▲ EXPERIMENTAL FIGURE 23-22 Mutations in human tumors that inactivate the function of p53 protein are highly concentrated in a few residues. Colored boxes represent sequences in the p53 gene that are highly conserved in evolution. Vertical lines represent the frequency at which point mutations are found at each residue in various human tumors.

These mutations are clustered in conserved regions II-V. The locations in the protein sequence of the most frequently occurring point mutations are labeled. In these labels, R = arginine; G = glycine. [Adapted from C. C. Harris, 1993, Science 262:1980, and L. Ko and C. Prives, 1996, Genes & Develop. 10:1054.]

S phase (see Figure 21-32, step 4a). The p53 protein is a sensor essential for the checkpoint control that arrests cells with damaged DNA in G1. Although p53 has several functions, its ability to activate transcription of certain genes is most relevant to its tumor-suppressing function. Virtually all p53 mutations abolish its ability to bind to specific DNA sequences and activate gene expression. Mutations in the p53 tumor-suppressor gene occur in more than 50 percent of human cancers (Figure 23-22).

Cells with functional p53 become arrested in G1 when exposed to DNA-damaging irradiation, whereas cells lacking functional p53 do not. Unlike other cell-cycle proteins, p53 is present at very low levels in normal cells because it is extremely unstable and rapidly degraded. Mice lacking p53 are viable and healthy, except for a predisposition to develop multiple types of tumors. Expression of the p53 gene is heightened only in stressful situations, such as ultraviolet or y irradiation, heat, and low oxygen. DNA damage by y irradiation or by other stresses somehow leads to the activation of ATM, a serine kinase that phosphorylates and thereby stabilizes p53, leading to a marked increase in its concentration (Figure 23-23). The stabilized p53 activates expression of the gene encoding p21CIP, which binds to and inhibits mammalian G1 cyclin-CDK complexes. As a result, cells with damaged DNA are arrested in G1, allowing time for DNA repair by mechanisms discussed later. If repair is successful, the levels of p53 and p21CIP will fall, and the cells then can progress into the S phase.

When the p53 G1 checkpoint control does not operate properly, damaged DNA can replicate, perpetuating mutations and DNA rearrangements that are passed on to daughter cells, contributing to the likelihood of transformation into metastatic cells. In addition, p21CIP and two other proteins induced by p53 inhibit the cyclin B-CDK1 complex required for entry into mitosis, thus causing cells to arrest in G2 (see Figure 21-32, step 4d). p53 also represses expression

▲ FIGURE 23-23 G1 arrest in response to DNA damage. The kinase activity of ATM is activated in response to DNA damage due to various stresses (e.g., UV irradiation, heat). Activated ATM then triggers two pathways leading to arrest in Gv Phosphorylation of p53 stabilizes it, permitting p53-activated expression of genes encoding proteins that (1) cause arrest in G-i and in some cases G2, ( 2) promote apoptosis, or ( 3) participate in DNA repair. In the other pathway phosphorylated Chk2 in turn phosphorylates Cdc25A, thereby marking it for degradation and blocking its role in CDK2 activation. See the text for a discussion.

of the genes encoding cyclin B and topoisomerase II, which also are required for the G2 n mitosis transition. Thus if DNA is damaged following its replication, p53-induced G2 arrest will prevent its transmission to daughter cells.

The active form of p53 is a tetramer of four identical sub-units. A missense point mutation in one of the two p53 alleles in a cell can abrogate almost all p53 activity because virtually all the oligomers will contain at least one defective subunit, and such oligomers cannot function as a transcription factor. Oncogenic p53 mutations thus act as dominant negatives, with mutations in a single allele causing a loss of function. As we learned in Chapter 9, dominant-negative mutations can occur in proteins whose active forms are multimeric or whose function depends on interactions with other proteins. In contrast, loss-of-function mutations in other tumor-suppressor genes (e.g., RB) are recessive because the encoded proteins function as monomers and mutation of a single allele has little functional consequence.

Under stressful conditions, the ATM kinase also phospho-rylates and thus activates Chk2, a protein kinase that phos-phorylates the protein phosphatase Cdc25A, marking it for ubiquitin-mediated destruction. This phosphatase removes the inhibitory phosphate on CDK2, a prerequisite for cells to enter the S phase. Decreased levels of Cdc25A thus block progression into and through the S phase (see Figures 23-23 and 21-32, step 4b). Loss-of-function mutations in the ATM or Chk2 genes have much the same effect as p53 mutations.

The activity of p53 normally is kept low by a protein called Mdm2. When Mdm2 is bound to p53, it inhibits the transcription-activating ability of p53 and catalyzes the addition of ubiquitin molecules, thus targeting p53 for protea-somal degradation. Phosphorylation of p53 by ATM displaces bound Mdm2 from p53, thereby stabilizing it. Because the Mdm2 gene is itself transcriptionally activated by p53, Mdm2 functions in an autoregulatory feedback loop with p53, perhaps normally preventing excess p53 function. The Mdm2 gene is amplified in many sarcomas and other human tumors that contain a normal p53 gene. Even though functional p53 is produced by such tumor cells, the elevated Mdm2 levels reduce the p53 concentration enough to abolish the p53-induced G1 arrest in response to irradiation.

The activity of p53 also is inhibited by a human papillomavirus (HPV) protein called E6. Thus HPV encodes three proteins that contribute to its ability to induce stable transformation and mitosis in a variety of cultured cells. Two of these—E6 and E7—bind to and inhibit the p53 and Rb tumor suppressors, respectively. Acting together, E6 and E7 are sufficient to induce transformation in the absence of mutations in cell regulatory proteins. The HPV E5 protein, which causes sustained activation of the PDGF receptor, enhances proliferation of the transformed cells.

The activity of p53 is not limited to inducing cell-cycle arrest. In addition, this multipurpose tumor suppressor stimulates production of pro-apoptotic proteins and DNA-repair enzymes (see Figure 23-23).

Apoptotic Genes Can Function as Proto-Oncogenes or Tumor-Suppressor Genes

During normal development many cells are designated for programmed cell death, also known as apoptosis (see Chapter 22). Many abnormalities, including errors in mitosis, DNA damage, and an abnormal excess of cells not needed for development of a working organ, also can trigger apop-tosis. In some cases, cell death appears to be the default situation, with signals required to ensure cell survival. Cells can receive instructions to live and instructions to die, and a complex regulatory system integrates the various kinds of information.

If cells do not die when they should and instead keep proliferating, a tumor may form. For example, chronic lym-phoblastic leukemia (CLL) occurs because cells survive when they should be dying. The cells accumulate slowly, and most are not actively dividing, but they do not die. CLL cells have chromosomal translocations that activate a gene called bcl-2, which we now know to be a critical blocker of apoptosis (see Figure 22-31). The resultant inappropriate overproduction of Bcl-2 protein prevents normal apoptosis and allows survival of these tumor cells. CLL tumors are therefore attributable to a failure of cell death. Another dozen or so proto-oncogenes that are normally involved in negatively regulating apoptosis have been mutated to become oncogenes. Overproduction of their encoded proteins prevents apopto-sis even when it is needed to stop cancer cells from growing.

Conversely, genes whose protein products stimulate apop-tosis behave as tumor suppressors. An example is the PTEN gene discussed in Chapter 14. The phosphatase encoded by this gene dephosphorylates phosphatidylinositol 3,4,5-trisphosphate, a second messenger that functions in activation of protein kinase B (see Figure 14-27). Cells lacking PTEN phosphatase have elevated levels of phosphatidylinositol 3,4,5-trisphosphate and active protein kinase B, which promotes cell survival and prevents apoptosis by several pathways. Thus PTEN acts as a pro-apoptotic tumor suppressor by decreasing the anti-apoptotic effect of protein kinase B.

The most common pro-apoptotic tumor-suppressor gene implicated in human cancers is p53. Among the genes activated by p53 are several encoding pro-apoptotic proteins such as Bax (see Figure 22-32). When most cells suffer extensive DNA damage, the p53-induced expression of pro-apoptotic proteins leads to their quick demise (see Figure 23-23). While this may seem like a drastic response to DNA damage, it prevents proliferation of cells that are likely to accumulate multiple mutations.

HWhen p53 function is lost, apoptosis cannot be induced and the accumulation of mutations required for cancer to develop becomes more likely. Tumors marked by loss of p53 or another gene needed for apoptosis are difficult to treat with chemical or radiation therapy, since the resulting DNA damage is not translated into programmed cell death. I

Failure of Cell-Cycle Checkpoints Can Also Lead to Aneuploidy in Tumor Cells

It has long been known that chromosomal abnormalities abound in tumor cells. We have already encountered several examples of oncogenes that are formed by translocation, amplification, or both (e.g., c-myc, bcr-abl, bcl-2, and cyclin D1). Another chromosomal abnormality characteristic of nearly all tumor cells is aneuploidy, the presence of an aberrant number of chromosomes—generally too many.

Cells with abnormal numbers of chromosomes form when certain cell-cycle checkpoints are nonfunctional. As discussed in Chapter 21, the unreplicated-DNA checkpoint normally prevents entry into mitosis unless all chromosomes have completely replicated their DNA; the spindle-assembly checkpoint prevents entry into anaphase unless all the replicated chromosomes attach properly to the metaphase mitotic apparatus; and the chromosome-segregation checkpoint prevents exit from mitosis and cytokinesis if the chromosomes segregate improperly (see Figure 21-32, steps 1-[3]). As advances are made in identifying the proteins that detect these abnormalities and mediate cell-cycle arrest, the molecular basis for the functional defects leading to aneuploidy in tumor cells will become clearer.

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