15.4.1 Cell-Cycle Sensitivity and DNA Damage Checkpoints
Mammalian cells respond to ionizing radiation by delaying their progression through the cell cycle. Such delays allow for the repair of DNA damage in cells prior to undergoing either DNA replication or mitosis and are thought to prevent genetic instability in future cell generations (Kastan and Bartek, 2004). There is a rapid decrease in the mitotic index in an irradiated cell population, as both lethally damaged and surviving cells ceased to enter mitosis, while cells already in mitosis continued to progress to the G1 phase. After a period of time, which depends on both the cell type and the radiation dose, surviving cells reenter mitosis (Fig. 15–9); this time is known as the mitotic delay. Mitotic delay appears to be due largely to a block of cell-cycle progression in G2 phase, although cells in G1 and S phases are also delayed to a lesser extent in their progression through the cell cycle. There is typically 3 to 4 hours of G2 delay per 1 Gy radiation in diploid cells. Cells may continue to experience delays in their progression through the next and subsequent cell cycles. As a result of radiation-induced delays in the cell cycle, cell populations can be partially synchronized by irradiation.
The effects of radiation on the progression of cells into mitosis after the treatment. A) At time zero, the cells are placed in medium containing colcemid, a drug that arrests cells in mitosis, and the percentage of cells that accumulate in mitosis is plotted as a function of time. The decline in the curves at late times is a result of cells escaping the drug-induced block or dying. The mitotic delay caused by a radiation dose of 5.5 Gy displaces the curves for the radiation-treated cells to the right. B) Cells are irradiated when in different phases of the cell cycle and the mitotic delay observed is plotted as a function of radiation dose. (Whitmore GF, Till JE & Gulyas S, unpublished data.)
Cells in different phases of the cell cycle have different radiosensitivity (Terasima and Tolmach, 1961; Wilson, 2004). This is illustrated for Chinese hamster cells by the survival curves shown in Figure 15–10A. If a single radiation dose is given to cells in different phases (ie, a vertical cut is taken through the curves in Fig. 15–10A), then a pattern of cell survival as a function of cell-cycle position is obtained (Fig. 15–10B). Figure 15–10 shows that Chinese hamster cells in late S phase have the highest probability of survival after radiation (ie, are the most resistant), and that cells in mitosis (M phase) are the most sensitive. Although many cell lines appear to have a resistant period in S phase following irradiation in vitro, cell lines have variability in sensitivity just before mitosis in the G2 phase of the cell cycle (Wilson, 2004). For example, some oncogene-transfected cells show increased resistance in the G2 phase, whereas other cells, including DNA repair-deficient cells, show similar sensitivity throughout all phases of the cell cycle (Kao et al, 2001).
The effect of position in the cell cycle on cellular radiosensitivity. A) Survival curves for Chinese hamster cells irradiated in different phases of the cell cycle. B) Cells were selected in mitosis and irradiated with a fixed dose as a function of time of incubation after synchronization. The pattern of cell survival reflects the changing cellular sensitivity as the cells move through the cell cycle. C) Diagram indicating the active repair mechanisms during the various cell cycle phases and relative radiosensitivity. HR, Homologous recombination which occurs during the S and G2 phases of the cell cycle; NHEJ, nonhomologous end-joining recombination that occurs in all phases of the cell cycle; RS, relative radiosensitivity. Dark shading indicates activity of the particular repair pathway. Dark red shading indicates most radiosensitive portions of the cell cycle. (From Rothkamm et al, 2003.)
The molecular biology of the mammalian cell cycle and its response to DNA damage (including that of ionizing radiation) is discussed in detail in Chapter 5, Section 5.4 and Chapter 9, Section 9.3. The ATM (ataxia-telangiectasia mutated) protein plays a role in initiating checkpoint pathways in all 3 cell-cycle phases (Shiloh, 2003). G1 cell-cycle arrest following irradiation centers around an intact ATM-p53/CDC25A-RB pathway and decreased activity of CYCLIN D and E complexes. This leads to continued hypophosphorylation of the retinoblastoma (RB) protein at the G1–S interface and blocking of the initiation of DNA replication. Consequently, radiation-induced G1 arrest is abrogated in cells that lack functional p53, ATM, or RB proteins (Fei and El-Deiry, 2003; Cuddihy and Bristow, 2004). Although somewhat controversial, it is likely that loss of p53 protein function (and an abrogated G1 checkpoint) do not lead to radiosensitivity or radioresistance in comparison with those cells having normal p53 protein function; in contrast, mutant p53 has been reported to confer a radiosensitive phenotype (Choudhury et al, 2006). The S-phase checkpoint is controlled though ATM-mediated phosphorylation of the BRCA1, NBS1, and FANCD2 proteins that modify the activity of transcription factors (ie, E2F) and DNA replication proteins (RPA [replication protein A], PCNA [proliferating cell nuclear antigen]) during S-phase and DNA replication (Iliakis et al, 2003; see Chap. 5, Sec. 5.4).
There are 2 "checkpoints" in irradiated G2 cells. The G2/M checkpoint occurs early after radiation, is transient, is ATM-dependent and dose-independent (between 1 and 10 Gy). This checkpoint controls the entry into mitosis of cells that were in G2-phase at the time of irradiation. The "G2 accumulation checkpoint" is independent of ATM, but dependent on dose and ensures that cells that pass through earlier cell-cycle phases with DNA damage do not enter mitosis (Xu et al, 2002) The G2 arrest following exposure to ionizing radiation probably allows damaged DNA to be repaired prior to mitosis since DNA repair activity has been detected during the radiation-induced G2 delay and has been related to cellular radiosensitivity (Nagasawa et al, 1994; Kao et al, 2001). Molecular alterations that have been associated with the onset and duration of the G2 delay following radiation treatment include (a) cytoplasmic sequestration or decreased expression and stability of the CYCLIN-B protein and (b) inhibitory phosphorylation of the p34CDC2 protein after inactivation of the CDC25C phosphatase following radiation-induced activation of CHK1 or CHK2. These effects result in prevention of the formation of nuclear cyclin-B-p34CDC2 complexes, which are required for G2 progression (see Chap. 9, Sec. 9.2.3). Cells lacking either ATM or ATR-CHK1 function exhibit a defective G2 checkpoint after irradiation (Xu et al, 2002 and Chap. 5, Sec. 5.4).
Tumor cells often exhibit an aberrant G1 cell-cycle checkpoint because of defects in the ATM-p53-RB pathways, while the G2 cell-cycle checkpoints remain intact. There have been attempts to develop drugs that abrogate the G2 checkpoint (eg, caffeine, methylxanthines, or 7-hydroxystaurosporine [UCN-01]) in tumor cells to potentiate the cytotoxicity of ionizing radiation over that of normal cells. These drugs lead to the induction of premature mitosis and mitotic catastrophe in the treated cells. For example, UCN-01 preferentially sensitizes p53-mutated, radioresistant tumor cells to ionizing radiation. Identification of the targets of caffeine and UCN-01 (ie, ATM/ATR and CHK1/2) has led to interest in the development of this class of agent for the radiosensitization of tumors (Tenzer and Pruschy, 2003; Choudhury et al, 2006; Mitchell et al, 2010).
15.4.2 Cellular and Molecular Repair
The repair of cellular damage between radiation doses is the major mechanism underlying the clinical observation that a larger total dose can be tolerated when the radiation dose is fractionated. The shoulder of the survival curve reflects accumulation of sublethal damage that can be repaired (Elkind and Sutton, 1960; Fig. 15–11). When Chinese hamster cells were incubated at 37°C (98.6°F) for 2.5 hours between the first and second radiation treatments, the original shoulder of the survival curve was partially regenerated, and it was completely regenerated when the cells were incubated for 23 hours between the treatments (Fig. 15–11A). When the interval between 2 fixed doses of radiation was varied (Fig. 15–11B), there was a rapid rise in survival as the interval was increased from zero (single dose) to about 2 hours. This was followed by a decrease before the survival rose again to a maximum level after about 12 hours. This pattern of recovery is a result of 2 processes. Repair of sublethal damage (SLDR) accounts for the early rise in survival. Because cells that survive radiation tend to be synchronized in the more resistant phases of the cell cycle, their subsequent progression (inevitably into more sensitive phases) leads to a reduction in survival at 4 hours. Continued repair and repopulation explain the increases in survival at later times (see Chap. 16, Sec. 16.6). This pattern of SLDR has been demonstrated for a wide range of cell lines.
Illustration of the repair of sublethal damage that occurs between 2 radiation treatments. A) Survival curves for a single-dose treatment or for treatments involving a fixed first dose followed, after 2.5 or 23 hours of incubation (at 37°C), by a range of second doses. B) Pattern of survival observed when 2 fixed doses of irradiation are given with a varying time interval of incubation (at 37°C) between them. (Adapted from Elkind and Sutton, 1960.)
The repair capacity of the cells of many tissues in vivo has been demonstrated using cell survival and functional assays in vivo (Withers and Mason, 1974). An increase in total dose is required to give the same level of biological damage when a single dose (D1) is split into 2 doses (total dose D2) with a time interval between them (Fig. 15–12). The difference in dose (D2 – D1) is a measure of the repair by the cells in the tissue.
Repair of radiation damage in vivo. A) Survival curves for murine intestinal crypt cells γ-irradiated in situ with a single dose (red line) or with 2 equal fractions given 3 hours apart (blue line). (Modified from Withers and Mason, 1974.) B) Average skin reaction following x-irradiation of mouse skin with a single dose (red line) or 2 fractions given 24 hours apart (blue line). In both cases, an increase in total dose is required to give the same level of biological damage when a single dose (D1) is split into 2 doses (total dose D2) with a time interval between them. The difference in dose (D2 – D1) is a measure of the repair by the cells in the tissue.
The capacity of different cell populations to undergo SLDR is reflected by the width of the shoulder on their survival curve—that is, the Dq or D2 – D1 value. Survival curves for bone marrow cells have little to no shoulder, presumably because of their propensity to undergo radiation-induced apoptosis. Cells that demonstrate little or no evidence of cellular repair and that do not undergo radiation-induced apoptosis (such as fibroblasts derived from the radiosensitive disorders ataxia telangiectasia [AT] and Nijmegen breakage syndrome [NBS]; see Chap. 5, Sec. 5.5) also lack a shoulder to their survival curve and accumulate increased levels of DNA breaks (Fig. 15–13A) (Shiloh, 2003; Jeggo and Lavin, 2009). This relationship is shown in Figure 15–13 where the increased radiosensitivity is associated with an increased accumulation of residual and lethal DNA double strand breaks. Other cells (eg, jejunal crypt cells) can demonstrate a large SLDR for repair capacity (D2 – D1 value of 4 to 5 Gy – see Fig. 15–12A).
The relationship between radiosensitivity and DNA double strand breaks. In addition to aberrant cell-cycle checkpoints, increased radiosensitivity and subtle DNA double-strand break repair defects are associated with the AT and NBS disorders. Shown in (A) is the relative radiation survival for normal diploid fibroblasts versus that of AT or NBS fibroblasts. Note increased radiosensitivity following clinically relevant doses of 1 to 2 Gy. Shown in (B) are the DNA double-strand break rejoining curves (based on pulse-field gel electrophoresis) for AT cells relative to normal cells. The number of DNA double-strand breaks remaining is plotted against time following irradiation. Although the 2 sets of data initially have similar rates of DNA rejoining, the AT cells have increased numbers of residual DNA double-strand breaks relative to controls at later times postirradiation. (Modified from Girard et al, 2000.)
The effect of a given dose of radiation on human tissues and cells differs widely for exposures given over a short time (acute irradiation) and over an extended period of time (chronic irradiation given at a low-dose rate). Dose rates above approximately 1 Gy/min can be regarded as acute (single-dose) treatment and result in survival curves similar to that in Figure 15–7. As the total dose of X- or γ-rays is delivered at decreasing dose rates, the DNA damage in the cell diminishes progressively because of repair of the damage during the treatment. As a result, the shape of the radiation survival curve changes from one exhibiting a shoulder at high dose rates to one approaching linearity at low dose rates (Fig. 15–14A, B).
Survival curves for a series of human cancer cells lines irradiated under acute (high; >1 Gy/min) (A) or continuously (low; ~1Gy/h) (B) dose rates. (From Steel, 1991.) C) Schematic to illustrate the influence on the survival curve following continuous low-dose rate irradiation, of the processes of cellular repair, redistribution, and repopulation. (Data compiled by Dr. J.D. Chapman, Fox Chase Cancer Center, Philadelphia.)
The magnitude of the dose-sparing effect may be calculated as the relative survival under conditions of low-dose rate irradiation compared to survival under conditions of acute-dose rate irradiation. Cell lines with a greater capacity to repair sublethal damage will demonstrate a large dose-sparing effect relative to those cells that have limited capacity to repair the damage. In addition to cellular repair, low-dose rate irradiation can trigger the G1, S, and G2 checkpoints, slowing down the progression of the cells through the cycle. However, if the dose rate is low enough, the cells will continue to divide and repopulate. Because cells in late S phase are often the most radioresistant, they will preferentially survive, but eventually will move into the more sensitive phases of the cell cycle during radiation, a process termed redistribution. The process of repopulation leads to relative radioresistance of the cell population whereas cell-cycle redistribution leads to relative radiosensitization. Most of the effect of cellular repair occurs in the range of dose rates of 1.0 to 0.01 Gy/min. Below approximately 0.1 Gy/min, the effects of cell-cycle progression (redistribution and the G2 block; see Sec. 15.4.1) become apparent; below approximately 0.01 Gy/min, the effects of cell repopulation will start to become evident as the radiation damage is not severe enough to trigger cell-cycle arrest (see Fig. 15–14C). Repair, repopulation, and redistribution are important for understanding dose fractionation in clinical radiotherapy, as described in Chapter 16, Section 16.6.
Cell survival can be increased by holding cells after irradiation under conditions of suboptimal growth, such as low temperature, nutrient deprivation, or high cell density. The latter conditions may reflect those experienced by G0-G1 populations of cells in growth-deprived regions of tumors (Malaise et al, 1989; see Chap. 12, Secs. 12.2 and 12.3). The property is a result of the repair of potentially lethal damage (PLDR), which usually results in a change in the slope of the cell-survival curve. Such repair may contribute to increased radiation survival observed in vivo for some transplantable cell lines when compared to the radiosensitivity of the same cells growing in vitro.
The molecular components of DNA repair pathway(s) are described in Chapter 5, Section 5.3. Data from a number of studies indicate that double-strand breaks (DSBs) are responsible for the majority of lethal damage induced by ionizing radiation (Jeggo and Lavin, 2009). The main pathways of repair of DNA-DSBs include homologous recombination (HR), which is maximally operational during S- and G2-phases, and nonhomologous end-joining (NHEJ), which is operational throughout the cell cycle (Rothkamm et al, 2003; Valerie and Povirk, 2003), as diagrammed in Figure 15–10.
There is no simple relationship between expression of DNA repair genes and relative radiosensitivity amongst normal or tumor cells that do not have a recognized genetic defect in DNA repair (Jeggo and Lavin, 2009). However, DNA repair capacity can influence cellular radiosensitivity, as indicated by the extreme radiosensitivity of cells from patients with DNA repair deficiency syndromes such as AT and the NBS (see Fig. 15–13 and Chap. 5, Sec. 5.5). Similarly, cells deficient in the BRCA1 or BRCA2 proteins, can have decreased HR-related repair and cell survival following radiation (Powell and Kachnic, 2003). A reduced capacity for repair of DNA DSBs is also observed among (radiosensitive) fibroblasts derived from severe combined immunodeficiency (SCID) mice in which deficient NHEJ is caused by a mutation in the enzyme DNA-PKcs, which is involved in recruiting repair proteins to the break site (see Chap. 5, Sec. 5.3.5). Indeed, mouse cells made deficient for NHEJ (ie, mouse knockouts for DNA-PKcs or Ku70 genes) have exquisite radiosensitivity and defective rejoining of DNA-DSBs (see Fig. 15–15).
The role of NHEJ in DNA DSB repair and cellular radiosensitivity. A) The Ku70 protein, with the Ku80 and DNA-PKcs proteins, forms an important DNA-PK complex that initially catalyzes the repair of DNA-DSBs (see Chap. 5, Sec. 5.3.5 for details). As shown in (A), cells that are deficient in NHEJ proteins (eg, Ku70–/– fibroblasts) show exquisite radiosensitivity relative to normal wild-type (WT) cells. This is also true for Ku80- and DNA-PKcs-deficient cells. This increased radiosensitivity is a result of a reduced capacity for DNA break rejoining such that NHEJ-deficient cells have increased residual DNA breaks following irradiation leading to increased cell killing. This is illustrated in (B) (upper panel) where the number of remaining DNA DSBs at 400 minutes following irradiation is increased in Ku70–/– cells relative to WT cells. This is consistent with a DNA rejoining defect in the Ku70–/– cells. Note that the induction of DNA DSBs is similar between the 2 types of cells (lower panel), which, shows the number of DNA breaks induced for a given dose measured immediately following irradiation. (Modified from Ouyang et al, 1997.)
Broadly, cells can fall into 3 categories of sensitivity to ionizing radiation (Fig. 15–16A): Group I represents the "normal" case and Li-Fraumeni cells with p53-mutations and cells from patients with defects in DNA repair pathways not involved in DSB repair, such as nucleotide excision repair (NER), fall in this category. The G1/S checkpoint may be lost owing to p53-mutations, but this has no effect on the sensitivity of most cells to IR. Group II includes cells that have defects in HR or in "mediator" proteins, such as RNF168 (see Chap. 5, Sec. 5.5 and Table 5–1). Group III is the most radiosensitive and includes cells with defects in NHEJ and with mutations in the NBS1, ATM, or DNA ligase IV genes from DNA repair disorders (Girard et al, 2000). These cells are sensitive as a consequence of errors in end-processing and end-joining of DSB, defects in chromatin modifications that prevent efficient rejoining of DSBs, and inefficient ATM-mediated G2/M-checkpoints (Beucher et al, 2009) (see Chap. 5, Sec. 5.5 and Table 5–1).
Schematic of clonogenic survival curves showing differences in survival following IR (A) and UV (B) radiation for cells deficient in genes involved in DNA repair or DNA damage signaling. These data are derived from experiments using DNA repair-deficient cells from DNA instability syndromes (details of these syndromes are as discussed in Chapter 5 [see Sec. 5.3.5]).
Most cells that show sensitivity to IR are not sensitive to UV radiation. Cells derived from people with Cockayne syndrome or xeroderma pigmentosum patients with NER defects are exquisitely sensitive to UV irradiation (Fig. 15–16B). This is consistent with the different types of damage caused by IR versus UV radiation and the different DNA repair pathways that are used (eg, DNA DSBs and single-strand breaks, repaired by HR and NHEJ versus cyclobutane pyrimidine dimers (CPDs) and 6-4PPs repaired by NER, respectively (see Chap. 5, Sec. 5.3 for details). It also highlights the repair of DSBs as the primary determinant of survival following IR.
Greater understanding of the relationship between deficient DNA repair and radiosensitivity has led to strategies designed to radiosensitize tumor cells. In human fibroblasts, small silencing RNAs (siRNAs; see Chap. 2, Sec. 2.4.3) or small molecule inhibitors have been used to decrease expression of endogenous DNA-PKcs or ATM, which results in defective DSB repair and an increase in residual (unrejoined) DNA DSBs, which leads to increased radiation cell killing (Peng et al, 2002; Thoms and Bristow, 2010). Similarly antisense RNA or pharmacological approaches (ie, the drug imatinib, which inhibits the interaction between the protein product of the c-abl oncogene and the DNA repair protein RAD51; see Chap. 17, Sec. 17.3.1) have been used to decrease expression of DNA repair proteins with resultant radiosensitization (Collis et al, 2001). Inhibiting the repair of DNA base damage and single-strand DNA breaks with inhibitors of poly (ADP-ribose) polymerase (PARP; see Chap. 17; Sec. 17.3.2) can also lead to radiosensitization (Chalmers et al, 2010). However, the degree of radiosensitization and DSB repair may differ in vitro and in vivo because of the additional effects of the microenvironment, which may lead to differential DSB induction and altered expression and function of DSB repair pathways (Chan et al, 2009; Jamal et al, 2010).
There may be a therapeutic advantage to targeting DNA repair in combination with radiotherapy in that cell kill in tumor cells can be increased relative to cell kill in normal tissues. In some tumor cells, DNA repair pathways may be nonfunctional so that the tumor cells are radiosensitized if there are tumor-specific defects in HR or NHEJ. Furthermore, some drugs, such as PARP inhibitors, may be selectively toxic to HR-defective tumor cells based on synthetic lethality, which could be used to decrease the number of tumor clonogens prior to or during radiotherapy (Thoms and Bristow, 2010).
15.4.3 Intracellular Signaling, Gene Expression, and Radiosensitivity
Intrinsic changes in gene expression in tumors can influence response to radiation, and irradiation can modify the expression of some genes. Biochemical processes in cells, such as DNA, RNA, or protein synthesis, respiration, or other metabolism can be inhibited by irradiation, but this usually requires quite large doses of the order of 10 to 100 Gy. Clinical doses of radiation can affect the expression of a number of genes involved in the response of cells to stress and this may change their properties. Aberrant expression of oncogenes or tumor-suppressor genes may increase the intrinsic cellular radioresistance of human and rodent cells (Haffty and Glazer, 2003). For example, increased radiation survival has been observed in selected cell lines following the transfection of a single oncogene, such as activated Ras, Src, or Raf (Kasid et al, 1996; Gupta et al, 2001). This has led to studies designed to radiosensitize tumor cells by the inhibition of oncogene function using inhibitors of intracellular signaling pathways or antisense RNA to decrease oncogene overexpression (Kasid and Dritschilo, 2003). Figure 15–17 indicates pathways that may be targeted and the preclinical and clinical data to support such targeting in tumor cell radiosensitization strategies (Begg et al, 2011)
Targeting signal transduction. Growth factor receptor activation by mutation or overexpression, or mutations in oncogenes (such as RAS) or tumor suppressor genes (such as PTEN) can lead to signaling through the phophatidylinositol-3 kinase (PI3K)-AKT, mitogen-activated protein kinase (MAPK)-extracellular signal regulated kinase (ERK), nuclear factor-κB (NF-κB), and transforming growth factor-β (TGFβ) pathways. Such signaling can affect radiosensitivity by decreasing apoptosis (left) or increasing DNA repair (right). AKT, MAPK, and NF-κB signaling can all lead to phosphorylation and inactivation of proapoptotic proteins or activation of antiapoptotic proteins. Altering apoptosis does not always lead to changes in clonogenic cell survival (dashed arrow with question mark). Activation of the AKT and MAPK pathways leads to the activation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), a central protein in DSB repair by NHEJ. Direct inhibition of DNA repair may also lead to radiosensitization by targeting the ATM and DNA-PKcs kinases or PARP proteins. DNA-PKcs can also be activated by the receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR) after it is translocated to the nucleus. There is a strong correlation between DNA repair capacity, particularly for DSBs, and radiosensitivity. Ionizing radiation can also activate the PI3K, MAPK, and NF-κB pathways. Inhibition of signaling pathways following irradiation can therefore reverse (Begg et al, 2011) tumor cell radioresistance. Some inhibitors have also been shown to affect tumor vasculature, leading to improved perfusion and reduced hypoxia (see also Chap. 16, Sec. 16.2.5). Asterisk indicates activation. (Taken from Begg et al, 2011.)
When the Ras oncogene undergoes mutation, it is permanently activated in the guanosine triphosphate (GTP)-bound signaling state, providing proliferative signals in the absence of growth factor ligands, leading to altered cell growth, transformation, and, occasionally, radioresistance (see Chap. 8, Sec. 8.2.3). However, increased radioresistance is more commonly observed in cells transfected with an activated Ras gene in combination with a nuclear cooperating oncogene, such as c-Myc or mutant p53 (McKenna et al, 1990; Bristow et al, 1996). Downstream to RAS, the RAS-MEK-ERK and phosphatidylinositol-3 kinase (PI3K)-AKT/PKB pathways (see Chap. 8, Sec. 8.2.5) have been linked to tumor radioresistance (Bussink et al, 2008). Using antisense oligonucleotides or silencing RNA against human Raf leads to increased cancer cell radiosensitivity (Woods Ignatoski et al, 2008; Kidd et al, 2010). RAS-mediated radioresistance in rat cells appears to be dependent on PI3K and RAF signaling pathways, and less on the MEK signaling pathway (Affolter et al, 2012). Inhibitors of RAS and PI3K signaling, such as LY294002 and wortmannin, significantly enhanced the response to radiation in lung, bladder, colon, breast, prostate, head and neck squamous cell carcinoma (HNSCC), and cervical cancer cells (Xiao et al, 2010). Although inhibitors of RAS protein prenylation or function (farnesyl transferase inhibitors) have been reported to enhance radiation-induced cytotoxicity among preclinical models of human breast, lung, colon, and bladder cancer cells expressing mutated H- or K-ras genes, the use of these agents in the clinic has been limited by the lack of biomarkers that reflect a precise drug targeting of RAS versus other intercommunicating signaling pathways (Rengan et al, 2008).
Activation of the PI3K-AKT/PKB pathway is associated with 3 major mechanisms of tumor radioresistance: intrinsic radioresistance, tumor-cell proliferation, and hypoxia. Activation of this pathway can be caused by stimulation of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR; see Chap. 8, Sec. 8.2.1). In clinical trials, an independent association has been noted between expression of activated AKT and outcome of treatment of head and neck cancer with radiotherapy (Bussink et al, 2008). More recent data implicate the Akt gene in DSB repair, as AKT can stimulate the accumulation of DNA-PKcs at DNA-DSBs and promote DNA-PKcs activity during NHEJ, and itself can bind to DSBs in vitro following irradiation (Fraser et al, 2011; Toulany et al, 2012). Consequently, there is interest in the use of AKT inhibitors as clinical radiosensitizers given the overexpression of activated AKT in many tumors.
Tyrosine kinase activity of EGFR is increased following cellular exposure to radiation and addition of exogenous epidermal growth factor (EGF) to cell culture renders cells relatively radioresistant. Both EGFR and the related HER-2/neu receptor are overexpressed in a wide variety of epithelial tumors (head and neck squamous cell cancers (HNSCC), gliomas, breast, lung, colorectal and prostate cancers) and this overexpression has been associated with poor clinical outcome following radiotherapy due in part to increased tumor cell repopulation during therapy (Verheij et al, 2010). Targeting EGFR or human epidermal growth receptor 2 (HER-2)/neu signaling using monoclonal antibodies or specific inhibitors (eg, cetuximab-EGFR or trastuzumab-HER-2) leads to radiosensitization, in vitro and in vivo. These drugs are being tested in combination with radiotherapy and chemotherapy in randomized, multicenter clinical trials (Verheij et al, 2010; see Chap. 16, Sec. 16.2.5). Nuclear EGFR is now thought to also be part of the DNA-damage repair complex that interacts with proteins involved with the NHEJ repair pathway during ATM activation and chromatin relaxation during the sensing and repair of DSBs. Thus, inhibition of EGFR signaling during radiotherapy may lead to both decreased tumor cell repopulation and increased cell kill as a consequence of defective DSB repair (Dittmann et al, 2011; Yu et al, 2012).
The radiosensitivity of cells may be influenced by the addition of exogenous growth factors or hormones in receptor-positive cells before or after irradiation. The insulin-like growth factor-1 receptor (IGF-1R) is a cell-surface receptor with tyrosine kinase activity that has been linked to increased radioresistance. IGF-1R is expressed at low levels in AT cells; this may, in part, contribute to their radiosensitivity, as reintroduction of IGF-1R, or addition of exogenous insulin-like growth factor (IGF), can increase their radioresistance (Peretz et al, 2001). Other data implicate a role for IGF-1R directly in DSB repair, at least in part via the HR pathway and small molecule IGF-1R kinase inhibitors can decrease radioresistance in tumor cells (Turney et al, 2012).
15.4.4 Radiation-Induced Changes in Gene Expression
Irradiation can modify intracellular signaling through modification of the activity of tyrosine kinases, mitogen-activated protein (MAP)-kinases, stress-activated protein (SAP)-kinases, and RAS-associated proteins (Ruiter et al, 1999; Dent et al, 2003; Schmidt-Ullrich et al, 2003). Tyrosine phosphorylation is involved in several DNA damage response pathways; an example is activation of the c-ABL pathway, which phosphorylates RAD51, a DNA repair protein at sites of DNA damage. Genes induced by radiation include those encoding cell-cycle-related proteins (eg, growth arrest after DNA damage [GADD] genes, p34CDC2, CYCLIN B, p53), growth factors, and cytokines (eg, platelet-derived growth factor [PDGF], transforming growth factor beta [TGF-β], bFGF, tumor necrosis factor alpha [TNF-α]), and enzymes (eg, plasminogen activator). Liberation of inflammatory cytokines such as TNF-α and interleukin-1 (IL-1) by cells following radiation damage may lead to a continuing cascade of cytokine production, which may be responsible for the acute inflammation and late-onset fibrosis observed in some irradiated tissues (see Chap. 16, Sec. 16.5.3).
Induction of the expression of early response genes (ie, within seconds to minutes) by irradiation can be initiated by damage to the plasma membrane or to nuclear DNA (Criswell et al, 2003). Some early response genes, such as the early growth response factor (EGR-1) and p21 Cdk-inhibitor proteins (see Chap. 9, Secs. 9.2 and 9.3), contain radiation-responsive regulatory domains in their promoter regions, which can facilitate their rapid induction by radiation (Hallahan et al, 1995). These sequences might be used in radiation-induced gene therapy as vectors to drive expression of suicide genes (eg, TNF-α) for tumor therapy. Synthetic enhancers of gene expression designed for use with radiation utilize short motifs of sequence CC(A/T)6GG (ie, radiation-responsive elements) derived from the EGR-1 gene (Datta et al, 1992; Marples et al, 2002). Such constructs can be responsive to radiation at doses of 1 to 5 Gy. These tumor-targeting vectors might be used in clinical situations where the radiation volume can be tightly controlled to spare normal tissues using conformal radiotherapy (see Chap. 16, Sec. 16.2.2) and have shown promise in animal models (Mauceri et al, 2009).
Approaches using complementary DNA (cDNA) microarrays (see Chap. 2, Sec. 2.2.12) have led to the discovery that radiation-induced gene expression can be cell-type specific and while induction of some genes is dose-dependent across a range of doses, others are activated specifically at either low (ie, 1 to 3 Gy) or high doses (ie, 10 Gy) (Khodarev et al, 2001; Nuyten and van de Vijver, 2008; Rashi-Elkeles et al, 2011). Furthermore, gene expression following a given radiation dose can be substantially higher when solid tumors are irradiated in vivo than when the same cell line is irradiated in culture. However, studies of biopsies from human tumors have demonstrated that differences in radiation-induced gene expression are greater between patients' tumors than within the tumor of a given patient (Hartmann et al, 2002). This observation supports the concept that "molecular profiling" of the tumors and normal tissues in individual patients may be able to predict radiation response.
MicroRNAs (miRNAs) are emerging as a class of endogenous gene modulators that control protein levels, thereby adding a new layer of regulation to the DNA damage response (see Chap. 2, Sec. 2.4.3). There is increasing interest in an association between miRNA expression in tumors and chemo- and radiosensitivity, both with regards to predicting and modulating sensitivity. These include the miRNAs of the Let-7 family, miR-21 and miR-200b, as inhibition of these miRNAs leads to increased radiosensitivity (Hu and Gatti, 2010).
The translation of preclinical to clinical testing for molecularly-based compounds is limited by multiple factors including the appropriate scheduling and toxicity of the agent, the interpatient and intratumoral variability of the expression of the molecular target and its role as a predictor of treatment response, and molecular crosstalk among redundant, parallel intracellular signaling pathways (Haffty and Glazer, 2003). Some of these limitations might be bypassed by simultaneous determination of multiple pathways using genomic and proteomic analyses (see Chap. 2) of both normal and tumor tissues as the basis for selection of the best molecular agents to be combined with radiation treatments (Ma et al, 2003; Ishkanian et al, 2010; Begg, 2012). The majority of proteins involved in DNA repair undergo posttranslational modification or novel protein–protein interactions following irradiation at sites of DNA damage. Because such modifications would not be detected using cDNA microarray analyses, which detect alteration of messenger RNA expression rather than altered protein levels, proteomic anaysis (see Chap. 2, Sec. 2.5) of tissue samples or sera may be required to determine molecular pathways that lead to radio-response in patients (Bentzen et al, 2008).
Ionizing radiation causes damage to cells and tissues by depositing energy as a series of discrete events.
Different types of radiation have different abilities to cause biological damage because of the different densities of the energy deposition events produced.
The RBE of densely ionizing (high-LET) radiation is greater than that of low-LET radiation. Radiation can cause damage to any molecule in a cell, but damage to DNA is most crucial in causing cell lethality expressed by loss of proliferative potential.
Depending on cell type, cells may die by a permanent (terminal) growth arrest, undergo interphase death or lysis during radiation-induced apoptosis, or undergo up to 4 abortive mitotic cycles before mitotic catastrophe.
Several assay procedures have been developed for assessing the clonogenic capacity of both normal and malignant cells, and these have been used to obtain radiation survival curves for a wide range of cell types.
For X- and γ-rays, survival curves for most mammalian cells have a shoulder region at low doses, while at higher doses the survival decreases approximately exponentially with dose.
Following treatment with low-LET radiation, cells can repair some of their damage over a period of a few hours; thus if the treatment is prolonged or fractionated, it is less effective than if given as a single acute dose.
Cells in S phase are often more resistant than cells in the G2/M phases, but there is variability between cell types.
The accurate and timely rejoining of DNA DSBs are correlated to the relative radiation survival of both normal and tumor cells. Defects in the DNA repair pathways in tumor cells may be useful in targeting repair-defective cancer cells using synthetic lethality or molecular-targeting treatment strategies.
Cell-cycle checkpoints in cells are activated following irradiation (to allow time for DNA repair) and the molecular events relating to G1- and G2-phase cell-cycle arrest appear to involve ATM, p53, and CYCLIN-CDK complexes that are associated with cell-cycle regulation.
There is an association between the aberrant expression of RAS, RAF, and p53 proteins and cellular response to radiation.
Future treatments involving radiation will increasingly utilize molecular-targeted drugs that increase tumor radiosensitivity by interfering with the G1, S, and G2 cell-cycle checkpoints, or modify intracellular signaling following DNA damage. Genomic and proteomic assays may be useful to discover abnormal signaling pathways in tumors to help use molecular-targeted drugs for radiosensitization in selected patients.