aphidicolin and afimoxifene

Whilst many studies have examined the role of the MAP Kinases in regulating the G1-->S transition, much less is known about the function of these pathways in regulating other cell cycle transitions. Stimulation of the conditional mutant Delta MEKK3:ER* in asynchronous hamster (CCl39) and rat (Rat-1) fibroblasts resulted in the strong activation of endogenous JNK and p38 but only a weak activation of ERK. Activation of Delta MEKK3:ER* inhibited cell proliferation through a combination of an initial G1 and G2 cell cycle arrest, followed by a delayed onset of apoptosis. When cells were synchronized in S phase with aphidicolin and then released, activation of Delta MEKK3:ER* resulted in the up-regulation of p21(CIP1) and a pronounced inhibition of cyclin A/CDK2 and cyclin B1/CDK1 kinase activity. Analysis of mitotic figures indicated that cells failed to enter mitosis, arresting late in G2. Delta MEKK3:ER*-mediated CDK inhibition and G2 arrest did not absolutely require p21(CIP1), since both events were observed in Rat-1 cells in which p21(CIP1) is transcriptionally silenced due to promoter methylation. Rather, CDK inhibition was associated with a down-regulation of cyclin A and B1 expression. Finally, application of the p38 inhibitor SB203580 partially restored cyclin B associated kinase activity and allowed cells to proceed through mitosis into the next G1 phase, suggesting that activation of the p38 alpha/beta 2 pathway can promote a G2 cell cycle arrest.

Topics: Animals; Aphidicolin; Apoptosis; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cells, Cultured; Cricetinae; Cyclin A; Cyclin B; Cyclin B1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; DNA Methylation; Enzyme Activation; Enzyme Inhibitors; Fibroblasts; G1 Phase; G2 Phase; Gene Silencing; Genes, Synthetic; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase Kinase 3; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinase 11; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; Pyridines; Rats; Receptors, Estrogen; Recombinant Fusion Proteins; Sequence Deletion; Tamoxifen