The normal adult liver has the unique ability to regenerate after poisoning, surgical resection or viral damage. However, repeated growth cycles as observed in chronic Hepatitis B Virus (HBV) or Hepatitis C Virus infection, can result in the failure of the normal controls on liver regeneration, leading to liver cancer. The alpha-fetoprotein (AFP) gene, an oncofetal antigen, is expressed in the developing fetus and under conditions of liver regeneration and tumorigenesis in the adult. The level of AFP gene expression during liver regeneration in mice is regulated by a genetically unlinked autosomal locus, Afr2. Inbred C57BL/6 mice express 8 to 10 fold less AFP during liver regeneration than wild type C3H/He mice. Lower AFP expression in regenerating liver reflects other regulatory processes, as C57BL/6 mice are less susceptible to liver carcinogenesis than C3H/He mice. The mechanisms that regulate growth may also regulate AFP gene expression making it an ideal surrogate marker to monitor the condition of the liver.
Analysis of expression of a panel of transgenic mice allowed us to identify the region of the AFP gene locus required for Afr2 regulation and Afr2 Response Element or ARE. Subsequent work in hepatoma cell lines, primary hepatocytes and in mice shows that this sequence does not function as a standard transcription factor recognition site in the same fashion as much of the AFP gene locus, although there are many sequences known to be recognized by transcription factors.
 |
|
Figure 1. Genetic mapping of the Afr2 gene locus. 730 mice were analyzed to determine Afr2 phenotype and genetic composition. The Afr2 gene locus must reside between PCNA and D2Mit107, an anonymous marker identified by the Mouse Genome Project. |
Using a genetic approach we have shown that the leading candidate for Afr2 is MCM8. MCM8 shares sequence homology with a family of genes that form the hexameric helicase required for DNA replication, but neither function nor role for MCM8 has been identified. The discovery that Afr2 is MCM8 suggests that regulation occurs either through DNA replication or transcription, both of which would have an impact of tumorigenesis. Based on the hypothesis that MCM8 functions an MCM family member specifically during liver regeneration, experiments to determine the activity and function of MCM8 are ongoing. Interestingly, if MCM8 functions similarly to other members of the family, the polymorphisms identified in the C3H/He allele could lead gene product that is regulated in a different fashion than the C57BL/6 allele (See figures 1, 2 and 3).
|
|
|
 |
|
|
|
Figure 2. Pattern of gene expression for Afr2 candidate genes. RT-PCR was used to determine the abundance of mRNA for each of the genes located within the Afr2 gene locus.
|
|
Figure 3. MCM8 expression over the course of liver regeneration. Mice were injected with CCl4 and sacrificed at the days indicated after injection. 100 mg of whole cell extracts were subject to western blot analysis with anti-MCM8 antiserum.
|
|
|
|
Current work is focusing on the activity and role of MCM8 asking if it functions as part of the replication helicase. In addition to studies in mouse liver and mammalian cells we are taking advantage of the availability of yeast strains deficient in each MCM subunits found in the replication helicase to determine if MCM8 can function in that complex. This experiment was made possible through the generosity of Dr. Byk Tye, Cornell University Ithaca NY and Dr. John Diffley Cancer Research, UK London Research Institute)
|
|
|
|
|
|
|
Figure 4. Another project in the lab is using DNA microarrays to identify additional genes regulated by Afr2. Red spots indicate genes expressed at higher levels in C3H/HeJ whereas green spots indicate genes expressed at higher levels in C57BL/6J mice.
|
PUBLICATIONS
Anderson, E.C. and M.H. Feuerman. Identification of MCM8 as Afr2. Manuscript in preparation.
Park, J. K. and M.H. Feuerman (2005). Afr2 regulation occurs cell-autonomously in vitro but is not conferred on episomal DNA in transient assays. DNA and Cell Biology 24(3): 189-198
Jin, D.K., E.C. Anderson, E. Gilbert and M.H. Feuerman (2005). AFP gene expression acute diethylnitrosamine intoxication is not Afr2 regulated. Cancer Letters 220 (2):211-220
Feuerman, M.H. (2002) Alpha-fetoprotein. Encyclopedia of Molecular Medicine. Editor: Thomas E. Creighton, EMBL. Editorial Board: C. Thomas Caskey, Michael R. Hayden, Haig H. Kakazian, Jr., George Klein, Hugo W. Moser, Anthony J. Pawson, Stuart H. Orkin, Bernard Roizman, R.V. Thakker, Hugh Watkins. Publisher John Wiley and Sons, New York
Jin, D.K., J. Vacher and M.H. Feuerman (1998). Alpha-fetoprotein Sequences Mediating Afr2 Regulation during Liver Regeneration. Proceedings of the National Academy of Science 95 (15): 8768-8772.
Jin, D.K. and M.H. Feuerman (1998). Chromosomal location of Afr2, regulator of alpha-fetoprotein gene expression during liver regeneration. Mammalian Genome. 9:256-258.
Serfas, M.S., E. Goufman, M.H. Feuerman, A.L. Gartel, and A.L. Tyner (1997). p53-independent induction of p21WAF1/CIP1 in pericentral hepatocytes following carbon tetrachloride intoxication. Cell Growth and Differentiation. 8:951-961.
