[Frontiers in Bioscience 14, 3724-3732, January 1, 2009]

ZHX2 and ZHX3 repress cancer markers in normal hepatocytes

Kazuya Yamada1,2, Hiroko Ogata-Kawata1, Kaoru Matsuura1, Norio Kagawa3, Katsuhiro Takagi2, Kosuke Asano2, Ayumi Haneishi2, Kaoru Miyamoto1

1Department of Biochemistry, Faculty of Medical Sciences, University of Fukui, Fukui 910-1193, Japan, 2Department of Health and Nutritional Science, Faculty of Human Health Science, Matsumoto University, Nagano 390-1295, Japan, 3Department of Biochemistry, Saarland University, D 66041 Saarbruecken, Germany

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Materials
3.2. Cells and cell culture
3.3. Probe DNAs
3.4. Northern blot analysis
3.5. Reverse transcription (RT)-polymerase chain reaction (PCR) analysis
3.6. Plasmid construction
3.7. DNA transfections
3.8. Library screening
4. Results
4.1. Analysis of gene expression of members of the ZHX family in isolated rat hepatocytes and various hepatoma cell lines
4.2. Members of the ZHX family regulate the expression of hepatocellular carcinoma biomarkers
4.3. ZHX2 represses transcription from the mouse alpha-fetoprotein (AFP) gene promoter
4.4. Screening of ZHX2- or ZHX3-interacting proteins
5. Discussion
6. Acknowledgements
7. References

1. ABSTRACT

ZHX2 and ZHX3 are the members of the ZHX transcriptional repressor family. To investigate the regulatory role of the repressors in hepatocytes and their involvement in carcinogenesis, the expression levels of ZHX2 and ZHX3 mRNAs were examined. The dRLh-84 hepatoma cells considerably expressed cancer marker genes PKM and HK II that are expressed in developing fetal tissues and cancer cells but repressed in normal hepatocytes. In dRLh-84 cells, the expression levels of ZHX2 and ZHX3 were very low compared with rat hepatocytes. Upon the reporter gene analysis utilizing the promoter region of these genes, ZHX3 repressed the transcription of the reporter luciferase gene from both promoters while ZHX2 only repressed that from HK II promoter. The promoter activity of alpha-fetoprotein was also repressed by the expression of ZHX2 in HLE hepatoma cells in a dose-dependent manner. We concluded that ZHX2 and ZHX3 were involved in the transcriptional repression of the hepatocellular cacinoma markers in normal hepatocytes, suggesting that the failure of the ZHX2 and/or ZHX3 expression might be a critical factor in the hepatocellular carcinogenesis.