Gene expression profiling following constitutive activation of MEK1 and transformation of rat intestinal epithelial cells

Abstract

Background: Constitutive activation of MEK1 (caMEK) can induce the oncogenic transformation of normal intestinal epithelial cells. To define the genetic changes that occur during this process, we used oligonucleotide microarrays to determine which genes are regulated following the constitutive activation of MEK in normal intestinal epithelial cells.

Results: Microarray analysis was performed using Affymetrix GeneChip and total RNA from doxycycline inducible RIEtiCAMEK cells in the presence or absence of doxycycline. MEK-activation induced at least a three-fold difference in 115 gene transcripts (75 transcripts were up-regulated, and 40 transcripts were down-regulated). To verify whether these mRNAs are indeed regulated by the constitutive activation of MEK, RT-PCR analysis was performed using the samples from caMEK expressing RIE cells (RIEcCAMEK cells) as well as RIEtiCAMEK cells. The altered expression level of 69 gene transcripts was confirmed. Sixty-one of the differentially expressed genes have previously been implicated in cellular transformation or tumorogenesis. For the remaining 8 genes (or their human homolog), RT-PCR analysis was performed on RNA from human colon cancer cell lines and matched normal and tumor colon cancer tissues from human patients, revealing three novel targets (rat brain serine protease2, AMP deaminase 3, and cartilage link protein 1).

Conclusion: Following MEK-activation, many tumor-associated genes were found to have significantly altered expression levels. However, we identified three genes that were differentially expressed in caMEK cells and human colorectal cancers, which have not been previously linked to cellular transformation or tumorogenesis.

Figure 1

Altered expression levels of caMEK-regulated genes involved in cell migration/invasion, cell adhesion, tumor suppression, anti-apoptosis, drug metabolism, and growth/proliferation. The microarray results from caMEK expressing cells (DOX(-)) compared to normal cells (DOX(+)) are expressed as fold difference ± S.D. Differentially expressed genes were verified through RT-PCR analysis. β-actin was used to indicate equal template in each lane.

Figure 2

Altered expression levels of caMEK-regulated genes from transcription factor, signal transduction, metabolic, transportation, cytoskeletal, and other pathways. The microarray results from caMEK expressing cells (DOX(-)) compared to normal cells (DOX(+)) are expressed as fold difference ± S.D. Differentially expressed genes were verified through RT-PCR analysis. β-actin was used to indicate equal template in each lane.

Figure 3

RT-PCR analysis of human colon cancer tissue and cell lines. (A) RT-PCR analysis was performed on 5 paired normal and tumor human colon cancer tissues. T indicates tumor tissue and N indicates corresponding normal adjacent mucosa. Gene-specific primers for PCR were designed by MacVector 7 software depending on the information from GeneBank. Amplication of the right target DNA was confirmed by sequence analysis. β-actin was used as an internal control to confirm equal amount of the templates. (B) RT-PCR analysis was performed on 5 human colon cancer cell lines (HCT116, HT29, LS174T, CaCO2, LoVo) with the indicated primer sets.