Fumiyuki Uematsu

Temporal correlation of pathology and DNA damage with gene expression in a choline‐deficient model of rat liver injury

Hepatocellular carcinoma (HCC) is the terminal event in chronic liver diseases with repeated cycles of cellular injury and regeneration. Although much is known about the cellular pathogenesis and etiological agents leading to HCC, the molecular events are not well understood. The choline‐deficient (CD) model of rodent HCC involves the consecutive emergence of a fatty liver, apoptosis, compensatory proliferation, fibrosis, and cirrhosis that is markedly similar to the sequence of events typified by human HCC. Moreover, oxidative stress is thought to play a pivotal role in the progression of the disease. Here, we hypothesize that gene expression profiling can temporally mirror the histopathology and oxidative DNA damage observed with this model. We show that clusters of highly co‐regulated genes representing distinct cellular pathways for lipid biosynthesis and metabolism, apoptosis, cell proliferation, and tissue remodeling temporally correlate with the well‐defined sequential emergence of pathological alterations in the progression of liver disease. Additionally, an oxidative stress signature was observed that was corroborated in a time‐dependent manner with increases in oxidized purines and abasic sites in DNA. Collectively, expression patterns were strongly driven by pathology, demonstrating that patterns of gene expression in advanced stages of liver disease are primarily driven by histopathological changes and to a much lesser degree by the original etiological agent. In conclusion, gene expression profiling coupled with the CD model of HCC provides a unique opportunity to unveil the molecular events associated with various stages of liver injury and carcinogenesis and to distinguish between causal and consecutive changes. Supplementary material for this article can be found on the HEPATOLOGYwebsite (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;42:1137–1147.)

Methylation of neutral endopeptidase 24.11 promoter in rat hepatocellular carcinoma

Neutral endopeptidase 24.11 (NEP), a cell‐surface enzyme expressed by epithelial cells that cleaves and inactivates biologically active small peptides, is downregulated in various cancers. NEP is encoded by a gene that contains a CpG island in the promoter region, whose hypermethylation appears related to decreased expression. Altered expression of NEP has also been reported in human hepatocellular carcinoma (HCC), suggesting its possible role in hepatocarcinogenesis. To elucidate the status of NEP in HCC, methylation in the promoter region of the gene that encodes NEP in male Fischer 344 rats with HCC, induced by a choline‐deficient, l‐amino acid‐defined diet, was investigated by methylation‐specific polymerase chain reaction, combined bisulfite restriction analysis, and bisulfite genomic sequencing. These analyses together showed the promoter to be frequently methylated in HCC in contrast to its unmethylated status in normal liver, the degree of methylation being inversely related to the level of mRNA expression evaluated by reverse transcription–polymerase chain reaction (P = 0.031). In two rat liver cell lines, RLC‐16 and RLC‐27, the promoter was heavily methylated and NEP mRNA expression was negative. However, administration of 5‐aza‐2′‐deoxycytidine caused NEP expression, suggesting that methylation of CpG is a factor regulating transcriptional expression. Together with the data from microarray analyses performed previously using the same animal model, the current results suggest that reduced expression of NEP or other ectopeptidases could impact on molecules involved in signal‐transducing systems, including G‐protein coupled receptors, via modified turnover of extracellularly active small peptides. (Cancer Sci 2006; 97: 611–617)

Distinct patterns of gene expression in hepatocellular carcinomas and adjacent non‐cancerous, cirrhotic liver tissues in rats fed a choline‐deficient, l‐amino acid‐defined diet

Gene expression profiles of HCC and surrounding non‐cancerous tissues in rats fed a CDAA diet for 70 weeks, as well as normal liver tissues, were explored using an oligonucleotide microarray for 3757 genes. A total of 146 genes were identified as differentially expressed; the affected functions including metabolism, apoptosis, cell cycling, RNA splicing, Wnt signaling, reactive oxygen species‐induced stress, and fibro/cirrhogenesis. The genes were found to fit into four distinct expression patterns after classification by hierarchical and k‐means clustering procedures. Notably, genes within the same functional category tended to be found within the same cluster, thus gene functions appeared to be related to their expression patterns. For example, genes encoding receptors (Fishers exact test, P < 0.01) and cytokines (Fishers exact test, P < 0.05) were both enriched in a cluster characterized by low expression in HCC compared to their surrounding tissues. While some of the receptors in this cluster had cell‐proliferative potential, others are known to be growth‐suppressive. It was noted, however, that four of the 10 receptor genes encode G‐protein‐coupled receptors, for which growth‐suppressive potential has been reported. The seven growth factors in the same cluster included two fibroblast growth factors. The current findings suggest the possibility that genes differentially expressed in this multistep carcinogenic model may be classified into relatively few clusters according to their expression patterns, and that these clusters may be associated with gene functional categories. (Cancer Sci 2005; 96: 414 – 424)

A Case Report of a Choroid Plexus Carcinoma Spontaneously Occurring in the Right Lateral Ventricle of a 14-Week-Old, Female Donryu Rat

We encountered a brain tumor arising in the right lateral ventricle of a 14-week-old, female Donryu rat and investigated its histological and immunohistochemical characteristics. Macroscopically, the tumor appeared as a grayish mass with a size of 10 mm in diameter, present in front of the right hemicerebrum and well circumscribed on the cut surface. Histological examination revealed the tumor to be a hypercellular mass occupying the front part of the right lateral ventricle and expanding into the area in front of the hemicerebrum, continuing to the ependymal area at its edge. The tumor was constituted by columnar- or pleomorphic-shaped, highly atypical cells of epithelial origin surrounding fibrovascular cores as single or multiple cell layers. Growth was papillary with high proliferating activity. Immunohistochemically, the tumor cells proved positive for cytokeratin but negative for vimentin, S100 protein or glial fibrillary acidic protein, a profile characteristic for the epithelial cells of the choroid plexus, whereas the ependymal cells were found to be positive for all 4 items. In conclusion, the present tumor was diagnosed as a rat choroid plexus carcinoma, only the third such case to be reported in the world literature, with particular features.

P1 Altered Expression of Gene Products Involved as a Complex in the Endogenous Hepatocarcinogenesis of Rats Fed a Choline-Deficient, L-Amino Acid-Defined Diet

Altered Expression of Gene Products Involved as a Complex in the Endogenous Hepatocarcinogenesis of Rats Fed a Choline-Deficient, L-Amino Acid-Defined Diet DAI NAKAE,1 FUMIYUKI UEMATSU,1 YUTAKA HATANAKA,2 YOICHI KONISHI,3 AKIHIKO MAEKAWA,3 MASAKAZU TAKAHASHI,3 AND MIDORI YOSHIDA,3 1Sasaki Institute, Sasaki Foundation, Chiyoda, Tokyo, Japan, 2DakoCytometaion Company Limited, City of Kyoto, Japan, and 3Nara Medical University, Sakai, Osaka, Japan