Gene expression profile and genomic alterations in colonic tumours induced by 1,2-dimethylhydrazine (DMH) in rats - PubMed (original) (raw)

Gene expression profile and genomic alterations in colonic tumours induced by 1,2-dimethylhydrazine (DMH) in rats

Angelo Pietro Femia et al. BMC Cancer. 2010.

Abstract

Background: Azoxymethane (AOM) or 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in rats shares many phenotypical similarities with human sporadic colon cancer and is a reliable model for identifying chemopreventive agents. Genetic mutations relevant to human colon cancer have been described in this model, but comprehensive gene expression and genomic analysis have not been reported so far. Therefore, we applied genome-wide technologies to study variations in gene expression and genomic alterations in DMH-induced colon cancer in F344 rats.

Methods: For gene expression analysis, 9 tumours (TUM) and their paired normal mucosa (NM) were hybridized on 4 x 44K Whole rat arrays (Agilent) and selected genes were validated by semi-quantitative RT-PCR. Functional analysis on microarray data was performed by GenMAPP/MappFinder analysis. Array-comparative genomic hybridization (a-CGH) was performed on 10 paired TUM-NM samples hybridized on Rat genome arrays 2 x 105K (Agilent) and the results were analyzed by CGH Analytics (Agilent).

Results: Microarray gene expression analysis showed that Defcr4, Igfbp5, Mmp7, Nos2, S100A8 and S100A9 were among the most up-regulated genes in tumours (Fold Change (FC) compared with NM: 183, 48, 39, 38, 36 and 32, respectively), while Slc26a3, Mptx, Retlna and Muc2 were strongly down-regulated (FC: -500; -376, -167, -79, respectively). Functional analysis showed that pathways controlling cell cycle, protein synthesis, matrix metalloproteinases, TNFalpha/NFkB, and inflammatory responses were up-regulated in tumours, while Krebs cycle, the electron transport chain, and fatty acid beta oxidation were down-regulated. a-CGH analysis showed that four TUM out of ten had one or two chromosomal aberrations. Importantly, one sample showed a deletion on chromosome 18 including Apc.

Conclusion: The results showed complex gene expression alterations in adenocarcinomas encompassing many altered pathways. While a-CGH analysis showed a low degree of genomic imbalance, it is interesting to note that one of the alterations concerned Apc, a key gene in colorectal carcinogenesis. The fact that many of the molecular alterations described in this study are documented in human colon tumours confirms the relevance of DMH-induced cancers as a powerful tool for the study of colon carcinogenesis and chemoprevention.

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Figures

Figure 1

Figure 1

Immunohistochemistry for NFkB/p65 and histology in a DMH-induced cancer. Panel A: adenocarcinoma sample challenged with an antibody against NFkB/p65. Panel B: magnification of the inset in panel A; note that positive cells in the tumour show immunoreactivity in both the cytoplasm and the nucleus. Panel C: paired normal mucosa of the tumor challenged with the same antibody. Panel D: H&E stained section of the same tumour. Original magnification in panels A and C: ×400, panel B: ×1000, panel D: ×100.

Figure 2

Figure 2

Semi-quantitative RT-PCR analysis of ten selected genes. For each up-regulated (first five rows) or down-regulated (last five rows) genes, three representative gel images of paired normal mucosa (N) and tumour (T) were provided. For each RT-PCR experiment, expression of β-actin was used as an internal control. P values refer to the t-test for paired samples (n = 7) to compare tumours with normal mucosa.

Figure 3

Figure 3

CGH analytics chromosome view of a region of chromosome 18 of all tumor samples analyzed. Colored curves represent Log2 ratio values for all nucleotide probes plotted as a function of their chromosomal position for each tumor sample. Thick straight vertical blue line and blue shaded area point out a deletion present in sample #9. The black arrow indicates the Apc gene position.

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