Functional characterization of human oncoprotein gankyrin in Zebrafish.
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.
Kim
So Yeon
SY
Department of Internal Medicine and WHO Collaborating Center of Viral Hepatitis, The Catholic University of Korea, Seoul 137-701, Korea.
Hur
Wonhee
W
Choi
Jung Eun
JE
Kim
Daniel
D
Wang
Jin Sang
JS
Yoon
Hye Yeon
HY
Piao
Lian Shu
LS
Yoon
Seung Kew
SK
D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D015870:Gene Expression; D006801:Humans; D017403:In Situ Hybridization; D023421:Models, Animal; D046988:Proteasome Endopeptidase Complex / Q000235:genetics* / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D015027:Zebrafish
C403591:PSMD10 protein, human; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D046988:Proteasome Endopeptidase Complex