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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 24.5Mutations Affecting Genome Stability

We have seen that the DNA of cancer cells contains insertions, deletions, and point mutations in multiple key growth and cell-cycle regulatory genes; in addition, several genes often are amplified and chromosome breaks and fusions (translocations) generate oncogenic chimeric proteins. Most cancer cells also lack one or more DNA-repair systems, which may explain the large number of mutations that they accumulate. Additionally, cancer cells are defective in one or more checkpoint controls, which normally prevent cells with damaged DNA or abnormal chromosomes from dividing or that cause such cells to undergo programmed death (see Figure 13-34).

Mutations in p53 Abolish G1 Checkpoint Control

The p53 protein is essential for the checkpoint control that arrests human cells with damaged DNA in G1, and mutations in the p53 gene occur in more than 50 percent of human cancers (Figure 24-21a). As detailed in Chapter 13, cells with functional p53 arrest in G1 when exposed to γ irradiation, whereas cells lacking functional p53 do not (see Figure 13-35). Unlike other cell-cycle proteins, p53 is expressed 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. Rather, p53 is activated only in stressful situations, such as ultraviolet or γ irradiation, heat, and low oxygen. DNA damage by γ irradiation or by other stresses somehow leads to the activation of certain kinases, including ATM, which is encoded by the gene mutated in ataxia telangiectasia, and a DNA-dependent protein kinase. Phosphorylation of p53 by these and other kinases results in its stabilization and thus in a marked increase in its concentration (Figure 24-21b).

Figure 24-21. The human p53 protein.

Figure 24-21

The human p53 protein. (a) Mutations in human tumors that inactivate the function of p53 protein. Hatched boxes represent sequences highly conserved in evolution. Vertical lines represent the frequency at which mutations are found at each residue in various (more...)

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. The most important protein relative to cellcycle control whose transcription is induced by p53 is the cyclin-kinase inhibitor p21, which binds to and inhibits mammalian G1 Cdk-cyclin complexes. As a result, cells with damaged DNA are arrested in G1 until the damage is repaired and the levels of p53 and p21 fall; the cells then can progress into the S phase. In most cells, accumulation of p53 also leads to induction of proteins that promote apoptosis. While this may seem like a drastic response to DNA damage, it prevents proliferation of cells that are likely to accumulate multiple mutations. When the p53 checkpoint control does not operate properly, damaged DNA can replicate, producing mutations and DNA rearrangements that contribute to the development of a highly transformed, metastatic cell. p53 also is important for G2-to-M checkpoint control, though the mechanism is only now being unraveled.

The active form of p53 is a tetramer of four identical subunits. A missense point mutation in one p53 of the two 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,” in contrast to tumor-suppressor genes such as RB. Since the Rb protein functions as a monomer, mutation of a single RB allele has little functional consequence.

Proteins that interact with and regulate p53 are also altered in many human tumors. The gene encoding one such protein, MDM2, is amplified in many sarcomas and other human tumors that maintain functional p53. Under normal conditions MDM2 protein binds to a site in the N-terminus of p53, both repressing the ability of p53 to activate transcription of p21 and other genes and mediating p53 degradation. Thus MDM2 normally inhibits the ability of p53 to restrain the cell cycle or kill the cell. Phosphorylation of p53 by ATM, as after γ irradiation, leads to displacement of bound MDM2 and thus stabilization of p53. 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. Enhanced MDM2 levels in tumor cells would cause a decrease in the concentration of functional p53 and abolish the ability of p53 to arrest a cell in response to irradiation.

The consequences of mutations in p53 and Mdm2 gene amplifications provide a dramatic example of the significance of cell-cycle checkpoints to the health of a multicellular organism. Germ-line p53 mutations are also known; p53 is mutated in the Li-Fraumeni syndrome of multiple inherited cancers.

Proteins Encoded by DNA Tumor Viruses Can Inhibit p53 Activity

We noted that human papillomavirus (HPV) is able to induce stable transformation and transient mitogenic stimulation of a variety of cultured cells. One HPV protein, E7, binds to and inhibits Rb, while another, E6, inhibits p53 (see Figure 24-21b). 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.

Similarly, SV40, a small transforming DNA monkey papovavirus, makes two early proteins called T (large T) and t (small t) formed by alternative splicing from the same reading frame. Large T, also called “T antigen,” is a 90-kDa protein found in the nucleus of infected cells; different domains of large T bind to p53 and Rb, inhibiting their function. Because large T inhibits both proteins, expression of only the SV40 large T protein is sufficient to induce transformation of cultured cells. (As discussed in Chapter 12, another domain of large T binds to the origin of SV40 DNA and initiates replication of SV40 DNA.) SV40 was isolated as a contaminant of the first poliovirus vaccine. Despite being injected into millions of children, there is no evidence that SV40 can induce human tumors or that SV40 DNA is found in human tumor cells. Nonetheless, both HPV and SV40 provide illuminating examples of mechanisms by which small DNA viruses induce cell transformation.

Some Human Carcinogens Cause Inactivating Mutations in the p53 Gene

As noted in Chapter 12, most carcinogenic agents cause DNA damage. Epidemiological studies established that cigarette smoking is the major cause of lung cancer, but precisely how this happens was not clear until the discovery that about 60 percent of human lung cancers contain inactivating mutations in the p53 gene. The chemical benzo(a)pyrene, found in cigarette smoke, undergoes metabolic activation in the liver to a potent mutagen that causes mainly G to T transversion mutations. When applied to cultured bronchial epithelial cells, activated benzo(a)pyrene induces inactivating mutations at codons 175, 248, and 273 of the p53 gene; these same positions are major mutational hot spots in human lung cancer (see Figure 24-21a). Thus, there is a direct link between a defined chemical carcinogen in cigarette smoke and human cancer; it is likely that cigarette smoke induces mutations in other genes as well.

Lung cancer is not the only major human cancer for which a clear-cut risk factor has been identified. Aflatoxin, a fungal metabolite found as a contaminant in moldy grains, induces liver cancer, a disease whose incidence is high in China. Aflatoxin induces a G to T transversion at codon 249 of p53, leading to its inactivation. Exposure to other chemicals has been correlated with minor cancers. However, hard evidence concerning dietary and environmental risk factors that would help us avoid breast, colon, and prostate cancer, leukemias, and other cancers is generally lacking.

Defects in DNA-Repair Systems Perpetuate Mutations and Are Associated with Certain Cancers

A link between carcinogenesis and failure of DNA repair is suggested by the finding that humans with inherited genetic defects in certain repair systems have an enormously increased probability of developing certain cancers (Table 24-1). One such disease is xeroderma pigmentosum, an autosomal recessive disease. Individuals with this disease get the skin cancers called melanomas and squamous cell carcinomas very easily if their skin is exposed to the UV rays in sunlight. Cells of affected patients are unable to repair UV damage or to remove bulky chemical substituents on DNA bases. Such damage commonly is repaired by the excision-repair mechanism (see Figure 12-26). The complexity of mammalian excision-repair systems is shown by the fact that mutations in at least seven different genes lead to xeroderma pigmentosum lesions, all having the same phenotype and the same consequences. Genetic evidence suggests that in yeast several RAD genes encode proteins required for excision repair. The predicted protein product encoded by the yeast RAD14 gene has considerable homology with the protein encoded by one of the genes that is mutated in some xeroderma pigmentosum patients, attesting to the conservation of the excision-repair system during evolution of eukaryotes.

Table 24-1. Human Hereditary Diseases Associated with DNA-Repair Defects.

Table 24-1

Human Hereditary Diseases Associated with DNA-Repair Defects.

Three Mut proteins participate in mismatch repair in bacteria (see Figure 12-24). Mutations in genes encoding the human homologs of the bacterial MutS or MutL repair proteins also have been associated with higher-than-normal cancer risks. Once cells lose the ability to repair mismatch errors in DNA, mutations in many other genes can accumulate, including those that are critical in controlling cell growth and proliferation. For instance, hereditary nonpolyposis colorectal cancer, one of the most common inherited predispositions to cancer, results from an inherited loss-of-function mutation in one allele of the gene encoding human MutSα, MutSβ, or MutL. Cells with one functional copy of any of these genes exhibit normal mismatch repair, but tumor cells frequently arise from those cells that have experienced a somatic mutation in the second allele and thus have lost the mismatch-repair system. Thus MutSα, MutSβ, and MutL are tumor-suppressor genes, and somatic inactivating mutations in these genes are also common in noninherited forms of colon cancer.

One gene frequently mutated in colon cancers because of the absence of mismatch repair encodes the type II receptor for TGFβ. The gene encoding the type II receptor contains an A10 sequence that frequently undergoes mutation to A9 or A11 because of “slippage” of DNA polymerase during replication. If unrepaired by the mismatch-repair system, these mutations cause a frame shift in the protein-coding sequence that abolishes production of the normal receptor protein. As noted earlier, such inactivating mutations make cells resistant to growth inhibition by TGFβ, thereby contributing to the unregulated growth characteristic of these tumors. This finding attests to the importance of mismatch repair in correcting genetic damage that might otherwise lead to uncontrolled cell proliferation. Additionally, recognition of certain DNA lesions by MutSα initiates a sequence of events that leads to cell death, and the absence of MutSα may allow the survival of cells with major DNA damage, cells that could progress to become tumors.

Chromosomal Abnormalities Are Common in Human Tumors

It has long been known that chromosomal abnormalities abound in tumor cells. Human cells ordinarily have 23 pairs of chromosomes, recognized by their well-defined substructure, but tumor cells are usually aneuploid (i.e., they have an abnormal number of chromosomes — generally too many), and they often contain translocations (fused elements from different chromosomes). Cells with abnormal numbers of chromosomes form when the S-phase or mitotic checkpoints are nonfunctional. The first condition normally prevents entry into mitosis unless all chromosomes have completely replicated their DNA, while the latter causes arrest in anaphase unless all the replicated chromosomes are appropriately lined up to be segregated properly (see Figure 13-34). Defects in these checkpoint controls are common in tumor cells; the molecular basis for these defects is now being uncovered as the checkpoint control proteins themselves are being identified.

As a rule, these chromosomal abnormalities are not the same from tumor to tumor: each tumor has its own set of anomalies. Certain anomalies recur, however, and they point to the presence of oncogenes (Figure 24-22, see Figure 9-38a). The first to be discovered, the Philadelphia chromosome, is found in hematopoietic cells of virtually all patients with the disease chronic myelogenous leukemia. This chromosome results from a translocation between chromosomes 9 and 22; which causes formation of the chimeric bcr-abl oncogene discussed earlier. We’ve also discussed the translocation of the c-myc gene seen in Burkitt’s lymphoma, resulting in abnormal expression of the Myc transcription factor. In this case, c-myc, normally located on chromosome 8, is moved to a site on chromosome 14 near an antibody-gene enhancer. In other lymphomas, a different chromosomal translocation brings the anti-apoptotic gene bcl-2 under the transcriptional control of an antibody enhancer. The resultant inappropriate overexpression of the Bcl-2 protein prevents normal apoptosis and allows survival of these tumor cells.

Figure 24-22. Chromosomal translocation in Burkitt’s lymphoma.

Figure 24-22

Chromosomal translocation in Burkitt’s lymphoma. This leads to overexpression of the Myc transcription factor.

Another common chromosomal anomaly in tumor cells is the localized reduplication of DNA to produce as many as 100 copies of a given region (usually a region spanning hundreds of kilobases). This anomaly may take either of two forms: the duplicated DNA may be tandemly organized at a single site on a chromosome, or it may exist as small, independent chromosomelike structures. The former case leads to a homogeneously staining region (HSR) that is visible in the light microscope at the site of the duplication; the latter case causes double minute chromosomes to pepper a stained chromosomal preparation (Figure 24-23). Again, oncogenes have been found in the duplicated regions. Most strikingly, the myc-related gene called N-myc has been identified in both HSRs and double minute chromosomes of human nervous system tumors.

Figure 24-23. Visible DNA amplifications.

Figure 24-23

Visible DNA amplifications. (a) Homogeneously staining regions (HSRs) in chromosomes from two neuroblastoma cells. In each set of three chromosomes, the left-most one is a normal chromosome 1 and the other two are HSR-containing chromosomes. The three (more...)

We noted that the p16 tumor-suppressor gene is inactivated in many human cancers. In many cases the gene has been deleted, but in others the sequence of the entire p16 gene is normal. To explain this, recall that gene expression is controlled at multiple levels, and that one of these involves the state of DNA methylation in long segments along a chromosome. Indeed, the p16 promoter is inactivated by hypermethylation in many human tumors, including lung cancer. How this epigenetic change happens is not known, but it does lead to nonproduction of this important cell-cycle control protein.

Telomerase Expression May Contribute to Immortalization of Cancer Cells

Telomeres, the physical ends of linear chromosomes, consist of tandem arrays of a short DNA sequence, TTAGGG in vertebrates. Telomeres provide the solution to the end-replication problem — the inability of DNA polymerases to completely replicate the end of a double-stranded DNA molecule. In the germ line and in rapidly dividing somatic cells, such as stem cells, telomerase, a reverse transcriptase that contains an RNA template, adds TTAGGG repeats to chromosome ends (see Figure 12-13). Because most human somatic cells lack telomerase, telomeres shorten with each cell cycle. Complete loss of telomeres leads to end-to-end chromosome fusions and cell death.

Most tumor cells overcome this barrier by expressing telomerase. Many workers believe that telomerase expression is essential for a tumor cell to become immortal, and specific inhibitors of telomerase have been suggested as cancer therapeutic agents. Surprisingly, however, mice homozygous for a deletion of the RNA subunit of the telomerase gene are both viable and fertile. When treated with carcinogens, these telomerase-null mice develop tumors to the same extent as normal mice, establishing that in mice, activation of telomerase is not essential for tumorigenesis. Whether the same applies to humans is a matter of controversy, since mice have much longer telomeres than humans and mouse cells may not require continuous telomerase expression.

SUMMARY

  •  The p53 protein is essential for the checkpoint control that arrests human cells with damaged DNA in G1. Replication of such cells would tend to perpetuate mutations. p53 functions as a transcription factor to induce expression of p21, an inhibitor of G1 Cdk-cyclin complexes.
  •  Mutations in the p53 gene occur in more than 50 percent of human cancers.
  •  Because p53 is a tetramer, a point mutation in one p53 allele can be sufficient to inhibit all p53 activity.
  •  MDM2, a protein that normally inhibits the ability of p53 to restrain the cell cycle or kill the cell, is overexpressed in several cancers.
  •  Defects in cellular DNA-repair processes found in certain human diseases are associated with an increased susceptibility for certain cancers (see Table 24-1).
  •  Chromosome abnormalities, including aneuploidy and translocations, are common in human tumors, and often result in duplications of oncogenes such as myc.
  •  Cancer cells, like germ cells and stem cells but unlike most differentiated cells, express telomerase, which may contribute to their immortalization.
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By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, W. H. Freeman and Company.
Bookshelf ID: NBK21551