Unknown,Transcriptomics,Genomics,Proteomics

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Aneuploidy, oncogene amplification, and epithelial to mesenchymal transition define spontaneous transformation of murine epithelial cells [transcriptome]

ABSTRACT: Human epithelial cancers are defined by a recurrent distribution of specific chromosomal aneuploidies. In our model system, mouse bladder and kidney epithelial cells spontaneously immortalize, transform and become tumorigenic after prolonged culture. We assessed genome and transcriptome alterations and found wide-spread aneuploidy, early transcriptional deregulation, and massive genomic dereguation of the cellular transcriptome. The results reveal a remarkable similarity with genome and transcriptome aberrations detected in human tumorigenesis, hence validating our newly derived cancer models. Epithelial cells were isolated from the C57BL/6 mouse bladder and kidney. These cells underwent spontaneous transformation in culture. We sought to identify the molecuar genomic alterations that occur during the transformation process and to compare these with the changes observed in human bladder and kidney cancers.

ORGANISM(S): Mus musculus

SUBMITTER: Hesed Padilla-Nash

PROVIDER: E-GEOD-45127 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

Project description:Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of multiple fluid-filled cysts that destroy the kidney architecture resulting in end-stage renal failure. Mutations in the ADPKD genes PKD1 and PKD2 account for nearly all the cases of ADPKD. Increased cell proliferation is one of the key features of the disease. Several studies indicated that polycystin-1, the product of PKD1, regulates cellular proliferation through various signaling pathways, but little is known about the role played by polycystin-2, the product of PKD2. Recently, it was reported that as with polycystin-1, polycystin-2 can acts as a negative regulator of cell growth by modulating the levels of the cyclin-dependent kinase inhibitor, p21 and the activity of the cyclin-dependent kinase 2, Cdk2. In this study we utilized different kidney cell lines expressing wild-type and mutant PKD2 as well as primary tubular epithelial cells isolated from a PKD transgenic rat to further explore the contribution of the p21/Cdk2 pathway in ADPKD proliferation. Surprisingly, over-expression of wild-type PKD2 in renal cell lines failed to inactivate Cdk2 activity and consequently had no effect on cell proliferation. On the other hand, expression of mutated PKD2 augmented proliferation only in the primary tubular epithelial cells of a rat model but this was independent of STAT-1/p21 pathway. Genome-wide expression analysis in these cells revealed that expression of the cyclin-dependent kinase inhibitor, p57 is downregulated, while p21 remains unchanged. This p57 reduction is accompanied by an increase in Cdk2 levels. Our results indicate the probable involvement of p57 on epithelial cell proliferation in polycystic kidney disease. In addition, it confirms that PKD2-induced proliferation is a multifactorial process. We used microarrays to study the modulation of gene expression by mutated PKD2 in the primary tubular epithelial cells of a transgenic rat mode Keywords: transgene Gene expression in the primary tubular epithelial of a transgenic rat mode with mutated PKD2 was compared with that of SD control rats

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Aneuploidy, oncogene amplification, and epithelial to mesenchymal transition define spontaneous transformation of murine epithelial cells [transcriptome]

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