The destruction box of human Geminin is critical for proliferation and tumor growth in human colon cancer cells.
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.
Yoshida
Kenichi
K
Division of Genetic Diagnosis, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, Japan. yoshidak@ims.u-tokyo.ac.jp
Oyaizu
Naoki
N
Dutta
Anindya
A
Inoue
Ituro
I
D000483:Alleles; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D002455:Cell Division; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D003110:Colonic Neoplasms / Q000473:pathology*; D004261:DNA Replication; D016193:G1 Phase; D064248:Geminin; D004854:Herpesvirus 4, Human / Q000502:physiology; D006801:Humans; D009154:Mutation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D014779:Virus Replication
C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; C577995:GMNN protein, human; D064248:Geminin; D012333:RNA, Messenger; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases