Previously, we demonstrated that expression of pololike kinase (PLK) is required for cellular DNA synthesis and that overexpression of PLK is sufficient to induce DNA synthesis. We now report that the endogenous levels of PLK, its phosphorylation status, and protein kinase activity are tightly regulated during cell cycle progression. PLK protein is low in G 1 , accumulates during S and G 2 M, and is rapidly reduced after mitosis. During mitosis, PLK is phosphorylated on serine, and its serine threonine kinase function is activated at a time close to that of p34 cdc2. The phosphorylated form of PLK migrates with reduced mobility on SDS-polyacrylamide gel electrophoresis, and dephosphorylation by purified protein phosphatase 2A converts it to the more rapidly migrating form and reduces the total amount of PLK kinase activity. Purified p34 cdc2 -cyclin B complex can phosphorylate PLK protein in vitro but causes little increase in PLK kinase activity.The processes of cell growth and division are stringently regulated to ensure fidelity of DNA replication and correct segregation of genetic information (1, 2). So essential are these processes to cellular homeostasis that many of the gene products regulating passage through the cell cycle are highly conserved both in amino acid sequence and function between organisms as evolutionarily divergent as yeast and man (3,4). Within the last few years, a variety of related enzymes known as cyclin-dependent protein kinases (CdKs) 1 have been identified that are activated and inactivated at specific times during cell cycle progression (5-8). The CdKs must form complexes with a cyclin family member to be activated (9, 10) and are also subject to both positive and negative phosphorylation by other protein kinases and dephosphorylation by protein phosphatases (11-17). Together, the family of CdKs constitutes critical components of the engine that propels the cell through the cycle. Many proteins that are phosphorylated in a cell cyclespecific fashion have been identified as putative targets of CdK regulation including nuclear lamins (18, 19), nucleolin (20, 21), and other matrix proteins, as well as cytoskeletal proteins (7,22). In addition to such architectural proteins, the tumor suppressor gene products p53 (23) and RB (24 -28) are both subject to regulatory phosphorylation by CdK family members (7). Thus, it is clear that reversible phosphorylation reactions play a major role in regulating cell cycle progression, and the list of enzymes known to be involved is rapidly expanding.A number of cell cycle-regulated kinases unrelated to the CdKs have also been identified in lower eukaryotes, including the homologous yeast CDC5 (29) and Drosophila polo (30). Polo was first identified as an embryonic lethal Drosophila mutation causing formation of monopolar and multipolar mitotic spindles and abnormal segregation of chromosomes (31). Saccharomyces cerevisiae CDC5 mutants likewise are impaired in mitotic spindle formation, but the CDC5 kinase also appears to have a function during S pha...
Multiple myeloma (MM) remains largely incurable despite conventional and high-dose therapies. Therefore, novel biologically based treatment approaches are urgently required. Here we demonstrate that expression of peroxisome proliferator-activated receptor gamma (PPARgamma) in MM cells and its agonists 15-d-PGJ2 and troglitazone completely abolished IL-6-inducible MM cell proliferation and induced apoptosis through affecting expression of multiple cell cycle or apoptosis genes, whereas PPARgamma antagonist GW9662 and PPARalpha agonist WY14643 did not display this inhibitory effect. These PPARgamma agonists significantly inhibited DNA binding and transactivation of STAT3 bound to the promoter of target genes in chromatin, but did not affect the expression of IL-6 receptor and phosphorylation of JAK/STAT3, MAPK, and PI3K/Akt. Interestingly, although inactivation of STAT3 by PPARgamma agonists is in a PPARgamma-dependent manner, the molecular mechanism by which two structurally distinct PPARgamma agonists suppress IL-6-activated STAT3 shows the divergent interactions between PPARgamma and STAT3 including direct or SMRT-mediated association.
Current antiestrogen therapy for breast cancer is limited by the mixed estrogenic and antiestrogenic activity of selective estrogen receptor modulators. Here we show that the function of zinc fingers in the estrogen receptor DNA-binding domain (DBD) is susceptible to chemical inhibition by electrophilic disulfide benzamide and benzisothiazolone derivatives, which selectively block binding of the estrogen receptor to its responsive element and subsequent transcription. These compounds also significantly inhibit estrogen-stimulated cell proliferation, markedly reduce tumor mass in nude mice bearing human MCF-7 breast cancer xenografts, and interfere with cell-cycle and apoptosis regulatory gene expression. Functional assays and computational analysis support a molecular mechanism whereby electrophilic agents preferentially disrupt the vulnerable C-terminal zinc finger, thus suppressing estrogen receptor-mediated breast carcinoma progression. Our results provide the proof of principle for a new strategy to inhibit breast cancer at the level of DNA binding, rather than the classical antagonism of estrogen binding.
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