Exp Mol Med.  2009 Jan;41(1):8-16. 10.3858/emm.2009.41.1.002.

Functional characterization of human oncoprotein gankyrin in Zebrafish

• Kim SY
• Hur W
• Choi JE
• Kim D
• Wang JS
• Yoon HY
• Piao LS
• Yoon SK

• KMID: 756281
• DOI: http://doi.org/10.3858/emm.2009.41.1.002

Abstract

Gankyrin is an oncoprotein containing seven ankyrin repeats that is overexpressed in hepatocellular carcinoma (HCC). Gankyrin binds to Mdm2, which results in accelerated ubiquitylation via degradation of p53, and it also plays an important role in cell proliferation. However, little is known about the relationships between p53 levels, cell proliferation, and gankyrin over-expression. In order to investigate the influence of gankyrin protein on p53 and Mdm2 in a zebrafish model, we injected human gankyrin (hgankyrin) containing expression vectors (pCS2-hgankyrin, pCS2-hgankyrin-EGFP) into zebrafish embryos. To measure p53 and Mdm2 expression in hgankyrin-injected embryos, RT-PCR, Northern blot and in-situ hybridization and BrdU immunostaining were used. In addition, to know the effect of hgankyrin on cell proliferation in vitro, cell viability assays such as MTT, trypan blue staining and RT-PCR following transfection of hgankyrin-containing vector into HEK 293 cell line were performed. In vivo results indicated that p53 mRNA levels decreased but those of Mdm2 were not decreased in the presence of hgankyrin. These results suggest that gankyrin downregulates p53 expression and not Mdm2 expression. In the study of cell proliferation, BrdU-positive cells were predominantly increased in the head and tail regions in hgankyrin-injected zebrafish. Additional in vitro studies using trypan blue staining and MTT assay showed that gankyrin-expressing HEK 293 cells proliferated at a faster rate, indicating that gankyrin promotes cell proliferation. Our results demonstrate that hgankyrin overexpression downregulates p53 expression and promotes cell proliferation in zebrafish. Gankyrin may play an important role in tumorigenesis via its effects on p53 and cell proliferation.

Keyword

cell proliferation; proto-oncogene proteins c-mdm2; PSMD10 protein, human; tumor suppressor protein p53; zebrafish

MeSH Terms

Animals
Cell Line
Cell Proliferation
Cell Survival
Gene Expression
Humans
In Situ Hybridization
Models, Animal
Proteasome Endopeptidase Complex/*genetics/*metabolism
Proto-Oncogene Proteins/*genetics/*metabolism
Proto-Oncogene Proteins c-mdm2/genetics/metabolism
Tumor Suppressor Protein p53/genetics/*metabolism
Zebrafish