Abstract
BackgroundWe previously analyzed human embryonic kidney (HEK) cell lines for the effects that simian virus 40 (SV40) small tumor antigen (ST) has on gene expression using Affymetrix U133 GeneChips. To cross-validate and extend our initial findings, we sought to compare the expression profiles of these cell lines using an alternative microarray platform. METHODS: We have analyzed matched cell lines with and without expression of SV40 ST using an Applied Biosystems (AB) microarray platform that uses single 60-mer oligonucleotides and single-color quantitative chemiluminescence for detection. RESULTS: While we were able to previously identify only 456 genes affected by ST with the Affymetrix platform, we identified 1927 individual genes with the AB platform. Additional technical replicates increased the number of identified genes to 3478 genes and confirmed the changes in 278 (61%) of our original set of 456 genes. Among the 3200 genes newly identified as affected by SV40 ST, we confirmed 20 by QRTPCR including several components of the Wnt, Notch, and Hedgehog signaling pathways, consistent with SV40 ST activation of these developmental pathways. While inhibitors of Notch activation had no effect on cell survival, cyclopamine had a potent killing effect on cells expressing SV40 ST. CONCLUSIONS: These data show that SV40 ST expression alters cell survival pathways to sensitize cells to the killing effect of Hedgehog pathway inhibitors.
Highlights
We previously analyzed human embryonic kidney (HEK) cell lines for the effects that simian virus 40 (SV40) small tumor antigen (ST) has on gene expression using Affymetrix U133 GeneChips
The total number of genes identified by both platforms was 185 or 41% of the 456 genes initially identified with the U133 GeneChips
When technical replicates were included in the Applied Biosystems (AB) analysis, 3478 unique genes were identified as affected by simian virus 40 small tumor antigen (SV40 ST) [see Additional file 1], increasing the number of confirmed genes from our previous analysis to 278 genes (61%) from the original set of 456 genes
Summary
We previously analyzed human embryonic kidney (HEK) cell lines for the effects that simian virus 40 (SV40) small tumor antigen (ST) has on gene expression using Affymetrix U133 GeneChips. METHODS: We have analyzed matched cell lines with and without expression of SV40 ST using an Applied Biosystems (AB) microarray platform that uses single 60-mer oligonucleotides and single-color quantitative chemiluminescence for detection. A number of studies comparing spotted cDNA two-color technology with the Affymetrix single-color technology have found fairly poor correlation of data between these two approaches [1,2,3]. A relatively new platform for microarray analysis developed by Applied Biosystems (AB) employs single 60-mer oligonucleotides, similar in length to Agilent, but uses single-color chemiluminescence detection technology as opposed to two-color Cy3/Cy5 labeling and laser fluorescence scanning. We show that the AB platform has substantially higher sensitivity, detecting four times as many gene changes in an identical experimental design, and over seven times as many genes when additional technical replicates were included.
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