We evaluated the genome-wide gene expression profiles of various cancer cell lines to identify the gastrointestinal tract cancer cell-related genes.
Gene expression profilings of 27 cancer cell lines and 9 tissues using 7.5K human cDNA microarrays in indirect design with Yonsei reference RNA composed of 11 cancer cell line RNAs were done. The significant genes were selected using significant analysis of microarray in various sets of data. The selected genes were validated using real-time PCR analysis.
After intensity-dependent, within-print-tip normalization by loess method, we observed that expression patterns of cell lines and tissues were substantially different, divided in two discrete clusters. Next, we selected 115 genes that discriminate gastrointestinal cancer cell lines from others using significant analysis of microarray. Among the expression profiles of five gastric cancer cell lines, 66 genes were identified as differentially expressed genes related to metastatic phenotype. YCC-16, which was established from the peripheral blood of one advanced gastric cancer patient, produced a unique gene expression pattern resembling the profiles of lymphoid cell lines. Quantitative real-time reverse transcription-PCR results of selected genes, including PXN, KRT8, and ITGB5, were correlated to microarray data and successfully discriminate the gastrointestinal tract cancer cell lines from hematologic malignant cell lines.
A gene expression database could serve as a useful source for the further investigation of cancer biology using the cell lines.
"Hs578T, Kato III and SKBR-3 were from American Type Culture Collection (ATCC), Manassas, USA. YCC16 cell line was from Yonsei Cancer Centre, Korea . The cells were cultured in the specified media (HiMedia); MCF-7 and YCC16: Minimal Essential Media( MEM), T47-D, ZR 75 & Kato III: RPMI 1640, Hs 578T: DMEM, SK-BR-3 & HBL-100: McCoy’s 5a medium, MDA-MB-231, MDA-MB-453 and MDA-MB-468: Leibovitz, AGS: DMEM-F12, with the supplements L-glutamine (2 mM), sodium pyruvate (1 mM), sodium bicarbonate (1.5 g/L), non-essential amino acids (0.1 mM), penicillin (100 μg/ml), streptomycin (100 μg/ml) (HiMedia) and 10% foetal bovine serum (20 % for Kato III) (Sigma). "
[Show abstract][Hide abstract] ABSTRACT: Genomic aberrations are common in cancers and the long arm of chromosome 1 is known for its frequent amplifications in breast cancer. However, the key candidate genes of 1q, and their contribution in breast cancer pathogenesis remain unexplored. We have analyzed the gene expression profiles of 1635 breast tumor samples using meta-analysis based approach and identified clinically significant candidates from chromosome 1q. Seven candidate genes including exonuclease 1 (EXO1) are consistently over expressed in breast tumors, specifically in high grade and aggressive breast tumors with poor clinical outcome. We derived a EXO1 co-expression module from the mRNA profiles of breast tumors which comprises 1q candidate genes and their co-expressed genes. By integrative functional genomics investigation, we identified the involvement of EGFR, RAS, PI3K / AKT, MYC, E2F signaling in the regulation of these selected 1q genes in breast tumors and breast cancer cell lines. Expression of EXO1 module was found as indicative of elevated cell proliferation, genomic instability, activated RAS/AKT/MYC/E2F1 signaling pathways and loss of p53 activity in breast tumors. mRNA-drug connectivity analysis indicates inhibition of RAS/PI3K as a possible targeted therapeutic approach for the patients with activated EXO1 module in breast tumors. Thus, we identified seven 1q candidate genes strongly associated with the poor survival of breast cancer patients and identified the possibility of targeting them with EGFR/RAS/PI3K inhibitors.
PLoS ONE 10/2013; 8(10):e77553. DOI:10.1371/journal.pone.0077553 · 3.23 Impact Factor
"The microarray hybridization was performed in an indirect-design; each of the cDNA targets generated from tissue total RNAs (Cy5-labeled) were competitively hybridized with cDNAs generated from the common reference RNA pool (Cy3-labeled) in each of the hybridization following a published protocols . Common reference RNA, which is biologically irrelevant to the samples being analyzed and only functions to provide a denominator in ratios in cDNA microarrays, was prepared by pooling equivalent amount of total RNAs from the following cell lines; AGS, MDA-MB231, HCT 116, SK-HEP-1, A-549, HL-60, MOLT-4, HeLa, Caki-2, U-87MG, SK-MEL-2 and Capan-2 . Hybridized slides were washed and then scanned using a Gene Pix 4000B laser scanner (Axon Instrument Inc, Union City, CA). "
[Show abstract][Hide abstract] ABSTRACT: To be able to describe the differences between the normal and tumor tissues of gastric cancer at a molecular level would be essential in the study of the disease. We investigated the gene expression pattern in the two types of tissues from gastric cancer by performing expression profiling of 86 tissues on 17K complementary DNA microarrays. To select for the differentially expressed genes, class prediction algorithm was employed. For predictor selection, samples were first divided into a training (n=58), and a test set (n=28). A group of 894 genes was selected by a t-test in a training set, which was used for cross-validation in the training set and class (normal or tumor) prediction in the test set. Smaller groups of 894 genes were individually tested for their ability to correctly predict the normal or tumor samples based on gene expression pattern. The expression ratios of the 5 genes chosen from microarray data can be validated by real time RT-PCR over 6 tissue samples, resulting in a high level of correlation, individually or combined. When a representative predictor set of 92 genes was examined, pathways of 'focal adhesion' (with gene components of THBS2, PDGFD, MAPK1, COL1A2, COL6A3), 'ECM-receptor interaction' pathway (THBS2, COL1A2, COL6A3, FN1) and 'TGF-beta signaling' (THBS2, MAPK1, INHBA) represent some of the main differences between normal and tumor of gastric cancer at a molecular level.
"Cell lines, tumor and normal tissue samples Cell lines used were: 12 NPC (C666-1, CNE-1, CNE-2, HK1, HNE1, HNE2, HNE3, HONE1, TW-01, BM1, 5-8F and 6- 10B) (Glaser et al., 1989; Qiu et al., 2004; Ying et al., 2005), 15 esophageal (EC1, EC18, EC109, HKESC1, HKESC2, SLMT- 1, KYSE30, KYSE70, KYSE140, KYSE150, KYSE180, KYSE270, KYSE410, KYSE510 and KYSE520) (Tang et al., 2001; Ying et al., 2006), nine breast (MCF7, ZR-75-1, MB-231; T47D, MB-468, MB-435, SKBR3, BT549, and YCC- B2) (Kim et al., 2005; Ying et al., 2005) and eight cervical carcinoma (HeLa, Caski, C33A, SiHa, 808, 866, 879 and 915) (Steenbergen et al., 2004; Ying et al., 2006). NP69, an SV40 Tantigen-immortalized nasopharyngeal epithelial cell line with many features of normal nasopharyngeal epithelial cells was used as a 'normal' control for NPC (Tsao et al., 2002). "
[Show abstract][Hide abstract] ABSTRACT: Identification of tumor suppressor genes (TSG) silenced by methylation uncovers mechanisms of tumorigenesis and identifies new epigenetic tumor markers for early cancer detection. Both nasopharyngeal carcinoma (NPC) and esophageal carcinoma are major tumors in Southern China and Southeast Asia. Through expression subtraction of NPC, we identified Deleted in Liver Cancer 1 (DLC1)/ARHGAP7 (NM_006094)--an 8p22 TSG as a major downregulated gene. Although expressed in all normal tissues, DLC1 was silenced or downregulated in 11/12 (91%) NPC, 6/15 (40%) esophageal, 5/8 (63%) cervical and 3/9 (33%) breast carcinoma cell lines. No genetic deletion of DLC1 was detected in NPC although a hemizygous deletion at 8p22-11 was found by 1-Mb array-CGH in some cell lines. We then located the functional DLC1 promoter by 5'-RACE and promoter activity assays. This promoter was frequently methylated in all downregulated cell lines and in a large collection of primary tumors including 89% (64/72) NPC (endemic and sporadic types), 51% (48/94) esophageal, 87% (7/8) cervical and 36% (5/14) breast carcinomas, but seldom in paired surgical marginal tissues and not in any normal epithelial tissue. The transcriptional silencing of DLC1 could be reversed by 5-aza-2'-deoxycytidine or genetic double knock-out of DNMT1 and DNMT3B. Furthermore, ectopic expression of DLC1 in NPC and esophageal carcinoma cells strongly inhibited their colony formation. We thus found frequent epigenetic silencing of DLC1 in NPC, esophageal and cervical carcinomas, and a high correlation of methylation with its downregulation, suggesting a predominant role of epigenetic inactivation. DLC1 appears to be a major TSG implicated in the pathogenesis of these tumors, and should be further tested as a molecular biomarker in patients with these cancers.
K. Bougoffa-Sadaoui, E. Gontier, M.S. Telliez, M. Lequart-Pillon, H. Ouadid-Ahidouch, F. Maiza-Benabdesselam
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