from a fetus with monosomy X (Turner syndrome) had decreased hybridization signals on most X-chromosome markers, compared with euploid female reference DNA. These results indicate that cffDNA extracted from AF can be analyzed using CGH microarrays to correctly identify fetal sex and aneuploidy. This technology facilitates rapid screening of samples for whole-chromosome changes and may augment standard karyotyping techniques by providing additional molecular information.

Amniocentesis↗
PubMed2006

Genome-wide detection of allelic imbalance in renal cell carcinoma using high-density single-nucleotide polymorphism microarrays.

OBJECTIVE: Renal cell carcinoma (RCC) appears in both a sporadic form and a hereditary form. Eighty-five percent of sporadic RCCs are of the clear-cell histologic type. The cytogenetic analysis of RCCs has revealed several recurring sites of chromosomal aberrations (non-disjunction, deletion or mitotic recombination) including segments of loss of heterozygosity (LOH) identifiable by polymorphic markers. In this pilot study, we performed a comprehensive genome-wide scan to identify LOH sites of RCCs in three Chinese patients using high-density single-nucleotide polymorphism microarrays (HuSNP arrays). DESIGN AND METHODS: Three sporadic clear-cell RCCs specimens were diagnosed histologically. Tumor genomic DNA was extracted from paraffin-embedded sections after microdissection to avoid gross contamination by non-tumor cells. Germline DNA was obtained from paired normal adjacent tissues. Affymetrix HuSNP mapping assay was performed according to the manufacturer's instructions. RESULTS: Using high-density single-nucleotide polymorphism microarrays, we were able to identify the previously described and new LOH sites in RCCs of the three Chinese patients. CONCLUSION: The high-density single-nucleotide polymorphism microarrays and assays offer significant operating cost benefits in sample preparation, processing, and data analysis for identification of LOH sites in cancer samples. In contrast to the typical microsatellite genotyping strategy, the entire genome scan is completed in one experiment taking less than 2 days.

Allelic Imbalance↗
PubMed2007

Identification and characterization of MFA1, the gene encoding Candida albicans a-factor pheromone.

In the opaque state, MTLa and MTLalpha strains of Candida albicans are able to mate, and this mating is directed by a pheromone-mediated signaling process. We have used comparisons of genome sequences to identify a C. albicans gene encoding a candidate a-specific mating factor. This gene is conserved in Candida dubliniensis and is similar to a three-gene family in the related fungus Candida parapsilosis but has extremely limited similarity to the Saccharomyces cerevisiae MFA1 (ScMFA1) and ScMFA2 genes. All these genes encode C-terminal CAAX box motifs characteristic of prenylated proteins. The C. albicans gene, designated CaMFA1, is found on chromosome 2 between ORF19.2165 and ORF19.2219. MFA1 encodes an open reading frame of 42 amino acids that is predicted to be processed to a 14-amino-acid prenylated mature pheromone. Microarray analysis shows that MFA1 is poorly expressed in opaque MTLa cells but is induced when the cells are treated with alpha-factor. Disruption of this C. albicans gene blocks the mating of MTLa cells but not MTLalpha cells, while the reintegration of the gene suppresses this cell-type-specific mating defect.

Base Sequence↗
PubMed2004

Detection of DNA copy number abnormality by microarray expression analysis.

Gene copy-number abnormalities (CNAs) are characteristic of solid tumors and are found in association with developmental abnormalities and/or mental retardation. The ultimate impact of CNAs is exerted by the altered expression of encoded genes. We have utilized high-density oligonucleotide arrays from Affymetrix to identify DNA CNAs via their impact on mRNA expression levels. In these studies, we have used three different trisomic cell lines (trisomy 9, trisomy 18, trisomy 21) as models of CNAs and have compared mRNA expression in those trisomic cells with that observed in diploid cell lines of matched tissue origin. Our data clearly show that genes from CNA chromosome regions are substantially over-represented ( P<0.000001 by chi-square analysis) in the differentially expressed subset from comparisons of all three trisomic cell lines with normal matching cells. In addition, we have been able to detect the origin of the duplication by a statistical scan for over-expressed genes. These data show that microarray detection of differential mRNA expression can be used to identify significant DNA CNAs.

Cell Line↗
PubMed2004

Molecular cloning and characterization of a novel human BTB domain-containing gene, BTBD10, which is down-regulated in glioma.

The broad-complex, tramtrack (ttk) and bric-a-brac/poxvirus and zinc finger proteins (BTB/POZ) domain is highly conserved in a large family of eukaryotic proteins and is crucial for the latter's diverse roles in mediating interactions among proteins that are involved in transcription regulation and chromatin structures. From a fetal brain cDNA library, we isolated a cDNA of 2489 base pairs (bp) encoding a novel human BTB domain-containing protein named BTBD10. The cDNA contained an open-reading frame (ORF) of 1428 bp encoding a putative 475-amino acid (aa) protein. The BTBD10 gene was located on human chromosome 11p15.2 and consisted of nine exons spanning about 75.2 kilobase pairs (kb) of the human genome. The cDNA microarray analysis showed that BTBD10 was down-regulated in all 18 glioma samples. The expression pattern of BTBD10 gene was examined by multiple tissue cDNA (MTC) panels (Clontech), which showed a ubiquitous expression pattern in the 16 tissues examined with high expression in adult brain, testis and small intestine and weak expression in the heart, lung, liver, kidney, pancreas, spleen, thymus, prostate, ovary and colon. The subcellular localization result revealed that BTBD10 was located specifically in the nucleus of HEK293 and COS7 cell lines, suggesting that it may function in transcriptional regulation. The different expression patterns of BTBD10 in different grades of glioma versus normal brain were also examined by RT-PCR and Northern blot. We also investigated the expression of BTBD10 in hepatocellular carcinoma, ovary cancer and lung cancer, and the results revealed no significant difference in these three tumors. All these data suggested that BTBD10 might play a role in glioma.

Adolescent↗
PubMed2005

Distinction in gene expression profiles demonstrated in parathyroid adenomas by high-density oligoarray technology.

OBJECTIVES: Somatic deletion of chromosome 11q13 is the most frequent genetic aberration in parathyroid adenoma. To gain further insight into the genetic etiology of parathyroid tumor development, we examined a comprehensive gene expression profile of parathyroid adenomas and normal parathyroid tissues. The results were then evaluated with respect to differences between adenomas and normal parathyroid tissue, and to the presence of loss of heterozygosity (LOH) in chromosomal region 11q13. DESIGN AND METHODS: Sporadic parathyroid adenomas and normal parathyroids were hybridized against HG-U95Av2 oligonucleotide arrays (Affymetrix) containing a total of 12,625 probe sets. Quantitative real-time PCR (QRT-PCR) was performed in a larger series of parathyroid adenomas, in order to con-firm the microarray results. RESULTS: Cyclin D1 and c-Jun showed increased expression in adenomas vs normal parathyroids by microarray analysis and QRT-PCR, suggesting an oncogenic role of these genes in parathyroid tumor development. At unsupervised hierarchical clustering, the adenomas fell into two groups: Group I adenomas were characterized by 11q13 LOH, while Group II adenomas lacked this abnormality. In addition, a t-test analysis identified largely non-overlapping genes with differential expression in the tumors subgroups; e.g. in Group I tumors the putative oncogene ENC 1 was found highly over-expressed vs Group II adenomas. CONCLUSIONS: The microarray analyses revealed partly distinctive and partly common expression profiles in parathyroid adenomas with and without 11q13 LOH. In addition, approximately half of the under-expressed genes were mapped to chromosome 11, in agreement with a dose effect following loss of this chromosome.

Adenoma↗
PubMed2005

Identification of chromosome arm 9p as the most frequent target of homozygous deletions in lung cancer.

Genome scanning at a 1-Mb resolution was undertaken in 29 lung cancer cell lines to clarify the distribution of homozygous (i.e., both allele) deletions along lung cancer genomes, using a high-resolution single nucleotide polymorphism array. Eighteen regions, including two known tumor suppressor loci, CDKN2A at 9p21 and FHIT at 3p14, were found homozygously deleted. Frequencies of deletions at the 18 regions were evaluated by genomic polymerase chain reaction in 78 lung cancer cell lines. Seven regions, 2q24, 3p14, 5q11, 9p21, 9p23, 11q14, and 21q21, were homozygously deleted in two or more cell lines. The CDKN2A locus at 9p21 was most frequently deleted (20/78, 26%), and the deletions were detected exclusively in non-small-cell lung carcinomas (NSCLCs). The PTPRD (protein tyrosine phosphatase receptor type D) locus at 9p23 was the second-most frequently deleted (8/78, 10%), and the deletions were detected in both small-cell lung carcinomas (SCLC) and NSCLC. In addition, the 9p24 region was deleted in a NSCLC. In total, 24 (31%) cell lines carried at least one deletion on chromosome arm 9p, while deletions on the remaining chromosome arms were observed at most in four (5%) cell lines. Deletions at 9p24, 9p23, and 9p21 were not contiguous with one another, and preferential co-occurrence or mutual exclusiveness for the deletions at these three loci was not observed. Thus, it was indicated that 9p is the most frequent target of homozygous deletions in lung cancer, suggesting that the arm contains multiple lung tumor suppressor genes and/or genomic features fragile during lung carcinogenesis.

Carcinoma, Non-Small-Cell Lung↗
PubMed2006

The effect of the Salmonella genomic island 1 on in vitro global gene expression in Salmonella enterica serovar Typhimurium LT2.

A Salmonella genomic island 1 (SGI1) isogenic strain pair was constructed using Salmonella enterica serovar Typhimurium LT2 (ST LT2). Real-time quantitative reverse transcriptase PCR revealed detectable mRNA transcripts for all 44 putative ORFs encoded within the SGI1. The highest levels of transcripts observed in SGI1 encoded ORFs were found in genes conferring antibiotic resistance to ampicillin, streptomycin/spectinomycin, and sulphonamides. Abundant mRNA transcripts, relative to gapA, were also noted for one putative regulatory ORF and seven ORFs of unknown function encoded within SGI1, whose products could represent factors contributing to increases in virulence and/or fitness of the organism. DNA microarray analysis revealed the differential expression of known factors that contribute to virulence in many pathogens. Twenty-two chromosomal genes were significantly upregulated in ST LT2 harboring SGI1, which included increased expression of iron and sialic acid utilization genes. Decreased expression was noted for 15 genes in ST LT2 harboring SGI1, including genes involved in chemotaxis and motility. This is the first report examining gene expression within the SGI1, as well as its potential effect on global gene expression, and sets the foundation for future studies involving the effect of SGI1 in other Salmonella spp.

Conjugation, Genetic↗
PubMed2005

Shaping time: chromatin structure and the DNA replication programme.

DNA is replicated according to a precise and reproducible temporal pattern. The S-phase programme has previously been analyzed in metazoan and yeast cells using different methods: cytological chromosome banding in human cells and DNA isotopic-labeling techniques in yeast. Microarray-based approaches for the analysis of the replication programme and chromatin structure are bringing us closer to a molecular understanding of the factors that determine replication time. In this article, I assess the impact of recent investigations and compare our knowledge of DNA replication-timing controls in yeast with those of metazoans.

Animals↗
PubMed2006

Oligonucleotide microarray analysis of genomic imbalance in children with mental retardation.

The cause of mental retardation in one-third to one-half of all affected individuals is unknown. Microscopically detectable chromosomal abnormalities are the most frequently recognized cause, but gain or loss of chromosomal segments that are too small to be seen by conventional cytogenetic analysis has been found to be another important cause. Array-based methods offer a practical means of performing a high-resolution survey of the entire genome for submicroscopic copy-number variants. We studied 100 children with idiopathic mental retardation and normal results of standard chromosomal analysis, by use of whole-genome sampling analysis with Affymetrix GeneChip Human Mapping 100K arrays. We found de novo deletions as small as 178 kb in eight cases, de novo duplications as small as 1.1 Mb in two cases, and unsuspected mosaic trisomy 9 in another case. This technology can detect at least twice as many potentially pathogenic de novo copy-number variants as conventional cytogenetic analysis can in people with mental retardation.

Child↗
PubMed2006

Specific effects of ribosome-tethered molecular chaperones on programmed -1 ribosomal frameshifting.

The ribosome-associated molecular chaperone complexes RAC (Ssz1p/Zuo1p) and Ssb1p/Ssb2p expose a link between protein folding and translation. Disruption of the conserved nascent peptide-associated complex results in cell growth and translation fidelity defects. To better understand the consequences of deletion of either RAC or Ssb1p/2p, experiments relating to cell growth and programmed ribosomal frameshifting (PRF) were assayed. Genetic analyses revealed that deletion of Ssb1p/Ssb2p or of Ssz1p/Zuo1p resulted in specific inhibition of -1 PRF and defects in Killer virus maintenance, while no effects were observed on +1 PRF. These factors may provide a new set of targets to exploit against viruses that use -1 PRF. Quantitative measurements of growth profiles of isogenic wild-type and mutant cells showed that translational inhibitors exacerbate underlying growth defects in these mutants. Previous studies have identified -1 PRF signals in yeast chromosomal genes and have demonstrated an inverse relationship between -1 PRF efficiency and mRNA stability. Analysis of published DNA microarray experiments reveals conditions under which Ssb1, Ssb2, Ssz1, and Zuo1 transcript levels are regulated independently of those of genes encoding ribosomal proteins. Thus, the findings presented here suggest that these trans-acting factors could be used by cells to posttranscriptionally regulate gene expression through -1 PRF.

DNA-Binding Proteins↗
PubMed2006

The YfgL lipoprotein is essential for type III secretion system expression and virulence of Salmonella enterica Serovar Enteritidis.

Salmonella enterica, like many gram-negative pathogens, uses type three secretion systems (TTSS) to infect its hosts. The three TTSS of Salmonella, namely, TTSS-1, TTSS-2, and flagella, play a major role in the virulence of this bacterium, allowing it to cross the intestinal barrier and to disseminate systemically. Previous data from our laboratory have demonstrated the involvement of the chromosomal region harboring the yfgL, engA, and yfgJ open reading frames in S. enterica serovar Enteritidis virulence. Using microarray analysis and real-time reverse transcription-PCR after growth of bacterial cultures favorable for either TTSS-1 or TTSS-2 expression, we show in this study that the deletion in S. enterica serovar Enteritidis of yfgL, encoding an outer membrane lipoprotein, led to the transcriptional down-regulation of most Salmonella pathogenicity island 1 (SPI-1), SPI-2, and flagellar genes encoding the TTSS structural proteins and effector proteins secreted by these TTSS. In line with these results, the virulence of the DeltayfgL mutant was greatly attenuated in mice. Moreover, even if YfgL is involved in the assembly of outer membrane proteins, the regulation of TTSS expression observed was not due to an inability of the Delta yfgL mutant to assemble TTSS in its membrane. Indeed, when we forced the transcription of SPI-1 genes by constitutively expressing HilA, the secretion of the TTSS-1 effector protein SipA was restored in the culture supernatant of the mutant. These results highlight the crucial role of the outer membrane lipoprotein YfgL in the expression of all Salmonella TTSS and, thus, in the virulence of Salmonella. Therefore, this outer membrane protein seems to be a privileged target for fighting Salmonella.

Animals↗
PubMed2005

Detection of chromosomal imbalances in children with idiopathic mental retardation by array based comparative genomic hybridisation (array-CGH).

Chromosomal aberrations are a common cause of multiple anomaly syndromes that include growth and developmental delay and dysmorphism. Novel high resolution, whole genome technologies, such as array based comparative genomic hybridisation (array-CGH), improve the detection rate of submicroscopic chromosomal abnormalities allowing re-investigation of cases where conventional cytogenetic techniques, Spectral karyotyping (SKY), and FISH failed to detect abnormalities. We performed a high resolution genome-wide screening for submicroscopic chromosomal rearrangements using array-CGH on 41 children with idiopathic mental retardation (MR) and dysmorphic features. The commercially available microarray from Spectral Genomics contained 2600 BAC clones spaced at approximately 1 Mb intervals across the genome. Standard chromosome analysis (>450 bands per haploid genome) revealed no chromosomal rearrangements. In addition, multi-subtelomeric FISH screening in 30 cases and SKY in 11 patients did not detect any abnormality. Using array-CGH we detected chromosomal imbalances in four patients (9.8%) ranging in size from 2 to 14 Mb. Large scale copy number variations were frequently observed. Array-CGH has become an important tool for the detection of chromosome aberrations and has the potential to identify genes involved in developmental delay and dysmorphism. Moreover, the detection of genomic imbalances of clinical significance will increase knowledge of the human genome by performing genotype-phenotype correlation.

Adolescent↗
PubMed2006

DEK Expression is controlled by E2F and deregulated in diverse tumor types.

Deregulation of the retinoblastoma (pRB) tumor suppressor pathway associated with aberrant activity of E2F transcription factors is frequently observed in human cancer. Microarray based analyses have revealed a large number of potential downstream mediators of the tumor suppressing activity of pRB, including DEK, a fusion partner of CAN found in a subset of acute myeloid leukaemia (AML) patients carrying a (6; 9) translocation. Here we report that the expression of DEK is under direct control of E2F transcription factors. Chromatin immunoprecipitation assays show that the DEK promoter is bound by endogenous E2F in vivo. The DEK promoter is transactivated by E2F and mutation of E2F binding sites eliminates this effect. Expression levels of DEK in human tumors have been investigated by tissue micro array analysis. We find that DEK is overexpressed in many solid tumors such as colon cancer, larynx cancer, bladder cancer, and melanoma.

Binding Sites↗