Abstract 4814: Detection of DNAH1 fragment closely related to human colorectal cancer by RLGS and functional analysis of DNAH1 in cancer cells
Authors: Matsuda Sachiko , Ueda Masakazu , Hashimoto Osamu , Itano Osamu , Hasegawa Hirotoshi , Kitagawa Yuko 
Published: June 2012

Abstract
[Introduction] Many cancer related genes in human cancer have been isolated by recent development of techniques in molecular biology, however, the existence of unknown genes still have been expected. Restriction landmark genomic scanning (RLGS) is one of the most powerful methods for the identification of a DNA methylation which correlate with protein expression level. This DNA analysis method is 2-dimensional gel electrophoresis of genomic DNA fragment digested with specific restriction enzyme. We have evaluated clinical samples such as breast cancer, hepatocellular carcinoma (HCC), glioma and colorectal cancer for more than 10 years. In this study, we evaluated RLGS to find genetic alterations relative to human colorectal cancer and cloned some of altered spots. We have found that one of these clone codes a part of dynein axonemal heavy chain 1 (DNAH1). Dyneins are large motor protein complexes that generate the force for the movement of eukaryotic cilia and flagella.
[Materials and Methods] High molecular weight genomic DNAs were extracted from 25 colorectal cancerous tissues and corresponding normal mucosae. To analysis alteration spot, each DNA was cleaved with the restriction enzyme Not I, size fractionated by 1st-dimensional electrophoresis using Pvu II, and the DNA fragments were then cleaved by Pst I as the third enzyme in gel and separated by 2-dimensional electrophoresis.
Various cell lines, such as A431, AsPC-1, TE10, MCF7, and WiDr were used for in vitro analysis. To examine gene expression of DNAH1, quantitative RT-PCR was performed using TaqMan® Gene Expression Assays. Cell migration was measured using Boyden chamber migration assay for vertical migration and EZ-TAXIScan (GE Healthcare Japan) for horizontal migration. AsPC-1 cells and AsPC-1 shRNA DNAH1 stable cells were used for xenograft model to evaluate function of DNAH1 in vivo.
[Results] 19 of 25 (76.0%) colorectal cancer cases showed a spot with the same alteration, which never appeared in normal mucosae. A 410-bp DNA fragment corresponding to this spot was cloned. This sequence was found to be 98% homologous to part of dynein axonemal heavy chain 1 (DNAH1).
Gene expression level of DNAH1 was correlated to cell motility in vitro migration assays. In addition, DNAH1 expression level and cell motility were altered by treatment of 5-azacytidin (a protein inhibitor of DNA methyltransferase) or siRNA of DNAH1. Histological and histopathological evaluation of xenogragt model showed that AsPC-1 is INF beta and shRNA DNAH1 stable AsPC-1 is INF alpha.
[Conclusion] We have identified the genomic alteration specific in human colorectal cancer by RLGS. The expression of homologpus gene to DNAH1 is closely related to colorectal cancer. Moreover, DNAH1 might not be express only in colorectal cancer but also other cancers. Expression level of DNAH1 related to cancer cell motility and metastasis in vivo and in vitro.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4814. doi:10.1158/1538-7445.AM2011-4814