Chapter 2 Molecular and Cellular Biology
The past decade has seen a revolution in two areas of cellular and molecular biology closely related to radiation therapy. The pathways involved in response to genomic stress, including mechanisms for sensing DNA damage and responding to cell cycle changes and DNA repair, have been elucidated by studies of model organisms, particularly the yeast Saccharomyces cerevisiae.1,2 In the second major area, understanding pathways of programmed cell death (PCD), which may play a major role in treatment response, studies of Caenorhabditis elegans were the most informative in demonstrating the significance of individual death genes.3 The challenge is to combine these two areas of knowledge to provide a rational understanding of how tumor cells can be made to enter the death pathways after processing DNA damage using the genomic stress response. Considerable technologic improvements also have enabled significant progress in translational work, including rational, in silico drug design and high-throughput screening of chemical libraries, which may permit the application of advances in molecular biologic knowledge to the development of drugs for use in combination with radiation therapy.
Essential Steps in Tumor Progression
Much evidence suggests that tumors are formed by stepwise progression of cells from a minimally altered state, where they are able to grow and form nodules or polyps (e.g., solid tumors) to a state of maximal transformation, characterized by multiply deviated cells that are capable of unlimited growth, manipulation of their local environment, invasion of surrounding tissues, and escape into the circulation to establish new colonies of secondary tumors, or metastases.4 Such phenotypic and genotypic progression involves an array of molecular and morphologic changes. In their landmark review, Hanahan and Weinberg5 suggested organizing these traits into six essential alterations in cell physiology: self-sufficiency in growth signals, insensitivity to growth inhibition, evasion of PCD, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis. Although the initial steps are believed to occur earlier in tumor progression, the exact order of occurrence varies among different types of malignant tumors, and some phenotypic transformations appear to require more than one molecular change.6,7 The occurrence in tumor cells of this series of radical, primarily genetic alterations in physiology is in part the result of an evolving instability of the tumor genome due to a breakdown in the DNA repair pathways that accompanies tumor progression.
In addition to progressing and acquiring enhanced malignant capabilities, most human tumors undergo further selections through exposure to various forms of cytotoxic therapy such as irradiation and chemotherapy, leading to the development of resistant phenotypes in the cells that survive the toxic insult.8 However, a new paradigm is emerging that may challenge our views of tumorigenisis. This is the concept of the cancer stem cell.9 According to this theory, tumors contain a small population of stem cells that divide slowly and give rise to more “differentiated” progeny that form the bulk of tumors. Tumor eradication would therefore involve killing this small, slowly dividing, and often treatment-resistant population. Studies in this area could revolutionize the cellular and molecular underpinnings of radiation biology.
All cancer treatments are aimed at reversing the hallmarks of neoplastic transformation and tipping the balance toward tumor cell death in both the bulk population and the stem cell fraction. Ionizing radiation kills cells almost exclusively through the generation of DNA double-strand breaks (DSBs).10 The evolving modification of the cellular phenotype during tumorigenesis leads to the development of resistance to therapy. Our major task is to explore the links among DSB response pathways, engagement of cell death mechanisms, and the outcome of radiation therapy.
Radiation Therapy and Radiation Biology
Ionizing radiation has long been known to be a potent modality in cancer therapy, killing cells and leading to tumor regression.11 Killing by ionizing radiation involves the oxygen-dependent generation of free radicals and subsequent damage to multiple molecular structures within the cell.10 The generation of DSBs is the dominant mechanism of direct cellular lethality. Indeed, it has been estimated that the existence of even one unrepaired DSB can lead to cell death.12
Within whole organisms, direct and indirect forms of lethality occur. Radiation can cause the death of hematopoietic stem cells in the bone marrow and stem cells in crypts of the small intestine, which leads to secondary damage caused by immunosuppression and infection, as the gastrointestinal lining is eroded.13,14 The challenge facing radiation therapy is to enhance cell killing within tumors while avoiding dose-related complications in organs containing rapidly renewing stem cell populations. Early studies using the clonogenic cell survival assay established the notion of optimizing radiobiologic parameters such as dose, fractionation, and cell cycle inhibition as a means of inhibiting reproductive cell death in critical normal tissues.15 The clonogenic cell survival assay is a useful measure of cell inactivation because the effects of ionizing radiation seen in vitro often mirror the responses of tumors in vivo.10 However, a weakness in the informative power of the method is that it does not allow for discrimination or elucidation of the various mechanisms leading to cell death. That kind of detailed information is essential for the successful combination of radiation therapy with other modalities, which may result in sensitization or protection, or both.
Irradiation activates a number of pathways that mediate reproductive death, including various forms of PCD (e.g., apoptosis, autophagy), replicative senescence, and necrosis, the default pathway that often dominates when other types of cell death are inhibited.16,17 Susceptibility to each of these forms of cell death changes over the course of tumor progression, and the tumor cell response to ionizing radiation is likely to reflect the aggregate of genetic changes that accompany tumor progression. The task of modern radiation biology is to examine mechanisms by which ionizing radiation gives rise to unrepaired DSBs and how such damage is coupled to the pathways of cell killing within the moving target of the evolving cancer cell.
DNA Double-Strand Break Response
All cells have evolved to live in an environment that is more or less mutagenic, and have evolved responses to permit survival when the genome is damaged.18 DNA may be damaged during the normal processes of DNA replication and segregation. Within tumor cells, these initial changes become progressively altered as cells compete for survival in the tumor milieu and are subjected to selection by various forms of cytotoxic therapy.19 The response to DSBs involves two principal components: arrest of cell proliferation to prevent replication and segregation of the damaged DNA and, when possible, repair of the lesion.19 A third component in the DSB response in multicellular organisms may be altruistic entry of irreversibly damaged cells into the PCD pathways.20,21 The latter effect is the desired response to cytotoxic therapy.
Mechanisms of Cell Cycle Arrest: The Cell Cycle and Checkpoints
The cell cycle is a series of molecular programs that read out in an invariant order leading to duplication of the genome and other cellular components and permits division into two cells, each with a full set of chromosomes and the organelles required for life.22 Orderly, high-fidelity, complete duplication of the genome is required for cell cycle progression. This is achieved through a surveillance system that detects DNA damage or unreplicated DNA and imposes cell cycle arrest at designated checkpoints, blocking the forward progress of the cell cycle engine (Fig. 2-1). Because ionizing radiation produces severe damage to DNA, checkpoints are essential for facilitating repair and subsequent cell survival after radiation exposure, and provide an ultimate target for strategies aimed at sensitizing cells to radiation therapy.
Much accumulated evidence indicates that the cell cycle is driven by sequential activation of a series of protein kinases that determine the order and rate of the metabolic events required in each stage. These cell division kinases, or cyclin-dependent kinases (CDKs) are activated by binding proteins originally designated as cell division cycle (CDC) molecules, now known as cyclins.22 The complexes between cyclins and CDK molecules are master regulators that determine the rates of the individual component reactions that regulate each stage of the cell cycle.23 Phosphorylation of cell cycle effector proteins by the cyclin-CDK complexes acts as a switch, turning them on in orderly fashion. In mammalian cells, many cyclins regulate individual stages of the cell cycle, including G1 cyclins (cyclins D and E), S phase-specific cyclins, and G2-specific cyclins (cyclins A and B), which associate, respectively, with G1-specific (CDK2, CDK4, and CDK6), S-specific, and G2-specific (CDC2) cyclin-dependent kinases. The events of G1 phase (i.e., synthesis of enzymes and other molecules involved in DNA replication), S phase (i.e., DNA replication), and G2/M phase (i.e., chromosome condensation, disappearance of the nuclear envelope, and mitosis) are regulated by the transient accumulation and subsequent degradation of the cyclins.22,23,24 Switching off involves the targeted degradation of the cyclins by a protein destruction machine called the proteasome.24 Targeted destruction of the cyclins after they have carried out their function ensures unidirectional, irreversible progression around the cell cycle.23 The timing of the various cell cycle phases under normal conditions is related to the time required for cyclin-CDK complexes to reach appropriate, critical concentrations and for the phosphorylation of key substrates.23
Irradiation leads to prolongation of the cell cycle and arrests in G1, G2, and S phases because of checkpoint activation25 (see Fig. 2-1). Although this phenomenon was observed many years ago, it has only recently been understood at the biochemical and genetic levels. Arrest of cells at the G1 checkpoint has been particularly well studied (see Fig. 2-1A). G1 arrest is caused by the accumulation of a CDK inhibitory protein, CDKN1A (formerly designated p21 or Cip1), and this prevents key events that are required for transit through G1, including phosphorylation of the retinoblastoma (RB1) protein and activation of the E2F transcription factors (e.g., E2F1, E2F2, E2F4, E2F6) necessary for accumulation of enzymes required to traverse S phase.26,27 CDKN1A is induced at the transcriptional level by TP53 (also known as p53), which accumulates in irradiated cells and binds to the CDKN1A promoter, thereby activating transcription. TP53 accumulation depends on the activation of a DSB sensor molecule, the protein kinase ATM (ataxia-telangiectasia, mutated).26,27 The exact mechanism involved in sensing DSBs and activation of ATM is unclear, although it involves ATM autophosphorylation.28
The common mechanism for regulating the G1 and G2 checkpoints is inhibition of the CDK components of the interphase cell cycle engine and a block to phosphorylation and activation of effector molecules23,27 (see Fig. 2-1). A variation on this theme is seen at the mitotic spindle checkpoint. Progression through mitosis requires switching off CDK activity through targeted degradation of cyclin B.29 Arrest in M phase, when the mitotic spindle is compromised, involves the TP53-dependent stabilization of cyclin B. The mitotic spindle checkpoint guarantees that replication is followed by cell division, ensuring prevention of aneuploidy. Not surprisingly, the polyploidy nature of many tumor cells is related to TP53 inactivation.29
Most evidence indicates that ATM (and its homologs in other organisms) functions as a sensor for DNA damage through a complex series of interactions with the damaged DNA. This ultimately leads to ATM activation and downstream signaling cascades involving the kinases CHK1 and CHK2 (also called CHEK1 and CHEK2), which couple to the cell cycle engine at the level of individual cyclin-CDK complex molecules1,2,19,27,30 (see Fig. 2-1B). The molecular details of these processes are evolving rapidly, and referenced reviews can provide more detailed insight into the mechanisms of DNA damage, sensing, and checkpoint engagement.
TP53: Cellular Triage after Ionizing Radiation Exposure
TP53 is a nuclear phosphoprotein with sequence-specific DNA binding activity. It can function as both a transcriptional activator and repressor and plays a triage role in deciding whether to undergo cell cycle arrest and repair or to enter the pathways of PCD or replicative senescence31,32 (Fig. 2-2). When cells are exposed to ionizing radiation or chemotherapeutic agents, the levels of wild-type TP53 protein are increased. TP53 then transcriptionally activates a number of genes, most notably CDKN1A, the CDK inhibitor that mediates many of the properties of TP53.33 In addition to G1 arrest, genotoxic stress and ionizing radiation can induce TP53-dependent PCD pathways, including caspase-dependent apoptosis.33 TP53 can transcriptionally activate the proapoptotic BAX gene, induce synthesis of JUN kinase, and repress transcription of the antiapoptotic gene BCL2, suggesting that TP53 is a central mediator of PCD processes.31,34
DNA damage in the presence of wild-type TP53 causes G1 arrest, followed by a period of DNA repair; if the damage is too great to be easily repaired, the cell is eliminated through TP53-dependent PCD pathways. However, approximately 50% of human tumors possess inactivating mutations in the TP53 gene.35 TP53 becomes inactivated in many tumors because of selection against its pro-apoptotic properties. The resultant loss of its central function as “sentinel of the genome” may be a type of collateral damage incurred in cells due to the selection advantage for survival accruing from the loss of TP53 apoptotic function.32,36 The loss of TP53 function is linked to poorer prognosis in malignant tumors such as lung, breast, colorectal, and hematopoietic tumors.37 Many tumor cell lines containing mutant TP53, including breast, glioma, and lymphoma cell lines, are more resistant to therapy than their wild-type TP53 counterparts.38,39
The loss of wild-type TP53 function in human malignant disease may be a key step in the progression of human cancer, and the TP53 status of cells may control the outcome of many tumor types in response to chemotherapy or radiation therapy.36 Although most of the other DSB response genes have molecular equivalents in yeast, TP53 does not. The TP53 gene appears to be required to determine the fate of damaged cells, which in mammalian cells may be PCD, a sacrifice that contributes to the well-being of the whole organism. TP53 monitors the degree of DNA damage and acts as a master switch, moving the cells from a state of cycle arrest and DNA repair to death or senescence pathways. Loss of TP53 in tumors compromises this critical surveillance and triage function. With the loss of this critical decision-point molecule, damaged and mutated tumor cells may survive to generate new and more malignant phenotypes (see Fig. 2-2).
ATM Gene: Master Regulator of the DNA Double-Strand Break Response
One of the many defects in ataxia-telangiectasia (AT) cells is increased chromosomal instability. Exposure to ionizing radiation also produces an increased number of chromosomal aberrations in AT cells compared with normal cells.40,41 This effect and the sequence similarity between ATM and the DNA repair protein DNA-PK suggested that AT cells might have deficient DNA repair. However, most studies have shown that AT cells do not have gross abnormalities in their ability to repair DNA damage. In general, it does not appear that the radiosensitivity of AT cells is caused by faulty DNA repair; it more likely results from an inability to detect the presence of DNA damage. Exposure to ionizing radiation causes normal cells to delay at the G1/S and G2/M transition phases of the cell cycle, and these checkpoints are thought to allow the cells to repair DNA damage before DNA synthesis or mitosis occurs.27 Both checkpoints are absent in AT cells, suggesting clues to the function of ATM.27
Although ATM and TP53 cooperate in radiation-induced apoptosis, there are ATM-independent pathways for the induction of TP53-dependent apoptosis, and many of the downstream effects of ATM are independent of TP53.27,28 Wild-type and knockout mice have been evaluated for acute and late toxicity after whole-body exposure to ionizing radiation. The ATM– and ATM/TP53-knockout mice had similar severe toxicity profiles, suggesting that TP53 does not play a role in acute radiation toxicity. Both TP53- and ATM-knockout mice preferentially developed lymphoid tumors, whereas ATM/TP53 double knockouts had an accelerated time to tumor formation and a broader spectrum of tumor types. Analysis of the acquired tumors in ATM-null/TP53-heterozygous mice revealed that three of seven had loss of the remaining TP53 allele. These studies showed that ATM and TP53 interact in a complex manner that is specific to cell type and outcome. This interaction most likely relies on a variety of other pathways (discussed later) and will require much additional work before a complete understanding of the ATM/TP53 relationship is obtained.
With isolation of the ATM gene it became possible to attempt correction of the cellular defects of the AT phenotype using gene transduction techniques. Several groups have reported that the introduction of ATM into AT cells resulted in reversal of AT defects.27,41 The transfection of full-length ATM into AT cells reversed DSBs, restored normal sensitivity to ionizing radiation, and decreased the number of chromosomal abnormalities. Transfection of full-length ATM also reversed the defective activation of CDKN1A (p21) and JUN kinase in response to ionizing radiation.42,43
Histones and Chromatin Structure
Native DNA exists in the cell in the form of chromatin complexed with a family of proteins called histones. Histones are involved in packaging DNA in the nucleus into a compact form. For gene transcription or DNA repair to occur, the histones are altered by post-translational modifications, most notably acetylation, that permit decondensation and access of factors to the DNA.44 The histone acetylase TIP60 is important both in this process and in the modification of ATM itself.26,45 For effective DNA repair, acetylation of histones by histone acetylases must occur to permit access of repair proteins to the sites of DSBs.46 DNA is then remodeled by ATP-dependent remodeling enzymes to permit repair molecules such as Mre11, RAD50, and NBS1 to access sites of DSBs.47,48 A novel histone, H2AX, found in low concentrations on chromatin, has been shown to play a crucial signaling role in the response to genomic stresses such as ionizing radiation. H2AX phosphorylation is one of the earliest events occurring after exposure to ionizing radiation, and ATM-dependent phosphorylation of H2AX may be involved in signaling to cell cycle checkpoints and DNA repair enzymes involved in recombination repair.49,50
Mechanisms of DNA Repair after Ionizing Radiation
Maintaining undamaged DNA is essential to cell survival. Cells have therefore evolved a wide range of mechanisms to halt cell cycle progression, survey DNA, and repair damage. Radiation causes a number of types of damage either by direct interaction with DNA or indirectly through effects on nearby water molecules and free radical generation. Types of damage include DNA base damage, damage to the deoxyribose sugar backbone, and physical breaks in one or both strands of the DNA. DNA damage induced by ionizing radiation tends to be clustered so that there is more than one damaged site in proximity along the double helix, known as locally multiply damaged sites.51
Among the less catastrophic forms of DNA damage induced by ionizing radiation are the singly damaged bases, which can be repaired by base excision repair. This is a process by which the damaged base is recognized and removed by an N-glycosylase, the apurinic or apyrimidinic (AP) site is cleaved by an AP endonuclease, a patch of DNA is excised, DNA is then resynthesized using the other strand as the template, and the repaired strand is ligated.18,52 In settings in which the base damage is not recognized by the N-glycosylase, another mechanism for repair exists, called nucleotide excision repair. In a manner somewhat similar to base excision repair, a damaged section of DNA is removed by incision and excision, a patch is resynthesized using the remaining strand, and the repaired strand is then ligated. Although no naturally occurring mammalian mutants have been identified that are defective in base excision repair, there are a number of different nucleotide excision repair mutants, which are exemplified by xeroderma pigmentosum and Cockayne’s syndrome.18 They are characterized by abnormalities in the repair of damage caused by ultraviolet light, although the clinical spectrum of these repair-deficiency syndromes varies. The abnormal genes are identified by finding the human gene that corrects rodent cell defects, called excision repair cross-complementing (ERCC) groups. For a comprehensive review on repair of single-strand breaks we recommend the article by Friedberg, Walker, and Siede.18
DNA Strand Breaks
Cellular lethality after radiation involves generation of DSBs.53,54,55 Misjoined or unrepaired DSBs lead to chromosome deletions, translocations, and acentric or dicentrics, with lethal consequences for the cell. As with the excision repair defects (e.g., ERCCs), there are several x-ray repair defects in the x-ray cross-complementation (XRCC) groups involved in the repair of DSBs. DNA single-strand repair is carried out in a manner similar to base damage repair, with the undamaged DNA strand serving as a template. DSB repair is more complicated because there is no adjacent, undamaged template available to form a template for repair of the broken strands. The ends of the broken DNA must be protected, and the damaged site must be reconstituted by the processes of homologous recombination and nonhomologous end joining (NHEJ).
DNA DSB repair has much in common with the recombinatorial processes involved in immunoglobulin and T-cell receptor gene rearrangement in the immune response. The XRCC groups that have been identified and are involved in DNA DSB repair include genes that produce DNA end-binding proteins XRCC6 (formerly designated KU70 or Ku-70) and XRCC5 (formerly designated KU80 or Ku-80) and a DNA-dependent protein kinase (DNA-PK). Defects in DNA repair genes are seen in severe combined immunodeficiency (SCID) mice, indicating the importance of recombination in the restoration of DNA integrity after DSBs and in the immune response.18,53
Double-Strand Break Damage and Repair
Experiments in yeast and human cells indicate that a single unrepaired DSB leads to cell death.53,55 However, such cells can detect such DSBs and repair them. DSB repair would be predicted to require three functional components: (1) a mechanism for detecting and gauging DNA damage, (2) a signal transduction system, and (3) an effector system for DNA repair. These components are discussed in the following paragraphs, commencing with the repair component. DSBs can be repaired by a number of mechanisms, but the most prevalent are homologous recombination and NHEJ. Human cells appear to differ from yeast in that while homologous recombination predominates in yeast, the opposite is true in mammalian cells.53,55
Genetic analysis has revealed the existence of a large number of genes that regulate DSB repair. One essential gene, involved in resistance to cell killing by ionizing radiation and the repair of DSB, is XRCC5, which encodes XRCC5, a protein that binds with high affinity to the ends of the double strands1,18 (Fig. 2-3A). XRCC5 functions in normal cellular processes that require DSB rejoining, most notably V(D)J rejoining in the immunoglobulin and T-cell receptor genes of immature B and T lymphocytes. XRCC5 exists in cells in a heterodimeric complex with XRCC6, the product of the XRCC6 gene. The XRCC5/XRCC6 heterodimer is required for the end-binding and repair functions.56 The XRCC (formerly designated KU) proteins recognize the ends of DSBs and protect them from further degradation before the onset of end-joining reactions required for DSB repair. An associated protein in the complex with important functions in DNA repair is DNA-PK, the product of the XRCC7 gene.56
