Chapter 110. Genome Instability, DNA Repair, and Cancer

The integrity of the genome of all living organisms is constantly threatened by exogenous and endogenous DNA-damaging agents. Exogenous DNA-damaging agents include physical agents, such as ultraviolet (UV) or ionizing radiation (IR), and a wide variety of chemical agents, such as components of cigarette smoke. Endogenous DNA damage arises from regular metabolic processes within the cell, mediated, for example, by reactive oxygen species. Maintaining the stability of the genome is of utmost importance to all living organisms. Therefore, since early evolution, all organisms ranging from prokaryotes to eukaryotes have been equipped with mechanisms that react to and repair DNA damage and thereby maintain genomic stability. The types of damage produced include alterations in the structure of nucleotides, DNA strand breaks, DNA cross-links, and DNA adducts. Different types of DNA-damaging agents induce different types of DNA damage (Table 110-1), which in turn require different responses and repair pathways for processing (Table 110-2).1

If DNA damage is not repaired adequately, it may lead to altered cell function, cell death, or the formation of mutations (alterations of the DNA sequence) in the damaged cells. These DNA damage-induced mutations will persist as long as the affected cell survives. At a cellular level, mutations in vital genes can lead to alterations of cell functions or malignant transformation. Accumulation of mutations may lead to organ dysfunction, aging, and cancer. Although most DNA damage is adequately repaired, none of the cellular responses is 100% effective in repairing all DNA damage under all circumstances.

A hereditary or acquired impairment in the way cells respond to DNA damage may result in genome instability with an increased rate of mutation formation. Numerous hereditary disorders are characterized by such genome instability (reviewed in Chapter 139). Many, but not all, of those are associated with an increased cancer risk and/or accelerated aging.

Exposure of the skin to UV radiation has multiple cellular and clinical effects, including an increase in skin cancer risk. The photocarcinogenesis cascade of events (Fig. 110-1) exemplifies the link between genome instability, DNA repair, and cancer. UV light produces a type of DNA damage involving the generation of photoproducts, which are alterations in the structure of nucleotides. The major DNA photoproducts are cyclobutane pyrimidine dimers (CPDs; Fig. 110-2) and 6,4-pyrimidine–pyrimidone dimers. Unrepaired CPDs or 6,4-pyrimidine–pyrimidone dimers may result in characteristic mutations: C to T single base and CC to TT tandem base substitution mutations.2–4 Such mutations are typical for UV light exposures and only rarely are induced by other mutagens. They have therefore been termed UV-signature mutations (see Fig. 110-2). Pleasance et al.5 sequenced all the genes in cells from a melanoma metastasis and reported that they harbor approximately 25,000 such mutations, accounting for about 70% of all mutations found. This clearly links the mutation burden in this cancer to prior UV-exposures.

DNA repair is an important cellular defense mechanism that prevents mutation formation at sites of DNA damage after UV exposure. However, it is not the only defense mechanism (see Fig. 110-1). Most mutations are generated during replication of damaged DNA. Therefore, a damage-induced arrest in cell cycling, which allows more time for repair, is another important cellular damage response that prevents mutation formation.6,7 Furthermore, programmed cell death (apoptosis) prevents the survival of cells with overwhelming DNA damage, and through that mechanism the frequency of cells with UV-induced mutations is also reduced.8 Other inherent defense mechanisms against the ultimately carcinogenic properties of UV light include increased melanogenesis and thickening of the epidermis and stratum corneum, which protect from future DNA damage. Other protection includes removal of mutated cells through host immune responses (see Fig. 110-1).

More than 100 DNA repair genes have been identified (http://sciencepark.mdanderson.org/labs/wood/DNA_Repair_Genes.html). The nucleotide excision repair (NER) pathway acts on DNA damaged by UV radiation, repairing CPDs and other photoproducts, as well as on DNA damaged by certain carcinogens (such as benzo-[a]-pyrene) (see Table 110-2). In NER, the damaged nucleotide is removed and replaced with undamaged DNA. A simplified schema of the NER system describing some of the many proteins that act in concert to repair UV-induced DNA damage is shown in Fig. 110-3. Defects in these repair genes can cause human diseases (Table 110-3), including xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD) (for details on these disorders, see Chapter 139). For instance, XP can be caused by a defect in any one of several genes involved in NER. Cells/patients with defects in the same gene are considered to be in the same complementation group, and in different complementation groups if different genes are affected. The term “complementation group” is based on cell fusion experiments, in which cells from different XP patients are fused to investigate if the DNA repair defect in the fused cells is corrected. If DNA repair in the fused cell is increased, each cell provides proteins that the other is lacking and the cells “complement” each other and are in different complementation groups. If DNA repair in the fused cells is not normalized, the cells do not “complement” each other, meaning that both cells harbor mutations in the same DNA repair gene. Seven such complementation groups have been identified (XP-A to XP-G), which correspond with mutations in seven distinct genes that can cause XP.

Transcribed genes are repaired faster than the rest of the genome. In the NER pathway, the first steps involving DNA damage recognition are different in nontranscribed (global genome NER) and transcribed genes (transcription-coupled NER). In nontranscribed genes and noncoding areas, which represent most of the genome, the XPE and XPC gene products bind to UV-damaged DNA, marking it for further processing. In contrast, DNA damage in transcribed genes appears to be sensed by a stalled RNA polymerase acting in conjunction with the CS complementation groups A and B (CSA and CSB) gene products. After the DNA damage recognition steps, global genome NER and transcription-coupled NER follow the same pathway. The XPA gene product functions in conjunction with replication protein A (RPA), the transcription factor IIH (TFIIH), XPF, and excision repair cross-complementing gene 1 (ERCC1). The following steps occur in both nontranscribed and transcribed gene repair:

• The XPB and XPD gene products partially unwind the DNA in the region of the damage, thereby exposing the lesion for further processing. These proteins are part of the TFIIH basal transcription factor (see Fig. 110-3B).
• The XPF gene product, in a complex with ERCC1, makes a single-strand nick at the 5′ side of the lesion, whereas the XPG gene product makes a similar nick on the 3′ side, which results in the release of a region of approximately 30 nucleotides containing the damage (see Fig. 110-3C).
• The resulting gap is filled by DNA polymerase using the other (undamaged) strand as a template in a process involving proliferating cell nuclear antigen (PCNA). DNA ligase 1 seals the region, restoring the original undamaged sequence (see Fig. 110-3D).

Other DNA repair pathways include base excision repair, recombination repair, and mismatch repair. Defects in mismatch repair are also associated with human diseases—Muir–Torre syndrome and human nonpolyposis colon cancer.1,9–11

Several cellular responses to DNA damage contribute to the maintenance of genome integrity, including: cell cycle arrest, apoptosis (programmed cell death), and DNA repair.6,12–19 These responses need to be carefully orchestrated, and there are many proteins involved in the signaling of DNA damage and the regulation of DNA damage responses. Different types of DNA-damaging agents and different types of DNA damage require different DNA damage responses. A simplified version of this complex pathway is presented in Figure 110-4. As with defects in DNA repair genes (see Table 110-3), defects in many of these DNA damage-signaling genes (boxed in Fig. 110-4) are also implicated in hereditary disorders of genome instability (Table 110-4; for further details see Chapter 139). These disorders are characterized by an increased cancer risk, which is due to the genome instability from impaired DNA damage signaling.

The tumor suppressor gene p53, termed the guardian of the genome,20 plays a pivotal role in regulating and orchestrating these responses and is mutated in many cancers, including cutaneous squamous cell carcinomas. Upstream regulators of p53 in the cellular DNA damage response pathway are ataxia telangiectasia mutated (ATM) and ataxia telangiectasia- and Rad3-related (ATR) genes. One of p53's several functions is the regulation of the cell cycle in response to DNA damage. After cell division (mitosis), cells have 23 pairs of chromosomes and are in the G1 phase of the cell cycle. The chromosomes then replicate during DNA synthesis, or S phase, and as a result have twice the number of chromosomes (G2 phase) just before mitosis (M phase). In response to damage, the cell may stop cycling (arrests) in specific cell cycle phases called cell cycle checkpoints. An important downstream effector in preventing cells from entering S phase (G1/S checkpoint) is p21.6,12 p53 also induces NER by transcriptionally inducing XPC, XPE/p48, and GADD45.21,22 This indicates that a cell's capacity to repair DNA damage can be upregulated in response to DNA damage.

If cells enter S phase with unrepaired DNA damage, or if cells are UV exposed during S phase, regular DNA polymerases stall at DNA photoproducts and fall off the DNA strand. For these instances, cells are equipped with several specialized DNA polymerases for translesional DNA synthesis.23 DNA polymerase eta is one of these; it is specialized to bypass DNA photoproducts but may introduce mutations while doing so.24 It is mutated in XP variant patients, who are clinically indistinguishable from other XP patients with defects in NER (see Table 110-3, Chapter 139).25,26 This demonstrates the importance of this second line of defense against the mutagenic and carcinogenic consequences of DNA photoproducts. p53 and p21 also downregulate the activity of this translesional DNA synthesis to maintain a low mutagenic activity at the price of reduced damage bypass.27

If this translesional DNA synthesis fails, cells can use recombination repair to resolve stalled replication forks.28 When invoked in response to damage from UV light, this third line of defense is mediated by activation of the Fanconi anemia/BRCA DNA damage response pathway.29 The exact mechanisms that initiate these DNA damage response signaling cascades is under investigation. Telomeres, which are repeats of TTAGGG that cap the ends of chromosomes and whose ends form a loop structure, are suggested to be important players in sensing DNA damage, for example, through opening of the telomere loop.30 Other sensors may be stalled DNA or RNA polymerases, or proteins that detect bending of the DNA helix at sites of DNA damage.

Apoptosis is a regulated physiologic process leading to cell death characterized by cell shrinkage, membrane blebbing, and DNA fragmentation. A group of cysteine proteases called caspases are central regulators of apoptosis. Triggers may be extrinsic or intrinsic to the cell (e.g., DNA damage) and involve separate initiator caspases (e.g., caspase 2 in response to DNA damage) but share the same downstream effector caspases.8

When a disorder of genome instability is suspected, the clinician is challenged with choosing the appropriate laboratory tests to secure a diagnosis and to provide guidance for affected patients and families. Table 110-5 lists some hallmark clinical features that may indicate the presence of such disorders and should prompt clinicians to initiate testing. Various laboratory tests for genome stability, DNA repair, and response to physical and chemical agents are listed in Table 110-6.

Use of Cultured Cells

Cells obtained directly from patients and grown in culture medium are termed primary cultures. Dermal fibroblasts generally grow easily in culture and can generally be established from a 2- to 4-mm sterile skin punch biopsy specimen. The inner surface of the upper arm has proven to be a suitable biopsy site because this area heals easily, the resulting scar is not readily visible, the site is shielded from UV radiation, and attempts to establish cultures from specimens are generally successful. The tissue is placed in sterile culture medium (or sterile saline) with antibiotics and transported to a cell culture laboratory at room temperature.

Human cell cultures are made available for research by the National Institutes of Health-funded Human Genetic Mutant Cell Repository (401 Haddon Ave, Camden, NJ 08103; telephone: 856–966-7377; http://ccr.coriell.org).

Diagnostic Tests of Genome Instability and DNA Repair

Tests to assess genome instability and/or DNA repair capacity may be divided into tests of intact cellular function and tests of chromosome integrity and breakage in response to DNA-damaging agents. Other tests measure the mechanism of impairment of a cell function such as DNA repair or characterize or determine the expression of defective genes (see Table 110-6).

Tests of Intact Cell Function

Tests of cellular function measure the capacity of the intact cell to recover from DNA damage. These tests do not provide information regarding the specific type of damage resulting in cellular injury or the mechanism of cellular recovery, but they do form the basis for identifying cells as hypersensitive to DNA-damaging agents and are often used as simple screening tests.

Cell Counts or Thymidine Incorporation

One of simplest tests after exposure to UV radiation or X-rays is the assessment of the growth rate in mass culture by using a microscope or an automated cell counter to count the number of cells or by measuring incorporation of radioactive thymidine into newly synthesized DNA (see Tables 110-6 and 110-7).

Colony-Forming Ability

A test of colony-forming ability assesses the capacity of a single cell to proliferate enough to form a visible colony (Fig. 110-5).

Tests of Chromosome Integrity and Breakage

Chromosome breakage is usually assessed in primary cultures of mitogen-stimulated peripheral blood leukocytes or in long-term cultures of fibroblasts or lymphoblastoid cell lines. Cell cycle progression is stopped at metaphase by treatment of the cells with an inhibitor of mitosis such as colchicine. In this procedure, the 23 pairs of metaphase chromosomes from a single cell are spread over a discrete area of the slide and stained (usually with Giemsa stain). Preparations may be analyzed for the number of chromosomes per metaphase, the morphology of the individual chromosomes, and the attachments or rearrangements of chromosomes in relation to each other.

Sister Chromatid Exchange

During DNA replication, chromatids occasionally exchange positions along the arms of a chromosome. This sister chromatid exchange (SCE) may be detected by permitting the cells to grow through two cycles of replication in medium containing the nucleic acid analog bromodeoxyuridine (BrUdR) (Fig. 110-6). SCEs are thought to be related to DNA recombination repair, although their precise significance is not understood.

Telomere Length

Shortened telomeres characterize cells from patients with dyskeratosis congenita. Several methods can be used to measure telomere length, including terminal restriction fragment (TRF) measurement on Southern blots, fluorescence in situ hybridization (FISH) with immunostaining, quantitative polymerase chain reaction (PCR), single telomere length analysis, and flow-cytometry with FISH (flow-FISH).31 Those tests are usually done on freshly isolated white blood cells, not cultured cells.

Tests of DNA Repair

Unscheduled DNA Synthesis

One of the most commonly used tests of NER is unscheduled DNA synthesis. This test has been used to measure DNA repair in intact human skin, in cultured epidermal or dermal cells, and in blood cells, and for prenatal diagnosis using amniotic fluid cells. Unscheduled DNA synthesis testing measures the repair-associated DNA synthesis in G1 or G2 cells (which usually do not synthesize DNA). Cells are treated with UV radiation or another DNA-damaging agent and then incubated in medium containing radioactive thymidine. During the process of NER, the damage is removed and the radioactive thymidine is incorporated into the repaired region. The cells are treated with fixative, coated with autoradiographic (photographic) emulsion, and kept in the dark for an appropriate interval, and then the emulsion is developed. UV radiation of normal fibroblasts results in a large increase in the number of grains seen over all the nuclei. In marked contrast, irradiation of the XP fibroblasts that cannot repair DNA damage results in very few grains over the nuclei.

RNA Synthesis Inhibition

After exposure to UV radiation, normal cells temporarily reduce RNA synthesis from active genes. Synthesis resumes when the damage is repaired. This resumption of RNA synthesis is delayed in cells from patients with CS and some forms of XP.

Host Cell Reactivation

The host cell reactivation assay relies on the fact that plasmids do not have the ability to repair damage to their DNA but depend on cellular repair systems. Thus, when transfecting plasmids with DNA damage into host cells, the DNA repair enzymes of the host cells need to repair the damage before the plasmids’ genes can be expressed. Therefore, damaged plasmids would be expected to be expressed at a higher level in cells with normal repair capacity. A nonreplicating plasmid that contains the gene for the firefly enzyme luciferase, constructed to permit expression in human cells,32 is widely used; generation of light provides a quantitative endpoint for its repair (Fig. 110-7).

Comet Assay

The comet assay is a single cell-based technique that allows detection and quantitation of DNA damage, in particular DNA strand breaks that were either introduced directly by the DNA-damaging agent or by repair endonucleases at sites of other types of DNA damage. For this assay, damaged cells are embedded in agarose, lysed, and exposed to an electric field. In the electrical field, the DNA migrates out of the nucleus forming a “comet” when stained. In presence of DNA strand breaks, DNA migrates out of the nucleus faster and with that generates a longer comet. Thus, the length of the comet is proportional to the fragmentation of the nuclear DNA. This assay can be modified for detection of single-strand or double-strand DNA breaks, UV damage, or oxidative DNA damage. Cells from patients with XP have defective repair in the post-UV comet assay.

Microsatellite Instability

Normal DNA has tens of thousands of regions with repeats of the dinucleotide CA or other short motifs up to five nucleotides long. In normal individuals each of these microsatellites (also called simple sequence repeats or short tandem repeats) has a uniform size. However, these sizes are highly variable among different individuals and are often used for DNA “fingerprinting.” The appearance of abnormally longer or shorter simple sequence repeats in different tissues or tumors from a patient is called microsatellite instability. This can be associated with a defect in mismatch repair genes.

Characterization and Expression of Defective Genes

Real-Time Polymerase Chain Reaction

Many disease-causing mutations, including those responsible for disorders of genome instabilty disrupt the gene product's function by changing its amino acid sequence. In some genome instability genes, disease-causing mutations create premature stop codons for protein synthesis. These mutations result not only in truncated but also in low levels of messenger RNA (mRNA) for the gene product through a process called nonsense-mediated message decay. The mRNA levels can be accurately measured by the use of quantitative reverse transcriptase real-time polymerase chain reaction. For example, low XPC mRNA levels have been found in cells from most XP patients with defects in this gene.33

Western Blotting

Some mutations result in reduced levels or size of encoded proteins, often through creation of premature stop codons.33 Reduced protein levels are most commonly detected by Western blotting. Cells are lysed and the proteins are extracted and separated by gel electrophoresis. The separated proteins are transferred to a membrane and probed with an antibody that is specific for the protein of interest. The intensity of the antibody staining reflects the amount of protein in the cells, and its location on the membrane is an indication of the size of the protein molecules. For example, undetectable or low levels of polymerase eta protein are present in cells from most XP variant patients.34

DNA Sequencing

Direct sequencing of defective genes is the gold standard for determination of the presence of a mutation. The final step in confirmation of a diagnosis may be DNA sequencing to determine the disease-causing mutation. By definition, disease-causing mutations alter the function of genes. However, not every alteration in the sequence of a gene alters the function of the encoded protein. In the human genome there are millions of single nucleotide polymorphisms, or changes in one nucleotide, that are not associated with disease and may not even change the amino acid composition of the encoded protein. In recessive disorders, each clinically unaffected parent has one normal allele and one potentially disease-causing mutation in the other allele. The affected child receives an allele with a disease-causing mutation from each parent. The two mutations must be in the same gene, although they need not necessarily be identical to each other.

Considerations in Diagnostic Testing

Diagnosis of disorders of genome instability or DNA repair is often a multistep process. A suggested sequence of steps is listed in Table 110-7. As new tests are developed and new information is obtained about these disorders, testing procedures may change accordingly. In addition, decisions regarding the extent of testing performed may be made with consideration of how much the tests cost and whether additional information would alter treatment. For example, DNA sequencing is rarely required for establishment of a diagnosis of XP in a child with classical clinical features and cells that are hypersensitive to killing by UV and have defective DNA repair. But if a family has one child affected with XP and is considering having additional children, DNA sequencing might offer them the possibility of prenatal diagnosis through DNA sequencing of a trophoblast biopsy specimen.

Genetic counseling is an important component of patient management for these genetic diseases. This function may be performed by the treating physician or by a trained genetic counselor.

In the United States, only laboratories certified in accordance with the Clinical Laboratory Improvement Act (CLIA) are allowed to perform these specialized tests in the context of patient care. Tests performed in research laboratories generally have limited use in clinical practice. However, a research laboratory may identify a disease mutation in cells from a patient that could then be confirmed in a CLIA-certified laboratory and used in clinical practice. A current listing of laboratory testing facilities for these diseases may be found at the Web site http://genetests.org, funded by the National Institutes of Health.