Project description:Background. Most colorectal cancers (CRC) arise in a progression through adenoma to carcinoma phenotypes as a consequence of altered genetic information. Clinical progression of CRC may occur in parallel with distinctive signaling alterations. We designed multidirectional analyses integrating microarray-based data with biostatistics and bioinformatics to elucidate the signaling and metabolic alterations underlying CRC development in the adenoma-carcinoma sequence. Methodology/Principal Findings. Studies were performed on normal mucosa, adenoma, and CRC samples obtained during surgery or colonoscopy. Collections of cryostat sections prepared from the tissue samples were evaluated by a pathologist to control the relative cell type content. RNA was isolated from 105 macro- and 40 microdissected specimens. The measurements were done using Affymetrix GeneChip HG-U133plus2, and probe set data were generated using two normalization algorithms: MAS5 and GCRMA with LVS. The data were evaluated using pair-wise comparisons and data decomposition into SVD modes. The method selected for the functional analysis used the Kolmogorov-Smirnov test. Based on a consensus of the results obtained by two tissue handling procedures, two normalization algorithms, and two probe set sorting criteria, we identified six KEGG signaling and metabolic pathways (cell cycle, DNA replication, p53 signaling pathway, purine metabolism, pyrimidine metabolism, and RNA polymerase) that are significantly altered in both macro- and microdissected tumor samples compared to normal colon. On the other hand, pathways altered between benign and malignant tumors were identified only in the macrodissected tissues. Conclusion/Significance. Multidirectional analyses of microarray data allow the identification of essential signaling alterations underlying CRC development. Although the proposed strategy is computationally complex and labor–intensive, it may reduce the number of false results. Gene expression profiles of 40 tissue samples representing distant full-thickness normal colon, normal colon mucosa dissected from tumor, adenoma, and carcinoma. Gene expression profiles of 105 tissue samples representing colon carcinomas, adenocarcinomas, and normal colon samples.

Project description:To characterize a series of mouse colon cells that have become spontaneously transformed over time in order to identify the chromosomal and genomic alterations that take place during the development of tumorigenesis. Colorectal cancer is the third common malignancy in the United States. The stepwise progression from adenoma to carcinoma is accompanied by specific genomic alterations. Existing mouse models of human colon cancer have been induced by chemicals, viruses, or through genetic manipulation. Presented here is a unique mouse model of spontaneous transformation designed to identify the sequential steps of tumorigenesis. Normal epithelial colon cells were selectively isolated from the large intestine from eight different isogenic five-six week old C57BL/6 mice. Primary colon cells were grown in vitro with reduced serum concentration. The cells were sequentially recovered from culture based on distinct morphological changes: first, when the cells were in the pre-immortal stage and established as adherent cultures; second, as the cells bypassed crisis, increased their mitotic activity and became immortal; third, at early transformation, as the cells first formed foci; fourth, mid-transformed where the cells had higher proliferation rates in later passages; and finally, late transformed with rapid proliferation, contact inhibition, and multiple foci. The cells within each stage were analyzed using the molecular cytogenetic techniques of spectral karyotyping (SKY) and array comparative genomic hybridization (aCGH), and gene expression profiling. The late transformed cells were injected into nude mice to assess their tumorigenic potential. Spectral karyotyping revealed many recurrent structural and numerical aberrations, specifically in the late transformed cells. Loss of chromosome 4 is a consistent chromosomal aberration observed in all stages of the transformed colon cells, as well as in other tissues during the process of spontaneous transformation. Array CGH identified a pattern of gains and losses, and showed deletions of APC and Trp53 as well as a gain of Kras similar that observed in human colorectal cancer. Gene expression profiling identified the deregulation of several genes known to be involved in the progression of human colon cancer. Tumors resulted from three of the eight late transformed cultures confirming tumorigenic potential. Ultimately, the molecular characterization of spontaneously transformed murine epithelial colon cells indeed recapitulated the step wise progression of human colon cancer, and will prove to be an invaluable system in which to test potential rational intervention strategies. Mouse colon epithelial cells were isolated from the colon of 8 isogenic C57BL\6 mice. These cells were grown in reduced serum and over time spontaneously became immortal and transformed. Our objective is to compare these changes to those that occur during human colon cancer development.

			Action	DRS
	SRR15239131_1.fastq.gz	Fastqsanger.gz
	SRR15239131_2.fastq.gz	Fastqsanger.gz
	SRR15239132_1.fastq.gz	Fastqsanger.gz
	SRR15239132_2.fastq.gz	Fastqsanger.gz
	SRR15239133_1.fastq.gz	Fastqsanger.gz

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