Anti-CDK2 monoclonal antibody, clone 3C7

Berberine has shown anticancer properties and has potential for a chemopreventive and/or chemotherapeutic agent for breast cancer. Berberine showed cytotoxicity to breast cancer cells, with an increase in the levels of p21/cip1 and p27/kip1, cyclin-dependent kinase inhibitors (CDKI), but mechanisms involved in up-regulating these molecules are largely unknown. Herein, we studied the key regulatory mechanisms involved in berberine-mediated up-regulation of p21/cip1 and p27/kip1. Berberine treatment for 24 and 48h decreased the number of cells by 44-84% (P<0.0001) and 38-78% (P<0.0001), and increased cell death by 12-17% (P<0.005) and 38-78% (P<0.0001) in MCF-7 and MDA-MB-231 cells, respectively. Cells were arrested in G1 phase by berberine which was accompanied with up-regulation of mRNA and protein level of both p21/cip1 and p27/kip1. Berberine decreased the expression of protein levels of cyclin D1, cyclin E, CDK2, CDK4, and CDK6 to cause G1 phase arrest. Berberine caused nuclear localization of p21/cip1 in both the cell lines. Our data for the first time showed that the post-translational stability of both the proteins was strongly increased by berberine as examined by cycloheximide chase assay. Inhibition of Akt was associated with berberine-mediated up-regulation of p21/cip1 and also led to a decrease in cell viability accompanied with significant G1 phase cell cycle arrest. Our study revealed that berberine not only up-regulates mRNA and protein levels of p21/cip1 and p27/kip1 but also increases their nuclear localization and post-translational protein stability. Further, Akt inhibition was found to mediate berberine-mediated up-regulation of p21/cip1 but not the p27/kip1.

To maintain tissue homeostasis, cells transition between cell cycle quiescence and proliferation. An essential G1 process is minichromosome maintenance complex (MCM) loading at DNA replication origins to prepare for S phase, known as origin licensing. A p53-dependent origin licensing checkpoint normally ensures sufficient MCM loading before S phase entry. We used quantitative flow cytometry and live cell imaging to compare MCM loading during the long first G1 upon cell cycle entry and the shorter G1 phases in the second and subsequent cycles. We discovered that despite the longer G1 phase, the first G1 after cell cycle re-entry is significantly underlicensed. Consequently, the first S phase cells are hypersensitive to replication stress. This underlicensing results from a combination of slow MCM loading with a severely compromised origin licensing checkpoint. The hypersensitivity to replication stress increases over repeated rounds of quiescence. Thus, underlicensing after cell cycle re-entry from quiescence distinguishes a higher-risk first cell cycle that likely promotes genome instability.