Abstract
Microtubule inhibitors are widely used in cancer chemotherapy. These drugs characteristically induce mitotic arrest and cell death but the mechanisms linking the two are not firmly established. One of the problems is that cancer cells vary widely in their sensitivity to these agents, and thus comparison of data from different systems is difficult. To alleviate this problem we sought to molecularly induce mitotic death and study its mechanisms, by expressing non-degradable cyclin B (R42A) in HeLa cells. However, this approach failed to induce significant mitotic arrest, Cdk1 activation, or phosphorylation of anti-apoptotic Bcl-2 proteins, all characteristics of cells treated with microtubule inhibitors. Furthermore, cyclin B1-R42A induced rapid cell death, and when expressed in synchronized cells, cell death occurred in G1 phase. Decreasing the plasmid concentration reduced transfection efficiency but restored mitotic arrest and eliminated non-specific death. These results show that inappropriate overexpression of cyclin B1 causes non-specific cell death and suggest caution in its use for the study of mitotic events.
Highlights
The cell cycle is regulated by the sequential and coordinated actions of cyclindependent protein kinases (Cdks) and their associated cyclin subunits [1, 2]
Based on immunoreactivity with the GFP antibody, and assuming both proteins are recognized with similar affinity, GFP at 27 kDa was expressed at a higher relative level than cyclin B1-R42A-GFP at 75 kDa
This may reflect in part different expression efficiencies, different turnover rates, and/or be related to the fact that GFP overexpression was generally well tolerated by the cells, whereas cyclin B1 overexpression was not and caused cell death, as described below
Summary
The cell cycle is regulated by the sequential and coordinated actions of cyclindependent protein kinases (Cdks) and their associated cyclin subunits [1, 2]. Nuclear cyclin B levels rise dramatically which leads to the activation of Cdk1/cyclin B1 complexes. Cdk1/cyclin B1 is the major regulator of the transition into and through mitosis. Cdk phosphorylates a variety of substrates during mitosis including nuclear lamins and linker histones which are involved in nuclear envelope breakdown and chromatin condensation, respectively [3, 4]. Mitotic exit requires inactivation of Cdk through the ubiquitination and degradation of cyclin B1 [5, 6]. Expression of a PLOS ONE | DOI:10.1371/journal.pone.0113283. Expression of a PLOS ONE | DOI:10.1371/journal.pone.0113283 November 21, 2014
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