Cell Cycle Checkpoints

The role of various CDKs, cyclins, and other gene products in regulating checkpoints at G1 to S, G2 to M, and mitotic spindle segregation have been described in detail elsewhere.156-158 Alterations of one or more of these checkpoint controls occur in most, if not all, human cancers at some stage in their progression to invasive cancer. Examples of some of these alterations are given below.

A key player in the G1-S checkpoint system is the retinoblastoma gene rb. Phosphorylation of the Rb protein by cyclin D-dependent kinase releases Rb from the transcriptional regulator E2F and activates E2F function. Inactivation of rb by genetic alterations occurs in retinoblas-toma and is also observed in other human cancers, for example, small cell lung carcinomas and osteogenic sarcomas.

The p53 gene product is an important cell cycle checkpoint regulator at both the G1-S and G2-M checkpoints but does not appear to be important at the mitotic spindle checkpoint because gene knockout of p53 does not alter mitosis. The p53 tumor suppressor gene is the most frequently mutated gene in human cancer, indicating its important role in conservation of normal cell cycle progression. One of p53's essential roles is to arrest cells in G1 after geno-toxic damage, to allow for DNA repair prior to DNA replication and cell division. In response to massive DNA damage, p53 triggers the apo-ptotic cell death pathway. Data from short-term cell-killing assays, using normal and minimally transformed cells, have led to the conclusion that mutated p53 protein confers resistance to genotoxic agents.

The spindle assembly checkpoint machinery involves genes called bub (budding uninhibited by benomyl) and mad (mitotic arrest deficient).158 There are three bub genes and three mad genes involved in the formation of this checkpoint complex. A protein kinase called Mps1 also functions in this checkpoint function. The chromosomal instability, leading to aneu-ploidy in many human cancers, appears to be due to defective control of the spindle assembly checkpoint. Mutant alleles of the human bubl gene have been observed in colorectal tumors displaying aneuploidy. Mutations in these spindle checkpoint genes may also result in increased sensitivity to drugs that affect microtu-bule function because drug-treated cancer cells do not undergo mitotic arrest and go on to die.

Maintaining the integrity of the genome is a crucial task of the cell cycle checkpoints. Two checkpoint kinases, called Chkl and Chk2

(also called Cdsl), are involved in checkpoint controls that affect a number of genes involved in maintenance of genome integrity (Fig. 4—9; see color insert). Chkl and Chk2 are activated by DNA damage and initiate a number of cellular defense mechanisms that modulate DNA repair pathways and slow down the cell division cycle to allow time for repair. If DNA is not successfully mended, the damaged cells usually undergo cell death via apoptosis (see below). This process prevents the defective

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Figure 4-9. Chkl and Chk2 as mediators of the checkpoint signaling network. Following their activation, Chkl and Chk2 phosphorylate unique (green and red, respectively) and overlapping (blue) downstream effectors that further propagate the checkpoint signaling. Depending on the type of stress, velocity of DNA damage, and cellular context, this leads to (l) switch to the stress-induced transcription program (E2Fl, Brcal, p53); (2) direct or indirect initiation of DNA repair (BRCAl, p53); (3) acute delay (degradation of Cdc25A) and/or sustained block (Cdc25C, p53, E2Fl); (4) apoptosis (Pmll, p53, E2Fl); and (5) modulation of the chromatin remodeling pathways (Tlk l/2). The known target sites of Chkl (green), Chk2 (red), and both Chkl and Chk2 (blue) on the individual substrates are shown. Some of the Chkl and Chk2 downstream effectors are classified as proto-oncogenes (PO) or tumor suppressors (TS), as indicated. (From Bartek and Lucas/59 reprinted with permission from Elsevier.)

Figure 4-9. Chkl and Chk2 as mediators of the checkpoint signaling network. Following their activation, Chkl and Chk2 phosphorylate unique (green and red, respectively) and overlapping (blue) downstream effectors that further propagate the checkpoint signaling. Depending on the type of stress, velocity of DNA damage, and cellular context, this leads to (l) switch to the stress-induced transcription program (E2Fl, Brcal, p53); (2) direct or indirect initiation of DNA repair (BRCAl, p53); (3) acute delay (degradation of Cdc25A) and/or sustained block (Cdc25C, p53, E2Fl); (4) apoptosis (Pmll, p53, E2Fl); and (5) modulation of the chromatin remodeling pathways (Tlk l/2). The known target sites of Chkl (green), Chk2 (red), and both Chkl and Chk2 (blue) on the individual substrates are shown. Some of the Chkl and Chk2 downstream effectors are classified as proto-oncogenes (PO) or tumor suppressors (TS), as indicated. (From Bartek and Lucas/59 reprinted with permission from Elsevier.)

genome from extending its paternity into daughter cells.

Upstream elements activating the checkpoint signaling pathways such as those turned on by irradiation or agents causing DNA doublestrand breaks include the ATM kinase, a member of the phosphatidylinositol 3-kinase (PI3K) family, that activates Chk2 and its relative ATR kinase that activates Chkl. There is also cross talk between ATM and ATR that mediates these responses (reviewed in Reference 159). Chkl and Chk2 phosphorylate CDC25A and C, which inactivate them. In its dephosporylated state CDC25A activates the CDK2-cyclin E complex that promotes progression through S phase. It should be noted that this is an example of dephosphorylation rather than phosphorylation activating a key biological function. This is in contrast to most signal transduction pathways, where the phosphorylated state of a protein (often a kinase) is the active state and the dephos-phorylated state is the inactive one (see Signal Transduction Mechanisms, below). In addition, Chkl renders CDC25A unstable, which also diminishes its activity (reviewed in Reference 160).

CDC25A also binds to and activates CDKl-cyclin B, which facilitates entry into mitosis. G2 arrest induced by DNA damage induces CDC25A degradation and, in contrast, G2 arrest is lost when CDC25A is overexpressed.

A number of proteins are now known to act as mediators of checkpoint responses by impinging on the Chkl and 2 pathways. These include the BRCT domain-containing proteins 53BP1, BRCA1, and MDC1. These proteins are involved in activation of Chk1 and Chk2 by acting through protein-protein interactions that modulate the activity of these checkpoint kinases. In genereal, these modulators are thought to be tumor suppressors.

Chk1 and 2 have overlapping roles in cell cycle regulation, but different roles during development. Chk1 but not Chk2 is essential for mammalian development, as evidenced by the early embryonic lethality of Chk1 knockout mice. Chk2-deficient mice are viable and fertile and do not have a tumor-prone phenotype unless exposed to carcinogens, and this effect is more evident later in life (reviewed in Reference 159). Rare germline mutations of Chk2 have been observed in cancer-prone patients with the Li-Fraumeni syndrome (LFS), thus Chk2 mutations maybe an alternative or overlapping genetic defect along with p53 mutations in these patients. Since LFS patients are susceptible to develop multiple types of tumors, including a predominant incidence of breast cancers and sarcomas, the Chk2 path may also be an important tumor suppressor for these tumors in non-LFS patients. Chk2 mutations have also been found in small subsets of "sporadic" human cancers, including carcinomas of the breast, lung, vulva, urinary bladder, colon, and ovary as well as in osteosarcomas and lym-phomas.159 In contrast, cancer-associated genetic defects in Chk1 are rare but have been observed in carcinomas of the colon, stomach, and endometrum.

As illustrated in Figure 4-9, there are interactions between the Chk kinases and the p53 pathway. Chk2 phosphorylates threonine-18 or serine-20 on p53, which attenuates p53's interaction with its inhibitor MDM2, thus contributing to p53 stabilization and activation. However, Chk2 and p53 only have partially overlapping roles in checkpoint regulation because not all DNA-damaging events activate both pathways. For example, some types of DNA damage that activate p53 do not activate Chk2 and vice versa. Thus, the two pathways are partly redundant and overlapping but not totally so, as evidenced by the fact that in Chk2-deficient cells, Chk1 can still phosphorylate and activate p53.

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