Transcription profiling of mouse parental vs tumor-derived immortalized kidney epithelial cell (iBMK) lines

ABSTRACT: Most tumors are epithelial-derived, and although disruption of polarity and aberrant cellular junction formation is a poor prognosticator in human cancer, the role of polarity determinants in oncogenesis is poorly understood. Using in vivo selection, we identified a mammalian orthologue of the Drosophila polarity regulator crumbs as a gene whose loss of expression promotes tumor progression. Immortal baby mouse kidney epithelial (iBMK) cells selected in vivo to acquire tumorigenicity displayed dramatic repression of crumbs3 (crb3) expression associated with disruption of tight junction formation, apicobasal polarity, and contact-inhibited growth. Restoration of crb3 expression restored junctions, polarity and contact inhibition, while suppressing migration and metastasis. These findings suggest a role for mammalian polarity determinants in suppressing tumorigenesis that may be analogous to the well-studied polarity tumor suppressor mechanisms in Drosophila. Experiment Overall Design: Gene Expression analysis was carried out using the Affymetrix Mouse 430 A 2.0 chips representing about 22,629 full-length genes and ESTs. Initial scaling was done using the Affymetrix Microarray Suite Expression Software version 5.0 and subsequent analysis was done using GeneSpring Software version 6.1 ( Silicon Genetics). (Tumor derived cell lines) /in vivo selected cell lines were compared to the parental cell lines from which they were derived. We had two parental cell line samples which were defined as replicates and treated as a single sample and 4 cell lines namely TD A, B, C and D derived from the parents. Three different filtering conditions were applied. First, we applied Expression Percentage Restriction retaining only those genes which had a raw expression value of 80.0 or more in at least 1 out of the 5 samples being compared. This enabled us to filter out the low-intensity values while retaining genes that may have a very low expression level in one sample but may be switched on in another sample. The cut-off value of 80.00 was determined by analyzing the raw data of each of the chips being compared and estimating the average background expression value. Of the 22,629 genes represented on the chips, 11,418 genes were retained. Secondly, we filtered on data quality selecting for genes with a Flag value of Present (P) in all the samples being compared. 10,126 genes were retained after applying this filter. We then carried out a pair-wise fold-change analysis comparing each tumor derived cell line to the parental cell-line. We retained genes that underwent at least 2 folds or more up-regulation or down-regulation in the tumor derived cell l ines, compared to the parentals. 1,010 genes were retained after this step. In order to identify genes that underwent statistically significant changes in gene expression in the tumor derived cell-lines when compared to the parentals we applied a statistical filter analysis and performed the Studentâ??s-t-test with a p value cut-off of 0.05. Multiple testing correction was turned off. We found 123 genes that were over-expressed and 5 that were underexpressed. We carried out two-way hierarchical clustering using the GeneSpring Software 6.1 using standard correlation as similarity metric. We also analyzed our data using Venn Diagrams in order to identify the statistically significant genes that were common to all 4 tumor derived cell-lines. We obtained 55 that were up-regulated while there were 5 which were down-regulated and common to all four cell-lines.

ORGANISM(S): Mus musculus

SUBMITTER: Cristina Karp 

PROVIDER: E-GEOD-10682 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress