Background and aims: Colorectal epithelial cells are prone to malignant transformation. Therefore, identification of differences in gene expression in the process from normal colonic crypts to adenomas with low grade dysplasia is essential for further insights into early tumorigenesis. To achieve this goal, a novel gene expression analysis strategy, screening for expressed transcripts in small histologically defined tissue samples, was performed.
Methods: First, laser mediated microdissection was used to isolate normal and adenomatous crypts from colonic cryosections. Then, nested RNA arbitrarily primed polymerase chain reaction (RAP-PCR) for differential display was performed to screen mRNA populations and to generate hybridisation probes for cDNA expression arrays. After evaluation of cDNA expression arrays, differential expression was confirmed at the protein level by immunohistochemistry.
Results: Evaluation of gene expression profiles of normal versus adenomatous colonic crypts of six different patients revealed, in general, dysregulation of up to 11% of all analysed genes (total number n=588): specifically, p21-rac1 was upregulated in four of six patients, mitogen activated protein kinase (MAPK) p38α in three of six patients, and interferon γ receptor in three of six patients. Conversely, FAST kinase was found to be downregulated in three of six patients, p53 in three of six patients, and thrombospondin 2 in three of six patients.
Conclusions: For the first time, distinct gene expression profiles of dysplastic areas within colonic adenomas, using the combination of laser mediated microdissection with RAP-PCR and cDNA expression array, were shown. In these samples, upregulation of proliferation associated genes (ras-oncogene related p21-rac1 and MAPK p38α) as well as downregulation of apoptosis related genes (FAST kinase and p53) most likely reflects specific alterations in adenomas with low grade dysplasia. Based on upregulation of p21-rac1 and MAPK p38α, activation of the MAPK pathway appears to be an early event in colonic carcinogenesis.
- laser mediated microdissection
- gene expression pattern
- low grade dysplasia
- RAS/MAPK pathway
- LMM, laser mediated microdissection
- RAP-PCR, RNA arbitrarily primed polymerase chain reaction
- MAPK, mitogen activated protein kinase
- IFGR, interferon gamma receptor
- SSC, standard saline citrate
- SDS, sodium dodecyl sulphate
- AEC, 3-amino-9-ethylcarbazole
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