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  • Original Paper
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The destruction box of human Geminin is critical for proliferation and tumor growth in human colon cancer cells

Abstract

A domain-specific disruption was performed on the destruction box sequence of endogenous Geminin gene, an inhibitor of the DNA replication initiation complex, in a human cancer cell line HCT116 resulting in the formation of a protein that was stable in the G1 phase of the cell cycle. Although the total amount of Geminin in asynchronous cultures was not elevated, the G1-specific stabilization of Geminin, diminished chromatin loading of minichromosome maintenance complex, inhibited DNA replication, and resulted in the accumulation of cells in G1. The mutated Geminin suppressed in vivo tumorigenicity and in vitro cell growth. Cells carrying this mutation failed to support the replication of a plasmid bearing the oriP replicator of Epstein–Barr virus. The DNA damage checkpoint pathway was activated in the mutated cells with increased levels of p53 protein and its target, the p21 protein. All these deficits were rescued by overexpression of Cdt1, a replication initiator protein that binds to Geminin. Therefore, alteration of the cell cycle-dependent regulation of endogenous Geminin in human cells without increasing total protein level inhibits DNA replication and suppresses tumor growth.

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Abbreviations

APC:

anaphase-promoting complex

ATM:

ataxia-telangiectasia-mutated

BrdU:

bromodeoxyuridine

EBV:

Epstein–Barr virus

cdk:

cyclin-dependent kinases

MCM:

minichromosome maintenance proteins

ORC:

origin recognition complex

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Acknowledgements

We are grateful to Yoko Kanamori for technical assistance, Haruo Onoda for histochemical staining and Shigeo Mori for help with histology. We thank the members of our laboratory for their helpful discussions during the course of this work. This study was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports and Culture, Japan.

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Correspondence to Kenichi Yoshida.

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Yoshida, K., Oyaizu, N., Dutta, A. et al. The destruction box of human Geminin is critical for proliferation and tumor growth in human colon cancer cells. Oncogene 23, 58–70 (2004). https://doi.org/10.1038/sj.onc.1206987

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