ABSTRACT
HeLa cells exposed to Escherichia coli cytolethal distending toxins (CDT) arrest their cell cycle at the G2/M transition. We have shown previously that in these cells the CDK1/cyclin B complex is inactive and can be reactivated in vitro using recombinant CDC25 phosphatase. Here we have investigated in vivo the effects of CDC25 on this cell cycle checkpoint. We report that overexpression of CDC25B or CDC25C overrides an established CDT-induced G2 cell cycle arrest and leads the cells to accumulate in an abnormal mitotic stage with condensed chromatin and high CDK1 activity. This effect can be counteracted by coexpression of the WEE1 kinase. In contrast, overexpression of CDC25B or C prior to CDT treatment prevents G2 arrest and allows most of the cells to progress through mitosis with only a low percentage of cells arrested in abnormal mitosis. The implications of these results on the biochemical nature of the CDT-induced cell cycle arrest are discussed.
Subject(s)
Bacterial Toxins/toxicity , Cell Cycle Proteins , Cell Cycle/drug effects , Cell Cycle/physiology , Nuclear Proteins , cdc25 Phosphatases/physiology , Escherichia coli , HeLa Cells , Humans , Protein-Tyrosine Kinases/physiology , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
Genistein, a natural isoflavone found in soybeans, exerts a number of biological actions suggesting that it may have a role in cancer prevention. We have previously shown that it potently inhibits OCM-1 melanoma cell proliferation by inducing a G(2) cell cycle arrest. Here we show that genistein exerts this effect by impairing the Cdc25C-dependent Tyr-15 dephosphorylation of Cdk1, as the overexpression of this phosphatase allows the cells to escape G(2) arrest and enter an abnormal chromatin condensation stage. Caffeine totally overrides the genistein-induced G(2) arrest, whereas the block caused by etoposide is not bypassed and that caused by adriamycin is only partially abolished. We also report that genistein activates the checkpoint kinase Chk2 as efficiently as the two genotoxic agents and that caffeine may counteract the activation of Chk2 by genistein but not by etoposide. In contrast, caffeine abolishes the accumulation of p53 caused by all the compounds. Wortmannin does not suppress the Chk2 activation in any situation, suggesting that the ataxia telangiectasia-mutated kinase is not involved in this regulation. Finally, unlike etoposide and adriamycin, genistein induces only a weak response in terms of DNA damage in OCM-1 cells. Taken together, these results suggest that the G(2) checkpoints activated by genistein and the two genotoxic agents involve different pathways.
Subject(s)
Caffeine/pharmacology , Cell Cycle/drug effects , Cell Division/drug effects , DNA Damage , Doxorubicin/pharmacology , Etoposide/pharmacology , Genistein/pharmacology , Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Cell Cycle Proteins/metabolism , Checkpoint Kinase 2 , Choroid Neoplasms , Enzyme Activation , G2 Phase , Humans , Melanoma , Tumor Cells, Cultured , cdc25 Phosphatases/metabolismABSTRACT
The cyclin-dependent kinase (CDK) inhibitor p21Cip1 consists of two domains that interact with CDKs and proliferating cell nuclear antigen (PCNA), respectively. We have investigated the interaction between p21Cip1 and PCNA using surface plasmon resonance (SPR) technology and compared the results with those obtained from other sources such as the yeast two-hybrid system. Whilst other methods are only semi-quantitative, the SPR technique allowed us to determine the kinetic parameters of the interaction. The apparent equilibrium constant KD calculated for these kinetic parameters was 3.2 x 10(-7) M. We further demonstrate the use of SPR to study the interaction between mutant proteins and to determine their actual KD. The interaction between p21Cip1/PCNA is shown to be dependent upon the trimeric conformation of PCNA since a point mutant that abolishes PCNA-PCNA interaction also abolishes PCNA's interaction with p21Cip1. Finally, we demonstrate that SPR can be used to characterise the interaction of p21Cip1 and PCNA in the presence of short competitive peptides.
