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Molecular Cancer Therapeutics 6, 789-801, March 1, 2007. doi: 10.1158/1535-7163.MCT-06-0596
© 2007 American Association for Cancer Research
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Reviews

Radiation-induced cell signaling: inside-out and outside-in

Kristoffer Valerie3, Adly Yacoub1,3, Michael P. Hagan3, David T. Curiel5, Paul B. Fisher4, Steven Grant1,2 and Paul Dent1,3

Departments of 1 Biochemistry, 2 Medicine, and 3 Radiation Oncology, Virginia Commonwealth University, Richmond, Virginia; 4 Departments of Pathology, Neurosurgery and Urology, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, College of Physicians and Surgeons, New York, New York; and 5 Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, and the Gene Therapy Center, University of Alabama at Birmingham, Birmingham, Alabama

Requests for reprints: Paul Dent, Department of Biochemistry, Virginia Commonwealth University, 401 College Street, Box 980035, Richmond, VA 23298. Phone: 804-628-0861; Fax: 804-828-6042. E-mail: pdent{at}vcu.edu


   Abstract
Top
 Abstract
Introduction
 Signal Transduction Pathway...
 Pathways for Ionizing Radiation...
 Conclusions
 References
 
Exposure of tumor cells to clinically relevant doses of ionizing radiation causes DNA damage as well as mitochondria-dependent generation of reactive oxygen species. DNA damage causes activation of ataxia telangiectasia mutated and ataxia telangiectasia mutated and Rad3-related protein, which induce cell cycle checkpoints and also modulate the activation of prosurvival and proapoptotic signaling pathways, such as extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH2-terminal kinase 1/2, respectively. Radiation causes a rapid reactive oxygen species–dependent activation of ERBB family and other tyrosine kinases, leading to activation of RAS proteins and multiple protective downstream signaling pathways (e.g., AKT and ERK1/2), which alter transcription factor function and the apoptotic threshold of cells. The initial radiation-induced activation of ERK1/2 can promote the cleavage and release of paracrine ligands, which cause a temporally delayed reactivation of receptors and intracellular signaling pathways in irradiated and unirradiated bystander cells. Hence, signals from within the cell can promote activation of membrane-associated receptors, which signal back into the cytosol: signaling from inside the cell outward to receptors and then inward again via kinase pathways. However, cytosolic signaling can also cause release of membrane-associated paracrine factors, and thus, paracrine signals from outside of the cell can promote activation of growth factor receptors: signaling from the outside inward. The ultimate consequence of these signaling events after multiple exposures may be to reprogram the irradiated and affected bystander cells in terms of their expression levels of growth-regulatory and cell survival proteins, resulting in altered mitogenic rates and thresholds at which genotoxic stresses cause cell death. Inhibition of signaling in one and/or multiple survival pathways enhances radiosensitivity. Prolonged inhibition of any one of these pathways, however, gives rise to lineages of cells, which have become resistant to the inhibitor drug, by evolutionary selection for the clonal outgrowth of cells with point mutations in the specific targeted protein that make the target protein drug resistant or by the reprogramming of multiple signaling processes within all cells, to maintain viability. Thus, tumor cells are dynamic with respect to their reliance on specific cell signaling pathways to exist and rapidly adapt to repeated toxic challenges in an attempt to maintain tumor cell survival. [Mol Cancer Ther 2007;6(3):789–801]


   Introduction
Top
 Abstract
 Introduction
Signal Transduction Pathway...
 Pathways for Ionizing Radiation...
 Conclusions
 References
 
Ionizing radiation is used as a primary treatment for many types of cancer. Although it has been appreciated for many years that radiation causes cell death, this therapeutic modality also has potential to enhance proliferation in the surviving fraction of cells and to promote the long-term resistance to multiple cytotoxic stresses (1, 2). Exposure of carcinoma cells to clinically relevant low doses of ionizing radiation causes DNA damage and the generation of reactive oxygen species (ROS), with subsequent rapid activation of wild-type p53, ataxia telangiectasia mutated (ATM), and ATM and Rad3-related protein as well as the activation of growth factor receptors in the plasma membrane [e.g., the ERBB family of receptors (3–6)]. Receptor activation within several minutes of exposure enhances the activities of RAS family transducer molecules that mediate signaling from the membrane environment to cause activation of multiple cytosolic signal transduction pathways, such as the RAF-1-mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)1/2-ERK1/2-p90rsk and phosphatidylinositol 3-kinase (PI3K)-phosphoinositide-dependent kinase-1-AKT-glycogen synthase kinase 3 pathways, which play a role in the long-term effects of cell survival from toxic stresses and the regulation of cell growth (Fig. 1 ; refs. 7–10). This minireview will attempt to connect some of the complex interplay between the primary effects of radiation exposure (DNA damage and ROS generation) to the rapid initial responses of cells due to DNA damage and ROS generation, particularly activation of signal transduction pathways and regulation of survival. In turn, the review will discuss the delayed secondary responses of irradiated tumor cells, as the effect of the initial wave of signaling pathway activation and transcriptional changes ripples outward to further modify cell biology, including the proteolytic processing and/or synthesis of paracrine growth factors, reactivation of receptors and pathways and transcription, and the possible long-term outcomes of these processes on cell signaling, cell survival, and proliferation.


Figure 1
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  		Figure 1. MAPK and PI3K signal transduction pathways in human carcinoma cells.

 

   Signal Transduction Pathway Structure
Top
 Abstract
 Introduction
 Signal Transduction Pathway...
Pathways for Ionizing Radiation...
 Conclusions
 References
 
A brief overview of signal transduction pathway structure is provided to assist with understanding some of the concepts in this article. "MAP-2 kinase" was first reported by the Sturgill laboratory in 1986 (11). Parallel studies identified an additional 44-kDa isoform of this enzyme, termed ERK1 (12). Because many growth factors could activate these enzymes, the acronym for this enzyme was changed to denote MAPK. The p42 (ERK2) and p44 (ERK1) MAPKs are activated by dual tyrosine and threonine phosphorylation; they activate another protein kinase (p90rsk) by catalyzing its serine/threonine phosphorylation (Fig. 1; refs. 13, 14). ERK1/2 are phosphorylated and activated by MAPK kinases termed MEK1/2 (15). MEK1 and MEK2 are dual specificity tyrosine/threonine kinases and are activated by dual serine phosphorylation. The protein kinase responsible for catalyzing MEK1/2 activation was initially described as RAF-1 (e.g., ref. 16). RAF-1 is a member of a family of serine-threonine protein kinases termed RAF-1, B-RAF, and A-RAF; all "RAF" family members phosphorylate and activate MEK1/2 (17). Thus, the RAF kinases act at the level of a MAPK kinase kinase. Different growth factors, in a cell type–dependent manner, use RAF-1 and/or B-RAF to activate the ERK1/2 pathway (18). Recently, studies have examined the roles of activating B-RAF mutations in tumorigenesis and the protection of tumor cells from toxic stresses (19–21). Activating mutations within the B-RAF kinase domain are noted in several malignancies and are more common than RAF-1 mutations (22). Ionizing radiation activates RAF-1 but not B-RAF (19).

The NH2 domain of RAF-1 reversibly interacts with RAS proteins in the plasma membrane, and the ability of RAF-1 to associate with RAS proteins is dependent on the RAS molecule being in the GTP-bound state (e.g., refs. 23–26). Growth factor receptors, such as ERBB1, stimulate GTP for GDP exchange in RAS (27, 28). RAF-1 activation is dependent on RAF-1 translocation to the plasma membrane followed by phosphorylation of RAF-1 S338, phosphorylation of RAF-1 Y341, and dephosphorylation of RAF-1 S259 (29–34). RAF-1 and B-RAF have also been proposed to dimerize during activation, with assistance from 14-3-3 proteins, in an analogous manner to receptor tyrosine kinases, which promote RAF protein activation (35). Thus, a signaling pathway can be delineated from receptor tyrosine kinases through the activation of RAS proteins, translocation of RAF proteins to the plasma membrane, and the feeding of signals into MEK1/2-ERK1/2-p90rsk.

Additional comparative cloning studies showed that the original "MAPK" pathway was in fact one of many MAPK pathways. Thus, the ERK1/2-MAPK pathway has served as a template around which other MAPK family pathways have been described: the c-Jun NH2-terminal kinase (JNK) 1/2/3, the p38 MAPK, and the "big MAPK" MEK5-ERK5 pathways (Fig. 1; refs. 36–38). In each pathway, a MAPK, a MAPK kinase, and a MAPK kinase kinase have been shown to exist and behave phenomenologically in a similar manner to proteins described in the original ERK1/2 cascade. From the standpoint of cell signaling, it should be noted that, in a similar conceptual manner, the PI3K signaling pathways, PI3K-phosphoinositide-dependent kinase-1-AKT-glycogen synthase kinase 3 and PI3K-phosphoinositide-dependent kinase-1-mammalian target of rapamycin-p70S6K, have been delineated (38–42).

The activities of multiple MAPK pathways and of the PI3K pathway are variably elevated and/or suppressed in a variety of transformed cells, and studies have linked enhanced basal levels of ERK1/2, AKT, and ERK5 pathway activity to increased rates of proliferation and protection of cells from toxic stresses (43). Similarly, reduced basal activity levels or abilities to activate the JNK1/2 and p38 MAPK pathways correlate with enhanced tumor cell viability. In this regard, exposure of cells to ionizing radiation promotes activation of RAS family proteins and downstream the ERK1/2, PI3K-AKT, and JNK1/2 pathways. Conceptually, the activation of protective AKT/ERK1/2 pathways counteracts toxic signaling from the JNK1/2 pathway (Fig. 1).


   Pathways for Ionizing Radiation–Induced Signal Transduction Processes
Top
 Abstract
 Introduction
 Signal Transduction Pathway...
 Pathways for Ionizing Radiation...
Conclusions
 References
 
There are at least two schools of thought as to how radiation exposure activates plasma membrane receptors and intracellular signaling pathways. First, radiation causes DNA damage, which activates ATM/ATM and Rad3-related protein that in turn promote activation of receptors/intracellular signaling pathways as well as stimulating cell cycle checkpoints, p53 activity, and DNA repair complex function. Second, radiation generates ionizing events in water in the cytosol that are amplified, thought to be mediated by mitochondria, which generate large amounts of ROS and reactive nitrogen species (RNS) that inhibit protein tyrosine phosphatase (PTPase) activities. In addition, radiation activates acidic sphingomyelinase and increases the production of ceramide. Inhibition of PTPases leads to a general derepression (activation) of receptor and nonreceptor tyrosine kinases and the activation of downstream signal transduction pathways. Radiation-induced ceramide has been shown to promote membrane-associated receptor activation by facilitating the clustering of receptors within lipid rafts (44).

Growth Factor Receptors and Intracellular Signaling Pathways
Activation of Growth Factor Receptors by Radiation. Multiple laboratories have shown that the epidermal growth factor receptor (also called ERBB1 and HER1) is rapidly activated in response to the irradiation of multiple tumor cell types in vitro (e.g., refs. 19, 45–49). Low-dose clinically relevant radiation exposure (1–2 Gy) activates ERBB1 and, by heterodimerization, other members of the ERBB receptor family (ERBB2, ERBB3, and ERBB4). Activation of ERBB1, ERBB2, and ERBB3 has been linked to downstream activation of intracellular signaling pathways, including the RAF-1-MEK1/2-ERK1/2 and the PI3K-phosphoinositide-dependent kinase-1-AKT pathways. Studies in the late 1990s argued that a 2 Gy radiation exposure caused similar levels of ERBB1 and ERK1/2 pathway activation to those observed by growth-stimulatory, epidermal growth factor concentrations (~0.1 nmol/L) 0 to 30 min after exposure (50–52).

An obvious scientific question was then asked (i.e., how did ionizing radiation promote such a rapid activation of ERBB1?). It was known that the activity of tyrosine kinases and proteins regulated by tyrosine phosphorylation (e.g., ERBB1 and RAF-1) is held in check by the actions of PTPases (53). The relative activity of a PTPase is approximately one order of magnitude higher than that of the substrate (i.e., kinase) it dephosphorylates (54). PTPase activity is sensitive to oxidation and/or nitrosylation of a key cysteine residue in the active site, and thus, any agent that generates ROS or RNS has potential to promote decreased PTPase activity and, hence, increased tyrosine phosphorylation of multiple proteins (55). Ionizing radiation induces small amounts of ROS by direct interaction with water; these ROSs are magnified in a Ca2+-dependent manner by mitochondria, generating more ROS and RNS, which can act to inhibit multiple PTPase activities. Inhibition of radiation-induced ROS and RNS generation, by use of ROS quenching agents, such as N-acetyl cysteine, or in cells lacking functional mitochondria (Rho zero cells), abrogates the suppression of PTPase activity by radiation (Fig. 2 ; refs. 56, 57). In general agreement with a role for PTPase inhibition in radiation-induced ERBB1 activation, expression of dominant-negative SHP2 abolishes radiation-induced phosphorylation of ERBB1; phosphorylation of ERBB1 Y992 in MDA-MB-231 mammary carcinoma cells was noted to be the most radioresponsive site in terms of fold induction following irradiation (56–59). ERBB1 Y992 phosphorylation has been linked to activation of phospholipase C{gamma} and the ERK1/2 pathway (60). In further support of a role for the modulation of PTPase activity and changes in tyrosine phosphorylation playing an important role in radiation responses downstream of growth factor receptors is that RAF-1, a protein whose activity is enhanced by tyrosine phosphorylation, becomes tyrosine phosphorylated and activated following radiation exposure. Of note, B-RAF, which lacks the sites of tyrosine phosphorylation in RAF-1 due to their substitution by acidic amino acid residues in B-RAF, is not potently activated following irradiation (19, 34, 48, 61). Thus, ionizing radiation has the potential to promote the tyrosine phosphorylation and activation of intracellular pathways via PTPase inhibition at the level of the receptor (ERBB family), a membrane proximal kinase (RAF-1), and, possibly, though not proven as yet, also at the level of the tyrosine phosphorylated MAPK proteins. It has been argued that ROS can inhibit MAPK phosphatase enzymes, which normally act to dephosphorylate the activating phosphotyrosine and threonine residues in MAPK proteins, and thus, loss of MAPK phosphatase function will tend to enhance the phosphorylation and activity of MAPK family enzymes (62).


Figure 2
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  		Figure 2. Proposed mode of signaling transduction pathway regulation following initial exposure to ionizing radiation: inside to outside signaling.

 
Other agents of physiologic relevance can also generate ROS and RNS and thereby promote activation of growth factor receptors via PTPase inhibition. For example, bile acids through ROS-dependent mechanisms can activate both ERBB1 and the insulin receptor in primary hepatocytes (63–65). Both mitochondria and NADPH oxidase enzymes have been linked to bile acid–induced ROS production (64–66). Activation of ERBB1 and the insulin receptor in hepatocytes can promote ERK1/2 and AKT activation and regulate cell survival as well as other processes, such as glycogen metabolism (67). Arsenic trioxide and hydrogen peroxide can generate ROS in cells and have also been noted to cause activation of growth factor receptors (68, 69). Collectively, these observations show that a wide variety of external stimuli can promote ROS generation in cells, leading to the initiation/activation of intracellular signaling pathways (Fig. 2).

After observations showing the initial radiation-induced activation of the ERBB receptors approximately 0 to 10 min after exposure, it became evident that the ERBB receptors also were reactivated approximately 60 to 180+ min after irradiation. The primary mode of receptor activation at these later times occurred via a paracrine/autocrine mechanism (Figs. 2 and 3 ; refs. 70, 71). The initial activation of ERBB1 and the ERK1/2 pathway was directly responsible for the cleavage, release, and functional activation of presynthesized paracrine ligands, such as pro-transforming growth factor {alpha} (TGF{alpha}), which fed back onto the irradiated tumor cell and potentially in vivo onto unirradiated distant tumor cells, thereby reenergizing the signaling system (72). Several studies have independently argued that ERK1/2 and/or p38 MAPK signaling can enhance plasma membrane metalloproteinase activities, which promote cleavage of the proforms/zymogens of multiple growth factor ligands into their functionally activated states (73): this has led to the clinical development of protease inhibitors, such as marimastat (74). Growth factors, such as insulin-like growth factor-I, can promote activation of ERBB1 via increasing the expression of ERBB1 paracrine ligands as well as promoting MAPK-dependent proteolytic processing of these ligands [e.g., heparin-binding epidermal growth factor (75)]. In irradiated HCT116 cells, the ERBB3/4 binding ligand heregulin, which in this cell type primarily interacts with ERBB3, can promote ERBB1, ERBB2, and AKT activation in a paracrine fashion 120 to 240 min after radiation exposure (76). Increasing the radiation dose from 2 Gy up to 10 Gy enhances both the amplitude and duration of the secondary activation of ERBB1 and the secondary activation of the intracellular signaling pathways, suggestive that radiation can promote a dose-dependent increase in the cleavage of pro-TGF{alpha} that reaches a plateau at ~10 Gy (70, 72). In contrast to the secondary receptor and pathway activations, primary receptor and signaling pathway activations seemed to have come to a plateau at 3 to 5 Gy.


Figure 3
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  		Figure 3. Proposed mode of signaling transduction pathway regulation following several hours after the initial exposure to ionizing radiation: outside to inside signaling with altered paracrine ligand activity and receptor reactivation. Long-term effects on cell growth and cell viability will be cell type and tumor origin dependent.

 
The expression of paracrine factors in tumor cells can change in the short term (hours) and in the long term (weeks) after irradiation, as potentially can the expression of the growth factor receptors that bind the factors. For example, in the instances of RAS-ERK1/2 signaling and p53 transcriptional function, the activities of which can be increased shortly following radiation exposure, in a variety of cells, these proteins act to enhance the expression of autocrine factors, such as heparin-binding epidermal growth factor and epiregulin (77). However, loss of p53 function can also alter ERBB1 expression; for example, comparing HCT116 wild-type and HCT116 p53–/– cells, ERBB1 expression is reduced and both wild-type and mutant p53 proteins have been shown to regulate the ERBB1 promoter (34, 78, 79). In MCF7 mammary carcinoma cells, exposed to multiple low doses of radiation, the expression of ERBB1 and TGF{alpha} was noted to rise and the expression of the estrogen receptor was noted to decline (80). These findings argue that the activation of ERBB family receptors and downstream pathways by radiation has the potential to be influenced in both the short and long term by the amount of prior radiation exposure a cell has received and the mutational status of p53 and RAS proteins. Collectively, these observations argue that radiation generates ROS/RNS signals within tumor cells that promote activation of growth factor receptors and signaling pathways that in turn promote the release of paracrine ligands from cells, leading to the reactivation of receptors and intracellular signaling pathways: "inside-out" followed by "outside-in" signaling.

Approaches to Radiosensitize Cells by Inhibition of Kinase Function. Signaling by ERBB family of receptors is, in general, believed to be pro-proliferative and cytoprotective, and inhibition of ERBB receptor function has been explored as a mode of cancer therapy (Table 1 ). Thus, when signaling from ERBB family receptors is blocked, either by use of inhibitory antibodies or small molecular weight inhibitors of receptor tyrosine kinases, tumor cell growth can be reduced and the sensitivity of these cells to being killed by noxious stresses increased (reviewed in refs. 81–83). In vitro and xenograft tumor animal model studies have strongly argued that inhibition of ERBB receptor function using single drug/antibody dosing has radiosensitizing effects (84–86). In some animal studies, however, ERBB receptor inhibitors have not radiosensitized ERBB1-expressing tumors (e.g., ref. 87). Furthermore, as a collective group, clinical trials in which modulation of ERBB receptor function was a primary goal for improved therapeutic outcomes have been considerably less successful in terms of tumor control than predicted based on in vitro studies (e.g., refs. 88, 89).


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  		Table 1. Inhibitors of ERBB family receptors, signal transducers, and kinase proteins downstream of the growth factor receptors

 
Several possible explanations could exist why a drug effect observed in vitro or in animals did not translate into as profound an antitumor effect in patients: (a) the required inhibitory concentration of the drug and the drug half-life are not achievable and are too short for a therapeutic effect, respectively, in patients; (b) the relative dependency (addiction) of cultured tumor cell isolates on ERBB receptor signaling, including expression of hyperactive ERBB receptor mutants (e.g., ERBB1 L858R), compared with actual tumors in patients may be biased based on in vitro studies with established cell lines and the development of drug-resistant ERBB receptor mutants in patients after long-term exposure to ERBB inhibitors (e.g., ERBB1 T790M) may preclude drug actions; (c) exposure of tumor cells in vitro to kinase and other inhibitors, such as tamoxifen, has argued that compensatory activation of parallel growth factor receptors, such as the insulin-like growth factor-I receptor and c-Kit, occurs to replace the loss of ERBB receptor signaling caused by drug exposure and acts to maintain tumor cell survival (90–92); (d) the ERBB inhibitors that are often used in therapy only inhibit one ERBB family member, such as ERBB1, and in a similar conceptual manner to point (c), other ERBB family members, such as ERBB2, may provide compensatory survival signaling to overcome loss of survival signaling from the inhibited receptor; and (e) the development of other somatic mutations in survival signaling with the tumor cell, such as loss of PTEN function, which may be selected for in tumor cells undergoing ERBB receptor inhibitor therapy, will lead to the development of tumor cells that are more resistant in general to inhibitors of growth factor receptors.

The role of RAS signaling in terms of regulating radiosensitivity directly downstream of plasma membrane receptor tyrosine kinases has also been investigated by many groups, with comparative data using cells from diverse genetic backgrounds arguing that mutated active H-RAS, K-RAS, and N-RAS proteins protect cells from the toxic effects of ionizing radiation by activating the PI3K pathway (93–99). In HCT116 colon cancer cells, expressing activated K-RAS D13, radiosensitivity was linked to signaling by the ERK1/2 pathway (100). Studies by others have also shown that HCT116 cells expressing active K-RAS use the ERK1/2 pathway as a primary signal to protect themselves from the toxic effect of radiation, and in these experiments, isogenic HCT116 cells expressing active H-RAS V12 (with expression of active K-RAS D13 deleted) were noted to use the PI3K pathway as a primary signal to protect themselves from radiation toxicity (101, 102). This suggests that different RAS family members, H-RAS and K-RAS, have the potential to generate qualitatively different radioprotective signals via activating different downstream signal transduction pathways.

As stated above, data from several groups have shown that a key radioprotective pathway downstream of receptors and RAS proteins is the PI3K pathway (Table 1). Inhibition of PI3K pathway function by use of small-molecule inhibitors radiosensitizes tumor cells expressing mutant active RAS molecules or wild-type RAS molecules that are constitutively active due to upstream growth factor receptor signaling (93–99). It is possible that PI3K inhibitors may also exert a portion of their radiosensitizing properties by suppressing the function of proteins with PI3K-like kinase domains, such as ATM, ATM and Rad3-related protein, and DNA-dependent protein kinase (DNA-PK). Expression of constitutively active p110 PI3K molecule is able to partially recapitulate the expression of mutant (active) H-RAS proteins in protecting cells from radiation toxicity. In cell lines where PI3K regulates radiosensitivity, inhibition of the ERK1/2 pathway did not significantly alter the radiosensitivity of cells, in agreement with data in HCT116 cells. ERK1/2 signaling has often been stated to play no role in controlling radiosensitivity; in some cell lines, inhibition of ERK1/2 has been linked to protection from radiation toxicity (103, 104).

In those cells where radiosensitizing effects have been observed by blocking ERK1/2 activation, the abilities of MEK1/2 inhibitors to enhance cell killing by radiation was originally linked to a derangement of radiation-induced G2-M growth arrest and enhanced apoptosis (105, 106). In DU145 human prostate cancer cells that express ERBB1 and the ligand TGF{alpha}, ionizing radiation increases the release of TGF{alpha} via ERBB1-ERK1/2 signaling. If radiation-induced ERBB1-ERK1/2 signaling is transiently blocked in DU145 cells by either the ERBB1 inhibitor AG1478 or a MEK1/2 inhibitor before and for 3 h after irradiation, then radiation-induced cell killing is decreased (Table 1). Moreover, if ERBB1 is strongly activated by epidermal growth factor or TGF{alpha} immediately after irradiation, then cell killing is increased. Thus, transient inhibition of radiation-induced ERK1/2 signaling, or suprastimulation of ERK1/2 signaling at the time of irradiation, radiosensitizes tumor cells. Removal of MEK1/2 inhibitor from the growth medium 24 and 48 h after irradiation results in a null effect on DU145 cell radiosensitivity, although inhibition of MEK1/2 modestly enhanced radiation-induced apoptosis at these time points. Data in general agreement with this concept were also obtained in LNCaP, PC3, and A431 squamous carcinoma cells (107). On the other hand, following irradiation, prolonged inhibition of ERK1/2 (more than approximately 60–72 h) significantly increases the apoptotic response of DU145 and A431 cells and reduces clonogenic survival. Therefore, the interruption of ERBB1 and ERK1/2 signaling can enhance or degrade carcinoma cell survival after irradiation depending on its timing and duration (reviewed in refs. 34, 72 and references therein).

Downstream Targets of Radiation-Induced Kinase Function. Growth factor–induced signaling from ERBB receptors through the PI3K-AKT and RAF-1-ERK1/2 pathways can increase expression of multiple antiapoptotic proteins, including BCL-XL, MCL-1, and c-FLIP isoforms, as well as the phosphorylation and inactivation of proapoptotic proteins, including BAD, BIM, and procaspase-9 (108–113). Radiation-induced ERK1/2 activation has also been linked to increased expression of the DNA repair proteins ERCC1, XRCC1, and XPC (reviewed in refs. 71, 72 and references therein). In contrast, radiation-induced activation of the JNK1/2 pathway has been linked to activation of proapoptotic protein function, including those of BAX and BAK, and the promotion of mitochondrial dysfunction (114, 115). Thus, as a general concept, activation of AKT and ERK1/2 will tend to suppress cell death processes, including those stimulated by activation of JNK1/2.

A downstream protein kinase effector of the ERK1/2 enzymes, p90rsk, phosphorylates the transcription factors cyclic AMP–responsive element binding protein and CAAT/enhancer binding protein ß, which can activate the promoters of several antiapoptotic proteins (e.g., refs. 116, 117), and recent studies have shown that radiation, via the ERK1/2 pathway, can enhance the DNA binding of cyclic AMP–responsive element binding protein, which plays a causal role in radioresistance (117). Transcriptional regulation of the ERCC1, XRCC1, and XPC DNA repair genes after irradiation seems to be via activator protein-1 and Sp1 sites (72). ERK1/2 signaling has also been linked to enhanced expression of MDM2, which can suppress the expression of p53 and thus diminish the proapoptotic signaling effects of wild-type p53, as has been argued in HCT116 cells (102). In contrast to potential radioprotective transcription factors downstream of ERK1/2, EGR-1 is an ERK1/2-dependent transcription factor that has been associated with enhanced cell killing following radiation exposure (118). The radiosensitizing effects of EGR-1 have been linked to increased tumor necrosis factor {alpha} and PTEN expression, whereas the radioprotective effects of EGR-1 have been linked to TGFß and enhanced growth arrest in late G1 phase of the cell cycle (119, 120). Thus, collectively, ERK1/2 may act to promote survival via increased activity of some transcription factors (cyclic AMP–responsive element binding protein and CAAT/enhancer binding protein ß) and decreased activity of others (p53) and may act in a cell type–dependent fashion to promote either survival or cell death through the activation of others (EGR-1 and activator protein-1).

DNA Damage and Activation of ATM, ATM and Rad3-Related Protein, and p53
Inside-Out Signaling in Response to Radiation and DNA Double-Strand Breaks. The basic mechanisms of how DNA damage in the form of double-strand breaks (DSB) trigger cell cycle check points and growth factor receptor–mediated signaling have been established during the past decade (see refs. 121, 122 for review). DNA damage surveillance proteins of the PI3K-like kinase family, including ATM, ATM and Rad3-related protein, and DNA-PK catalytic subunit (DNA-PKcs), as well as other key proteins sense the damage and signal through transducers and effectors that trigger G1-S, intra-S, and G2-M checkpoints. The actions of p53-p21 signaling also play a role in checkpoint control (15, 102). Triggering of the checkpoints permits the cell to assess the damage and repair the DNA before replication and mitosis. However, how the signal generated from DNA damage in the nucleus is transmitted to the cytoplasm to modulate cell growth and apoptosis/death is not clear. It is expected that the cell can repair minor to moderate DNA damage, recover, and reenter the cell cycle. Major damage would likely result in cell death and apoptosis depending on cell type and mutational status of p53 (102, 121). Thus, the net effect of multiple signaling pathways emanating in the nucleus and elsewhere in the cell would need to be coordinated to result in either increased DNA repair and survival or, alternatively, cell death/apoptosis. Indeed, such balance has been proposed to occur in response to DNA damage (see ref. 122 for review).

The four major MAPK signaling pathways in mammalian cells, ERK1/2, ERK5, JNK1/2, and p38, have been linked to prosurvival (ERK1/2 and ERK5) and apoptosis (JNK1/2 and p38), respectively (123). Several studies have shown that these pathways are modulated in response to radiation and other types of DNA damage, resulting in proliferative or apoptotic signaling depending on dose and cell line. For example, radiation activates ERBB1 and other members of the ERBB family through ligand-independent and ligand-dependent pathways at low and clinically relevant doses resulting in prosurvival signaling (52, 70, 124). Recently, an association between ERBB1 and DNA-PKcs was uncovered, which might link DNA damage and repair to the ERBB1-RAS-MEK1/2-ERK1/2 signaling pathway (125–127). DNA-PKcs is critical for nonhomologous end joining (128). In support of this notion, split-dose radiation survival experiments have revealed that expression of a truncated dominant-negative ERBB1 protein, ERBB1-CD533, reduces DNA repair, suggesting a direct role for ERBB receptor signaling in repair processes [ref. 129; in general agreement with data linking ERBB1-ERK1/2 signaling to the regulation of repair protein expression (72)]. Similarly, other studies have found an association between ATM and ERBB1 (130), insulin-like growth factor-I receptor (131, 132), and platelet-derived growth factor receptor (133). It has been known for some time that ATM can modulate signaling through the JNK1/2 pathway. ATM knockout mice show elevated MKK4-JNK1/2-c-Jun activation as shown by increased activator protein-1 transcription factor activity and cellular stress, suggesting that ATM regulates the JNK1/2 signaling pathway in a negative fashion (134). Recently, ATM was linked directly to ERK1/2 signaling, which was shown to be necessary in fibroblasts for triggering apoptosis in a p53-independent manner (135). We recently showed that ATM and MEK1/2-ERK1/2 signaling form a regulatory feedback loop that regulates homologous recombination repair.6 Using the MEK1/2-specific inhibitor PD184352, we noted that not only was homologous recombination repair reduced more than ~80% but that autophosphorylation of ATM at S1981 was completely abrogated in response to radiation, suggesting that MEK1/2-ERK1/2 signaling is closely linked to the activation of ATM and the regulation of homologous recombination repair (Table 1). Phosphorylation at S1981 is evidence for ATM activation (136). Furthermore, inhibiting the ATM kinase with KU-55933 reduced radiation-induced ERK1/2 phosphorylation by >70% (137). This suggests that ATM positively regulates signaling through ERK1/2, and MEK1/2-ERK1/2 positively affects ATM activation. This finding argues, for the first time, a close association between growth control through ERK1/2 signaling and DNA damage surveillance via ATM that modulates homologous recombination repair. The underlying mechanism(s) for this feedback control is not known but it is possible that ATM signals via a growth factor receptor, as other reports have indicated, or through a novel yet to be discovered signaling mechanism (130–133).

In another recent study supporting the link between ERK1/2 and ATM signaling, it has been shown that DSB signaling affects the ERK1/2 pathway differently depending on the extent of DNA damage.7 DNA DSBs were induced by bromodeoxyuridine/Hoechst 33258/UV-A treatment to generate DSB-specific signaling in favor over extranuclear signaling (138). Interestingly, at doses generating few DSBs (<2 Gy equivalents), a clear increase in ERK1/2 phosphorylation was noted, whereas at higher doses dephosphorylation of ERK1/2 occurred. The increase in ERK1/2 phosphorylation was found to be ATM dependent, whereas dephosphorylation was ATM independent.7 The protein phosphatase responsible for dephosphorylating ERK1/2 after high doses of DSBs has not yet been identified; this may represent a ROS-independent control mechanism for PTPase activities. This result suggests that pure DNA damage–induced signaling through the ERK1/2 pathway is bimodal and dependent on the extent of DNA damage. Low damage levels activate ERK1/2 signaling, whereas high levels inactivate. Thus, several lines of evidence link ATM with MEK1/2-ERK1/2 signaling and vice versa, suggesting that ATM, in its role of monitoring cellular homeostasis (139), modulates prosurvival ERK1/2 signaling in tumor cells. Collectively, these findings show the potential for inside (nuclear) to outside (cytoplasmic) signaling, which, based on paracrine signaling effects, could then lead to temporally delayed outside (paracrine) to inside (cytoplasmic) signaling events.

What is not fully understood at this time is how DSB signaling and ATM intersect with extranuclear (plasma membrane and cytoplasmic) signaling. Radiation is known to directly inactivate PTPases, resulting in increased protein kinase signaling through the pathway in which the phosphatase acts (140). Several serine/threonine protein phosphatases with close ties to ATM and DNA-PKcs have also been implicated in the radiation response (141). The serine/threonine protein phosphatase 5 is a negative regulator of ATM that plays an important role in apoptosis and c-Abl regulation and is critical for ATM activation and the intra-S phase checkpoint (142). Similarly, protein phosphatase 5 modulates DNA-PKcs activity and radiosensitivity (143). Protein phosphatase 5 is a negative regulator of ASK1, a MAPK kinase kinase that is upstream in the JNK1/2 and p38 signaling pathways (Fig. 1; ref. 144). Protein phosphatase 5 could thus be the link between ATM and JNK1/2 signaling. Protein phosphatase 2A (PP2A) also regulates ATM activation (145). PP2A was found to interact with ATM through one of its scaffolding subunits and negatively regulate ATM. In addition, PP2A is known to regulate RAF-1-MEK1/2-ERK1/2 signaling through both positive and negative mechanisms. Critical phosphoserine residues on RAF-1, which are dephosphorylated by PP2A, reduce RAF-1 catalytic activity and lower the ability of RAF-1 to associate with RAS, which keeps signaling through MEK1/2-ERK1/2 low (146). ERK1/2 phosphorylates these same serines through a negative feedback mechanism (146). Earlier studies showed that PP2A dephosphorylates ERK2 on T183 (15, 16), suggesting that PP2A regulates RAF-1-MEK1/2-ERK1/2 positively and negatively through a feedback loop.

Another protein phosphatase that has been associated with ERBB1-RAF-1-MEK1/2-ERK1/2 signaling is the cell cycle regulator and dual specificity phosphatase CDC25A (147, 148). Similar to PP2A, CDC25A is linked to ATM through the signaling cascade ATM-CHK2-CDC25A (128). Phosphorylation of CDC25A decreases protein stability, thereby increasing the de facto phosphorylation of CDC25A target proteins. Thus, reduced CDC25A expression could invoke positive control over RAF-1 signaling, promoting RAF-1 phosphorylation on tyrosine or serine/threonine residues that would enhance downstream ERK1/2 signaling. Altogether, these examples suggest that DSBs generated in the nucleus and linked to growth signaling pathways in the cytoplasm by ubiquitously targeting phosphatases acting on the RAF-1-MEK1/2-ERK1/2 cascade are plausible mechanisms of inside-out signaling. The task at hand is to show which phosphatases are critical in the inside-out signaling of specific pathways.

ATM is also closely associated with nuclear factor-{kappa}B (NF-{kappa}B) signaling and balancing prosurvival and apoptotic signaling in response to radiation and other chemotherapeutic agents inducing DSBs. In some cell systems, ATM has been shown to phosphorylate I{kappa}B kinase in response to DSBs (149), and ATM and DNA-PKcs activate a prosurvival ERK1/2-I{kappa}B kinase-NF-{kappa}B pathway in response to DSBs that opposes the apoptotic response following DNA damage (150). As noted previously, in response to ionizing radiation exposure, both proapoptotic and antiapoptotic signals are simultaneously induced, with the proapoptotic pathway mediated by p53 targets, such as BAX, and the antiapoptotic/prosurvival pathway by NF-{kappa}B targets, such as BCL-XL, which could constitute a mechanism of balancing between proapoptotic and antiapoptotic signals induced by ionizing radiation exposure (151). Furthermore, NF-{kappa}B essential modulator, the regulatory subunit of I{kappa}B kinase, has been shown to associate with ATM after the induction of DSBs (152). ATM phosphorylates NF-{kappa}B essential modulator at S85, which promotes ubiquitin-dependent nuclear export of NF-{kappa}B essential modulator. In addition, ATM is also exported to the cytoplasm in a NF-{kappa}B essential modulator–dependent manner where it associates with and causes the activation of I{kappa}B kinase. Therefore, NF-{kappa}B essential modulator links two critical kinases, ATM and I{kappa}B kinase, to the activation of NF-{kappa}B by DSBs. Altogether, the NF-{kappa}B inside-out signaling that balances prosurvival and apoptotic signaling has been clarified to a large extent. The full range of genes targeted by NF-{kappa}B important for cell survival and DNA repair is yet to be identified. In summary, much progress in our understanding how DSB signaling is transmitted and fed into various cytoplasmic signaling pathways has been made during the past few years but much remains to fully comprehend the intricacy of the cross-talk, regulation, and balancing between growth/survival, DNA repair, and apoptosis.


   Conclusions
Top
 Abstract
 Introduction
 Signal Transduction Pathway...
 Pathways for Ionizing Radiation...
 Conclusions
References
 
Ionizing radiation can activate multiple signaling pathways in cells and causes DNA damage. The ability of radiation to activate pathways depends on the generation of ROS and RNS, the presence of DNA damage, alterations in the expression of many growth factor receptors and their cognate binding paracrine factors, and changes in RAS mutational status. Thus, because a pathway is activated by radiation in one cell type does not mean that the same pathway will be activated in a different cell type. In some cell types, enhanced basal signaling by receptors, such as ERBB1, or by oncogenes, such as RAS proteins, may provide a direct overriding radioprotective signal. In many cell types, this may be via PI3K signaling into AKT or mammalian target of rapamycin-p70S6K; in others, potentially by NF-{kappa}B or ERK1/2. Radiation, however, causes the generation of ROS/RNS, which can stimulate the activities of pathways above the high basal levels caused by receptor overexpression or RAS mutation. Similarly, DNA damage–induced signaling events have been linked to the regulation of receptor expression and to activation of signaling pathways, such as NF-{kappa}B and ERK1/2, through ATM signaling. The activation of signaling pathways occurs in waves and is dependent on the dose of radiation exposure. Thus, tumor cells may incur DNA damage and ROS/RNS generation on initial exposure to radiation, which precipitates an inside (nucleus and cytoplasm) to plasma membrane (growth factor receptor) series of signals that lead to the activation of intracellular signal transduction pathways. Activation of intracellular pathways can promote increased levels of transcription of both proapoptotic and antiapoptotic proteins depending on the balance between the various signaling pathways that have become activated. In addition, however, activation of pathways, such as ERK1/2 and p38, can promote the cleavage/release/activation of presynthesized paracrine ligands, which can feedback onto irradiated and distant unirradiated tumor cells, thereby reinitiating growth factor receptor signaling and reactivating intracellular signal transduction pathways and transcription (Figs. 2 and 3). Thus, the signaling response of a low-dose irradiated tumor cell attempting to survive is in fact a very complicated series of cause-and-effect signals. Presumably, alterations in cell signaling function and transcriptional activity after this complicated signaling response will cause further ripple effects on the long-term biological behavior of tumor cells. With this in mind, it is noteworthy that repeated exposure of breast cancer cells can increase basal expression of survival signaling growth factor receptors.

Based on careful dissection of the complicated series of signaling changes within multiple pathways, it may in the future be possible to rationally combine multiple inhibitors of these processes to block cell survival, including inhibition of DNA damage sensing, receptor activation, paracrine ligand evolution, and intracellular signaling pathway, to achieve a better therapeutic response to radiotherapy.


   Footnotes
 
Grant support: PHS grants R01-CA88906, P01-CA72955, R01-DK52825, P01-CA104177, and R01-CA108520 and Department of Defense Awards BC980148 and BC020338 (P. Dent); PHS grants P01-CA72955, R01-CA63753, and R01-CA77141 and Leukemia Society of America grant 6405-97 (S. Grant); The Department of Radiation Oncology; Friede LLC; Jim Valvano Foundation for Cancer Research; and Goodwin Foundation. P. Dent is the holder of the Universal, Inc. Professorship in Signal Transduction Research.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

6 S. Golding et al. ERK positively regulates ATM, homologous recombination repair and the DNA damage response, submitted for publication. Back

7 A. Khalil et al. ATM-dependent and -independent ERK signaling in response to DNA double-strand breaks, submitted for publication. Back

Received 9/26/06; revised 10/26/06; accepted 12/13/06.


   References
Top
 Abstract
 Introduction
 Signal Transduction Pathway...
 Pathways for Ionizing Radiation...
 Conclusions
 References
 

  1. Willers H, Held KD. Introduction to clinical radiation biology. Hematol Oncol Clin North Am 2006;1:1–24.
  2. Bentzen SM, Atasoy BM, Daley FM, et al. Epidermal growth factor receptor expression in pretreatment biopsies from head and neck squamous cell carcinoma as a predictive factor for a benefit from accelerated radiation therapy in a randomized controlled trial. J Clin Oncol 2005;24:5560–7.
  3. Hammond EM, Giaccia AJ. The role of ATM and ATR in the cellular response to hypoxia and re-oxygenation. DNA Repair (Amst) 2004;3:1117–22.[Medline]
  4. Barzilai A, Yamamoto K. DNA damage responses to oxidative stress. DNA Repair (Amst) 2004;3:1109–15.[Medline]
  5. Abraham RT. Checkpoint signaling: epigenetic events sound the DNA strand-breaks alarm to the ATM protein kinase. Bioessays 2003;25:627–30.[CrossRef][Medline]
  6. Amundson SA, Bittner M, Fornace AJ, Jr. Functional genomics as a window on radiation stress signaling. Oncogene 2003;22:5828–33.[CrossRef][Medline]
  7. Astsaturov I, Cohen RB, Harari P. Targeting epidermal growth factor receptor signaling in the treatment of head and neck cancer. Expert Rev Anticancer Ther 2006;6:1179–93.[CrossRef][Medline]
  8. Chinnaiyan P, Allen GW, Harari PM. Radiation and new molecular agents, part II: targeting HDAC, HSP90, IGF-1R, PI3K, and Ras. Semin Radiat Oncol 2006;16:59–64.[CrossRef][Medline]
  9. Kim DW, Huamani J, Fu A, Hallahan DE. Molecular strategies targeting the host component of cancer to enhance tumor response to radiation therapy. Int J Radiat Oncol Biol Phys 2006;64:38–46.[CrossRef][Medline]
  10. Brunner TB, Hahn SM, McKenna WG, Bernhard EJ. Radiation sensitization by inhibition of activated Ras. Strahlenther Onkol 2004;180:731–40.[CrossRef][Medline]
  11. Sturgill TW, Ray LB. Muscle proteins related to microtubule associated protein-2 are substrates for an insulin-stimulatable kinase. Biochem Biophys Res Commun 1986;134:565–71.[CrossRef][Medline]
  12. Boulton TG, Cobb MH. Identification of multiple extracellular signal-regulated kinases (ERKs) with antipeptide antibodies. Cell Regul 1991;2:357–71.[Medline]
  13. Sturgill TW, Ray LB, Erikson E, et al. Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II. Nature 1988;334:715–8.[CrossRef][Medline]
  14. Wu J, Michel H, Rossomando A, et al. Renaturation and partial peptide sequencing of mitogen-activated protein kinase (MAP kinase) activator from rabbit skeletal muscle. Biochem J 1992;285:701–5.[Medline]
  15. Robbins DJ, Zhen E, Owaki H, et al. Regulation and properties of extracellular signal-regulated protein kinases 1 and 2 in vitro. J Biol Chem 1993;268:5097–106.[Abstract/Free Full Text]
  16. Dent P, Haser W, Haystead TA, et al. Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro. Science 1992;257:1404–7.[Abstract/Free Full Text]
  17. Bosch E, Cherwinski H, Peterson D, et al. Mutations of critical amino acids affect the biological and biochemical properties of oncogenic A-Raf and Raf-1. Oncogene 1997;15:1021–33.[CrossRef][Medline]
  18. Marais R, Light Y, Paterson HF, et al. Differential regulation of Raf-1, A-Raf, and B-Raf by oncogenic ras and tyrosine kinases. J Biol Chem 1997;272:4378–83.[Abstract/Free Full Text]
  19. Tombes RM, Auer KL, Mikkelsen R, et al. The mitogen-activated protein (MAP) kinase cascade can either stimulate or inhibit DNA synthesis in primary cultures of rat hepatocytes depending upon whether its activation is acute/phasic or chronic. Biochem J 1998;330:1451–60.[Medline]
  20. Schmidt-Ullrich RK, Mikkelsen R, Dent P, et al. Radiation-induced proliferation of the human A431 squamous carcinoma cells is dependent on EGFR tyrosine phosphorylation. Oncogene 1997;15:1191–7.[CrossRef][Medline]
  21. Dritschilo A, Huang CH, Rudin CM, et al. Phase I study of liposome-encapsulated c-raf antisense oligodeoxyribonucleotide infusion in combination with radiation therapy in patients with advanced malignancies. Clin Cancer Res 2006;4:1251–9.
  22. Grana TM, Rusyn EV, Zhou H, et al. Ras mediates radioresistance through both phosphatidylinositol 3-kinase-dependent and Raf-dependent but mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-independent signaling pathways. Cancer Res 2002;14:4142–50.
  23. Sharp S, Workman P. Inhibitors of the HSP90 molecular chaperone: current status. Adv Cancer Res 2006;95:323–48.[CrossRef][Medline]
  24. Moodie SA, Willumsen BM, Weber MJ, et al. Complexes of Ras GTP with Raf-1 and mitogen-activated protein kinase kinase. Science 1993;260:1658–61.[Abstract/Free Full Text]
  25. Van Aelst L, Barr M, Marcus S, et al. Complex formation between RAS and RAF and other protein kinases. Proc Natl Acad Sci U S A 1993;90:6213–7.[Abstract/Free Full Text]
  26. Leevers SJ, Paterson HF, Marshall CJ. Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature 1994;369:411–4.[CrossRef][Medline]
  27. Dent P, Sturgill TW. Activation of (His)6-Raf-1 in vitro by partially purified plasma membranes from v-Ras-transformed and serum stimulated fibroblasts. Proc Natl Acad Sci U S A 1994;91:9544–8.[Abstract/Free Full Text]
  28. Li N, Batzer A, Daly R, et al. Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signaling. Nature 1993;363:85–8.[CrossRef][Medline]
  29. Olivier JP, Raabe T, Henkemeyer M, et al. A Drosophila SH2-SH3 adaptor protein implicated in coupling the sevenless tyrosine kinase to an activator of Ras guanine nucleotide exchange, Sos. Cell 1993;73:179–91.[CrossRef][Medline]
  30. Stokoe D, McCormick F. Activation of c-Raf-1 by Ras and Src through different mechanisms: activation in vivo and in vitro. EMBO J 1997;16:2384–96.[CrossRef][Medline]
  31. Tzivion G, Luo J, Avruch J. A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity. Nature 1998;394:88–92.[CrossRef][Medline]
  32. Marais R, Light Y, Paterson HF, Marshall CJ. Ras recruits Raf-1 to the plasma membrane for activation by tyrosine phosphorylation. EMBO J 1995;14:3136–45.[Medline]
  33. Yip-Schneider MT, Miao W, Lin A, et al. Regulation of the Raf-1 kinase domain by phosphorylation and 14-3-3 association. Biochem J 2000;351:151–9.[CrossRef][Medline]
  34. Dhillon AS, Kolch W. Untying the regulation of the Raf-1 kinase. Arch Biochem Biophys 2002;404:3–9.[CrossRef][Medline]
  35. Yacoub A, Miller A, Caron RW, et al. Radiotherapy-induced signal transduction. Endocr Relat Cancer 2006;13 Suppl 1:S99–114.
  36. Tournier C, Whitmarsh AJ, Cavanagh J, et al. The MKK7 gene encodes a group of c-Jun NH2-terminal kinase kinases. Mol Cell Biol 1999;19:1569–81.[Abstract/Free Full Text]
  37. Han J, Lee JD, Bibbs L, Ulevitch RJ. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 1994;265:808–11.[Abstract/Free Full Text]
  38. Wang X, Tournier C. Regulation of cellular functions by the ERK5 signalling pathway. Cell Signal 2006;6:753–60.
  39. Vanhaesebroeck B, Alessi DR. The PI3K-PDK1 connection: more than just a road to PKB. Biochem J 2000;346:561–76.[CrossRef][Medline]
  40. Simpson L, Parsons R. PTEN: life as a tumor suppressor. Exp Cell Res 2001;264:29–41.[CrossRef][Medline]
  41. Cross DA, Alessi DR, Cohen P, et al. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 1995;378:785–9.[CrossRef][Medline]
  42. Andjelkovic M, Maira SM, Cron P, et al. Domain swapping used to investigate the mechanism of protein kinase B regulation by 3-phosphoinositide-dependent protein kinase 1 and Ser473 kinase. Mol Cell Biol 1999;19:5061–72.[Abstract/Free Full Text]
  43. Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet 2006;8:606–19.
  44. Galabova-Kovacs G, Kolbus A, Matzen D, et al. ERK and beyond: insights from B-Raf and Raf-1 conditional knockouts. Cell Cycle 2006;5:1514–8.[Medline]
  45. Gulbins E, Kolesnick R. Raft ceramide in molecular medicine. Oncogene 2003;22:7070–7.[CrossRef][Medline]
  46. Kavanagh BD, Lin PS, Chen P, et al. Radiation-induced enhanced proliferation of human squamous cancer cells in vitro: a release from inhibition by epidermal growth factor. Clin Cancer Res 1995;12:1557–62.
  47. Balaban N, Moni J, Shannon M, et al. The effect of ionizing radiation on signal transduction: antibodies to EGF receptor sensitize A431 cells to radiation. Biochim Biophys Acta 1996;1314:147–56.[Medline]
  48. Goldkorn T, Balaban N, Shannon M, et al. EGF receptor phosphorylation is affected by ionizing radiation. Biochim Biophys Acta 1997;3:289–99.
  49. Kavanagh BD, Dent P, Schmidt-Ullrich RK, et al. Calcium-dependent stimulation of mitogen-activated protein kinase activity in A431 cells by low doses of ionizing radiation. Radiat Res 1998;6:579–87.
  50. Contessa JN, Reardon DB, Todd D, et al. The inducible expression of dominant-negative epidermal growth factor receptor-CD533 results in radiosensitization of human mammary carcinoma cells. Clin Cancer Res 1999;2:405–11.
  51. Todd DG, Mikkelsen RB, Rorrer WK, et al. Ionizing radiation stimulates existing signal transduction pathways involving the activation of epidermal growth factor receptor and ERBB-3, and changes of intracellular calcium in A431 human squamous carcinoma cells. J Recept Signal Transduct Res 1999;6:885–908.
  52. Meyer RG, Kupper JH, Kandolf R, et al. Early growth response-1 gene (Egr-1) promoter induction by ionizing radiation in U87 malignant glioma cells in vitro. Eur J Biochem 2002;1:337–46.
  53. Bowers G, Reardon D, Hewitt T, et al. The relative role of ErbB1-4 receptor tyrosine kinases in radiation signal transduction responses of human carcinoma cells. Oncogene 2001;20:1388–97.[CrossRef][Medline]
  54. Dent P, Reardon DB, Wood SL, et al. Inactivation of raf-1 by a protein-tyrosine phosphatase stimulated by GTP and reconstituted by G{alpha}i/o subunits. J Biol Chem 1996;6:3119–23.
  55. Tonks NK. Protein tyrosine phosphatases and the control of cellular signaling responses. Adv Pharmacol 1996;36:91–119.[Medline]
  56. Van Montfort RL, Congreve M, Tisi D, et al. Oxidation state of the active-site cysteine in protein tyrosine phosphatase 1B. Nature 2003;423:773–7.[CrossRef][Medline]
  57. Mikkelsen RB, Wardman P. Biological chemistry of reactive oxygen and nitrogen and radiation-induced signal transduction mechanisms. Oncogene 2003;37:5734–54.
  58. Leach JK, Van Tuyle G, Lin PS, et al. Ionizing radiation-induced, mitochondria-dependent generation of reactive oxygen/nitrogen. Cancer Res 2001;10:3894–901.
  59. Sturla LM, Amorino G, Alexander MS, et al. Requirement of Tyr-992 and Tyr-1173 in phosphorylation of the epidermal growth factor receptor by ionizing radiation and modulation by SHP2. J Biol Chem 2005;15:14597–604.
  60. Barrett DM, Black SM, Todor H, et al. Inhibition of protein-tyrosine phosphatases by mild oxidative stresses is dependent on S-nitrosylation. J Biol Chem 2005;15:14453–61.
  61. Vega QC, Cochet C, Filhol O, et al. A site of tyrosine phosphorylation in the C terminus of the epidermal growth factor receptor is required to activate phospholipase C. Mol Cell Biol 1992;1:128–35.
  62. Kasid U, Suy S, Dent P, et al. Activation of Raf by ionizing radiation. Nature 1996;382:813–6.[CrossRef][Medline]
  63. Kamata H, Honda S, Maeda S, Chang L, Hirata H, Karin M. Reactive oxygen species promote TNF{alpha}-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases. Cell 2005;120:649–61.[CrossRef][Medline]
  64. Dent P, Han SI, Mitchell C, et al. Inhibition of insulin/IGF-1 receptor signaling enhances bile acid toxicity in primary hepatocytes. Biochem Pharmacol 2005;11:1685–96.
  65. Reinehr R, Becker S, Eberle A, et al. Involvement of NADPH oxidase isoforms and Src family kinases in CD95-dependent hepatocyte apoptosis. J Biol Chem 2005;29:27179–94.
  66. Fang Y, Han SI, Mitchell C, et al. Bile acids induce mitochondrial ROS, which promote activation of receptor tyrosine kinases and signaling pathways in rat hepatocytes. Hepatology 2004;4:961–71.
  67. Reinehr R, Becker S, Keitel V, et al. Bile salt-induced apoptosis involves NADPH oxidase isoform activation. Gastroenterology 2005;6:2009–31.
  68. Han SI, Studer E, Gupta S, et al. Bile acids enhance the activity of the insulin receptor and glycogen synthase in primary rodent hepatocytes. Hepatology 2004;2:456–63.
  69. Liu ZM, Huang HS. As2O3-induced c-Src/EGFR/ERK signaling is via Sp1 binding sites to stimulate p21WAF1/CIP1 expression in human epidermoid carcinoma A431 cells. Cell Signal 2006;2:244–55.
  70. Khan EM, Heidinger JM, Levy M, et al. Epidermal growth factor receptor exposed to oxidative stress undergoes Src- and caveolin-1-dependent perinuclear trafficking. J Biol Chem 2006;20:14486–93.
  71. Dent P, Reardon DB, Park JS, et al. Radiation-induced release of transforming growth factor {alpha} activates the epidermal growth factor receptor and mitogen-activated protein kinase pathway in carcinoma cells, leading to increased proliferation and protection from radiation-induced cell death. Mol Biol Cell 1999;8:2493–506.
  72. Shvartsman SY, Hagan MP, Yacoub A, et al. Autocrine loops with positive feedback enable context-dependent cell signaling. Am J Physiol Cell Physiol 2002;3:C545–59.
  73. Hagan M, Yacoub A, Dent P. Ionizing radiation causes a dose-dependent release of transforming growth factor {alpha} in vitro from irradiated xenografts and during palliative treatment of hormone-refractory prostate carcinoma. Clin Cancer Res 2004;17:5724–31.
  74. O-Charoenrat P, Wongkajornsilp A, Rhys-Evans PH, et al. Signaling pathways required for matrix metalloproteinase-9 induction by betacellulin in head and neck squamous carcinoma cells. Int J Cancer 2004;2:174–83.
  75. Zhang Q, Thomas SM, Xi S, et al. SRC family kinases mediate epidermal growth factor receptor ligand cleavage, proliferation, and invasion of head and neck cancer cells. Cancer Res 2004;17:6166–73.
  76. El-Shewy HM, Kelly FL, Barki-Harrington L, Luttrell LM. Ectodomain shedding-dependent transactivation of epidermal growth factor receptors in response to insulin-like growth factor type I. Mol Endocrinol 2004;11:2727–39.
  77. Caron RW, Yacoub A, Zhu X, et al. H-RAS V12-induced radioresistance in HCT116 colon carcinoma cells is heregulin dependent. Mol Cancer Ther 2005;2:243–55.
  78. Fang L, Li G, Liu G, Lee SW, Aaronson SA. p53 induction of heparin-binding EGF-like growth factor counteracts p53 growth suppression through activation of MAPK and PI3K/Akt signaling cascades. EMBO J 2001;8:1931–9.
  79. Sheikh MS, Carrier F, Johnson AC, et al. Identification of an additional p53-responsive site in the human epidermal growth factor receptor gene promotor. Oncogene 1997;9:1095–101.
  80. Nishi H, Senoo M, Nishi KH, et al. p53 homologue p63 represses epidermal growth factor receptor expression. J Biol Chem 2001;276:41717–24.[Abstract/Free Full Text]
  81. Schmidt-Ullrich RK, Valerie K, Chan W, Wazer DE, Lin PS. Expression of oestrogen receptor and transforming growth factor-{alpha} in MCF-7 cells after exposure to fractionated irradiation. Int J Radiat Biol 1992;61:405–15.[Medline]
  82. Shelton JG, Steelman LS, Abrams SL, et al. The epidermal growth factor receptor gene family as a target for therapeutic intervention in numerous cancers: what's genetics got to do with it? Expert Opin Ther Targets 2005;5:1009–30.
  83. Baselga J, Arteaga CL. Critical update and emerging trends in epidermal growth factor receptor targeting in cancer. J Clin Oncol 2005;11:2445–59.
  84. Harari PM. Epidermal growth factor receptor inhibition strategies in oncology. Endocr Relat Cancer 2004;4:689–708.
  85. Pino MS, Shrader M, Baker CH, et al. Transforming growth factor {alpha} expression drives constitutive epidermal growth factor receptor pathway activation and sensitivity to gefitinib (Iressa) in human pancreatic cancer cell lines. Cancer Res 2006;7:3802–12.
  86. Chinnaiyan P, Huang S, Vallabhaneni G, et al. Mechanisms of enhanced radiation response following epidermal growth factor receptor signaling inhibition by erlotinib (Tarceva). Cancer Res 2005;8:3328–35.
  87. Bianco C, Tortora G, Bianco R, et al. Enhancement of antitumor activity of ionizing radiation by combined treatment with the selective epidermal growth factor receptor-tyrosine kinase inhibitor ZD1839 (Iressa). Clin Cancer Res 2002;10:3250–8.
  88. Sarkaria JN, Carlson BL, Schroeder MA, et al. Use of an orthotopic xenograft model for assessing the effect of epidermal growth factor receptor amplification on glioblastoma radiation response. Clin Cancer Res 2006;7:2264–71.
  89. Raben D, Helfrich BA, Chan D, et al. ZD1839, a selective epidermal growth factor receptor tyrosine kinase inhibitor, alone and in combination with radiation and chemotherapy as a new therapeutic strategy in non-small cell lung cancer. Semin Oncol 2002;1:37–46.
  90. Harari PM, Huang S. Radiation combined with EGFR signal inhibitors: head and neck cancer focus. Semin Radiat Oncol 2006;1:38–44.[CrossRef]
  91. Kwak EL, Sordella R, Bell DW, et al. Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib. Proc Natl Acad Sci U S A 2005;21:7665–70.
  92. Thomas RK, Greulich H, Yuza Y, et al. Detection of oncogenic mutations in the EGFR gene in lung adenocarcinoma with differential sensitivity to EGFR tyrosine kinase inhibitors. Cold Spring Harb Symp Quant Biol 2005;70:73–81.[CrossRef][Medline]
  93. Jones HE, Gee JM, Barrow D, et al. Inhibition of insulin receptor isoform-A signalling restores sensitivity to gefitinib in previously de novo resistant colon cancer cells. Br J Cancer 2006;2:172–80.
  94. Yacoub A, Park MA, Hanna D, et al. OSU-03012 promotes caspase-independent but PERK-, cathepsin B-, BID-, and AIF-dependent killing of transformed cells. Mol Pharmacol 2006;2:589–603.
  95. Ihle NT, Paine-Murrieta G, Berggren MI, et al. The phosphatidylinositol-3-kinase inhibitor PX-866 overcomes resistance to the epidermal growth factor receptor inhibitor gefitinib in A-549 human non-small cell lung cancer xenografts. Mol Cancer Ther 2005;9:1349–57.
  96. Gupta AK, Bakanauskas VJ, Cerniglia GJ, et al. The Ras radiation resistance pathway. Cancer Res 2001;61:4278–782.[Abstract/Free Full Text]
  97. Gupta AK, McKenna WG, Weber CN, et al. Local recurrence in head and neck cancer: relationship to radiation resistance and signal transduction. Clin Cancer Res 2002;8:885–92.[Abstract/Free Full Text]
  98. Gupta AK, Bernhard EJ, Bakanauskas VJ, et al. RAS-mediated radiation resistance is not linked to MAP kinase activation in two bladder carcinoma cell lines. Radiat Res 2000;154:64–72.[CrossRef][Medline]
  99. Kim IA, Bae SS, Fernandes A, et al. Selective inhibition of Ras, phosphoinositide 3 kinase, and Akt isoforms increases the radiosensitivity of human carcinoma cell lines. Cancer Res 2005;17:7902–10.
  100. Brunner TB, Cengel KA, Hahn SM, et al. Pancreatic cancer cell radiation survival and prenyltransferase inhibition: the role of K-Ras. Cancer Res 2005;18:8433–41.
  101. Caron RW, Yacoub A, Li M, et al. Activated forms of H-RAS and K-RAS differentially regulate membrane association of PI3K, PDK-1, and AKT and the effect of therapeutic kinase inhibitors on cell survival. Mol Cancer Ther 2005;2:257–70.
  102. Caron RW, Yacoub A, Mitchell C, et al. Radiation-stimulated ERK1/2 and JNK1/2 signaling can promote cell cycle progression in human colon cancer cells. Cell Cycle 2005;3:456–64.