The Cell Cycle And Checkpoint Function

To understand the potential involvement of the cell cycle and its regulators in cell death, it is important to first define the "normal" cell cycle and the role of checkpoint function. Cell division requires the replication of genetic material and the partitioning of nuclear and cytosolic contents into two daughter cells, each essentially identical to the parent. In most cases, this is accomplished by the orderly progression through four phases, Gi (gap phase), S (DNA synthesis phase), G2 (gap phase), and M (mitosis phase) and is schematically represented in Fig. 1. Transition through the phases of the cell cycle is mediated by a family of serine/threonine protein kinases, called cyclin-dependent kinases (Cdks), which are expressed and become activated in a sequential fashion (31,32). The activation of the Cdks is controlled by a series of regulations, such as binding to their cognate cyclin partner (33,34), activating and inactivating post-translational modifications (35,36), and inhibition by protein-protein interaction with inhibitors (37,38). These regulations not only coordinate progression but also link the cell cycle, and cell replication, to both intracellular and extracellular signals.

Cdks promote progression through the cell cycle by phosphorylating key substrate targets, such as the G1 pocket proteins, pRb, p107, and p130 (39). The best characterized of these, pRb, is phosphorylated in mid-G1 by Cdk4/6, which complexes with cyclin D, and in late-G1 by Cdk2, which complexes with cyclin E. These phosphorylation events inactivate pRb resulting in the derepression of E2F transcription factors, which induce the expression of genes controlling S-phase and subsequent cell cycle progression (40). E2F-dependent expression of the phosphatase, Cdc25A, is required for the dephosphory-lation and activation of the cyclin A-Cdk2 complex, which marks the transition from G1-to S-phase and the onset of DNA synthesis (36,41). Later in S-phase, cyclin A complexes with Cdc2 (Cdk1), promoting the cell cycle through to the G2/M transition point. For mitosis to occur, cells must progress through the G2/M transition, which requires another set of Cdk-regulated events. The cyclin B-Cdc2 complex, originally defined as maturation-promoting factor (MPF), induces M-phase and mediates phosphorylation-induced changes in the microtubule network, actin filaments, and the nuclear lamina (42-45). One of the

Fig. 1. The cell cycle: A schematic representation of the four phases of the cell cycle. Specific cyclin-Cdk complexes regulate progression through Gl-phase by the phosphorylation and inactivation of the retinoblastoma protein (pRb). Inactivation of pRb derepresses E2F transcription factors, inducing the genes required for S-phase. Progression through G2- and M-phases of the cell cycle is also regulated by cyclin-Cdk activity.

Fig. 1. The cell cycle: A schematic representation of the four phases of the cell cycle. Specific cyclin-Cdk complexes regulate progression through Gl-phase by the phosphorylation and inactivation of the retinoblastoma protein (pRb). Inactivation of pRb derepresses E2F transcription factors, inducing the genes required for S-phase. Progression through G2- and M-phases of the cell cycle is also regulated by cyclin-Cdk activity.

targets of MPF is also the anaphase-promoting complex (APC), a multimeric ubiquitin ligase that initiates and coordinates chromatin separation, spindle disassembly, cytokinesis, and mitotic exit (46).

While Cdks are the workhorses of the cell cycle, promoting progression through the various phases of the cell cycle, they are regulated by two families of cyclin-dependent kinase inhibitors (CKIs), Kip/Cip and INK4 (37,38). Members of the Kip/Cip family of proteins (p21Cip1, p27Kip1, and p57Kip2) regulate the activity of all the G1 cyclin-Cdk complexes and, to a lesser extent, cyclin B-Cdc2, by associating with the preactivated cyclin-Cdk complexes. In contrast, members of the INK4 family (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) specifically interact with monomeric Cdk4 and Cdk6 thereby preventing their activation through D-type cyclin binding. The expression of many of the CKIs is regulated negatively by mitogenic stimuli and positively by growth suppression factors.

At each phase of the cell cycle, elaborate feedback mechanisms called checkpoints function as molecular switches to tightly control transition from one phase to the next, ensuring critical events in one phase of the cell cycle are completed before the next phase is started, and halting the cell cycle if damage to the mitotic apparatus is detected (47,48).

If the damage to the mitotic apparatus is irreparable or a loss of cell cycle coordination occurs, cell death results. For example, the uncoupling of M-phase-activating events from the S-phase checkpoint by the premature activation of cyclin and Cdk proteins triggers an attempt of aberrant chromosome segregation, termed mitotic catastrophe, which culminates in the induction of apoptosis and cellular demise (47,49,50).

Damage to DNA is another potent stimulator of checkpoint function. By attenuating cell cycle progression and DNA synthesis, checkpoints afford repair mechanisms extra time to remove DNA lesions and allow disrupted replication forks to recover. Furthermore, checkpoints may also play active roles in stimulating and coordinating the DNA repair and replication fork recovery (51). The end targets of the checkpoints therefore include components of the cell cycle, DNA replication, and DNA repair machinery. One of the most important sensors of DNA damage is the p53 tumor suppressor protein. In response to DNA damage p53 inhibits cell cycle progression at the G1 checkpoint, largely through the induction of the CKI, p21Cip1, and activates DNA repair enzymes (52,53). Although CKIs were originally thought to be specific for Cdks, p21Cip1 can interact and inhibit other classes of protein kinases. p21Cip1 can associate with and inhibit the apoptotic-inducing stress mitogen-activated protein kinases (MAPKs) of the JNK (SAPK) and p38 families (54) as well as the SAPK and p38 upstream activator, apoptosis signal-regulating kinase 1 (ASK1) (55,56). p21Cip1 has also been shown to directly inhibit the activation of caspase 3 (57) and indirectly inhibit caspases 8 and 10 (58), suggesting that its induction by p53 may not only be related to cell cycle arrest, but also be part of the cell survival decision. If DNA repair is not possible, p53 can directly induce a program of cell death by stimulating the expression of proapoptotic genes including bax and apaf-1 (48,59-61). Collectively, these checkpoints are specialized damage control mechanisms responsible for maintaining genetic integrity by minimizing the risk of DNA lesions being converted into inheritable mutations (62). The fact that checkpoints can halt cell cycle progression and direct a program of apoptosis clearly demonstrates that the cell cycle is a highly coordinated event and its proper regulation crucial for cell viability. Indeed, checkpoint function may play a critical role in the demise of terminally differentiated postmitotic neurons in which reactivation of cell cycle components and uncoordinated cell cycle reentry occurs.

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