Inhibition of protein kinase B activity induces cell cycle arrest and apoptosis during early G1 phase in CHO cells.

Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase.

Focal adhesion signaling and actin stress fibers are dispensable for progression through the ongoing cell cycle.

Prevention of cell spreading or disruption of actin filaments inhibits growth factor stimulated cell cycle re-entry from quiescence, mainly because of a failure to induce cyclin D expression. Ectopic cyclin D expression overrules anchorage-dependency, suggesting that cell spreading per se is not required as long as cyclin D is otherwise induced. We investigated whether cyclin D expression in cells exiting mitosis is sufficient to drive morphology-independent cell cycle progression in continuously cycling (i.e. not quiescent) cells. Disruption of post-mitotic actin reorganization did not affect substratum reattachment but abolished the formation of filopodia, lamellipodia and ruffles, as well as stress fiber organization, focal adhesion assembly and cell spreading. Furthermore, integrin-mediated focal adhesion kinase (FAK) autophosphorylation and growth factor stimulated p42/p44 mitogen activated protein kinase (MAPK) activation were inhibited. Despite a progressive loss of cyclin D expression in late G1, cyclin E and cyclin A were normally induced. In addition, cells committed to DNA synthesis and completed their entire cycle. Our results demonstrate that post-mitotic disruption of the actin cytoskeleton allows cell cycle progression independent of focal adhesion signaling, cytoskeletal organization and cell shape, presumably because pre-existing cyclin D levels are sufficient to drive cell cycle progression at the M-G1 border.

Role of signal transduction and actin in G1 phase progression.

Progression through the cell cycle of mammalian cells is dependent upon external factors such as growth- and ECM factors. These factors exert their effect predominantly during the G1 phase of the cell cycle. When cells are cultured in suspension or when growth factors are withdrawn from the medium, cells will stop cell cycle progression and enter a quiescent state. Cells will remain in this quiescent state until extracellular conditions change and cells are stimulated to re-enter the cell cycle. This stimulation is mediated by various signal transduction cascades such as the mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol 3-kinase (PI3-kinase) pathway. In Chinese hamster ovary cells at least two serum-dependent points exist during G1 phase that lead to diffent cellular responses. The first point is located immediately after mitosis and is suggested to link with apoptosis. The second point is located in late G1 phase and probably corresponds with cellular differentiation. Signal transduction is mutually related to the cytoskeleton, especially the actin microfilament system. The actin microfilament system influences signal transduction and several signal transduction pathways influence the actin structure. Here we describe the role of the MAPK and PI3-kinase activities and of actin microfilaments in progression through the cell cycle and their role in the two G1 checkpoints.