Radiation-induced Signal Transduction Research Articles

DNA damage response signaling pathways and targets for radiotherapy sensitization in cancer

Radiotherapy is one of the most common countermeasures for treating a wide range of tumors. However, the radioresistance of cancer cells is still a major limitation for radiotherapy applications. Efforts are continuously ongoing to explore sensitizing targets and develop radiosensitizers for improving the outcomes of radiotherapy. DNA double-strand breaks are the most lethal lesions induced by ionizing radiation and can trigger a series of cellular DNA damage responses (DDRs), including those helping cells recover from radiation injuries, such as the activation of DNA damage sensing and early transduction pathways, cell cycle arrest, and DNA repair. Obviously, these protective DDRs confer tumor radioresistance. Targeting DDR signaling pathways has become an attractive strategy for overcoming tumor radioresistance, and some important advances and breakthroughs have already been achieved in recent years. On the basis of comprehensively reviewing the DDR signal pathways, we provide an update on the novel and promising druggable targets emerging from DDR pathways that can be exploited for radiosensitization. We further discuss recent advances identified from preclinical studies, current clinical trials, and clinical application of chemical inhibitors targeting key DDR proteins, including DNA-PKcs (DNA-dependent protein kinase, catalytic subunit), ATM/ATR (ataxia–telangiectasia mutated and Rad3-related), the MRN (MRE11-RAD50-NBS1) complex, the PARP (poly[ADP-ribose] polymerase) family, MDC1, Wee1, LIG4 (ligase IV), CDK1, BRCA1 (BRCA1 C terminal), CHK1, and HIF-1 (hypoxia-inducible factor-1). Challenges for ionizing radiation-induced signal transduction and targeted therapy are also discussed based on recent achievements in the biological field of radiotherapy.

Raltitrexed increases radiation sensitivity of esophageal squamous carcinoma cells

BackgroundRadiation therapy remains an important therapeutic modality, especially for those patients who are not candidates for radical resection. Many strategies have been developed to increase the radiosensitivity of esophageal cancer, with some success.MethodsThis study was conducted to determine whether raltitrexed can enhance radiosensitivity of esophageal squamous cell carcinoma (ESCC). ESCC cell lines 24 h were incubated with raltitrexed or DMSO with or without subsequent irradiation. Cell Counting Kit assay-8 assay and clonogenic survival assay were used to measure the cell proliferation and radiosensitization, respectively. Flow cytometry was utilized to examine cell apoptosis and cell cycle distribution in different groups. Immunofluorescence analysis was performed to detect deoxyribonucleic acid (DNA) double-strand breaks. In addition, the expression levels of proteins that are involved in radiation induced signal transduction including Bax, Cyclin B1, Cdc2/pCdc2, and Cdc25C/pCdc25C were examined by western blot analysis.ResultsThe results indicated that raltitrexed enhanced radiosensitivity of ESCC cells with increased DNA double-strand breaks, the G2/M arrest, and the apoptosis of ESCC cells induced by radiation. The sensitization enhancement ratio of 1.23–2.10 was detected for ESCC cells with raltitrexed treatment in TE-13 cell line. In vitro, raltitrexed also increased the therapeutic effect of radiation in nude mice.ConclusionRaltitrexed increases the radiosensitivity of ESCC. This antimetabolite drug is promising for future clinical trials with concurrent radiation in esophageal cancer.

Sunitinib modulates the radiosensitivity of esophageal squamous cell carcinoma cells in vitro.

This study aims to explore the radiosensitivity of sunitinib on esophageal cancer cell lines. For in vitro studies, human esophageal squamous cell carcinoma (ESCC) cell lines were treated with sunitinib 24 hours before irradiation. ESCC cell lines were treated with sunitinib with or without radiation. Cell proliferation was detected by Cell Counting Kit 8 assay. Radiosensitization was evaluated by clonogenic survival assay. Cell apoptosis and cell cycle analysis were detected by flow cytometry. Deoxyribonucleic acid (DNA) double-strand breaks were performed by immunocytofluorescence analysis. Western blot analysis was used to determine the effect of sunitinib on radiation induced signal transduction. Sunitinib potently sensitized ESCC cells to radiation with a sensitization enhancement ratio of 1.13-1.72. Furthermore, sunitinib increased radiation induced DNA double-strand breaks, promoted the apoptosis of ESCC cells and induced the G2/M arrest. Radiosensitization was accompanied with enhanced apoptosis and regulated by the intrinsic pathway of apoptosis. Sunitinib sensitized ESCC cells to the cytotoxic effects of radiation. This compound is promising for future clinical trials with chemoradiation in esophageal cancer.

The radiosensitization effect of mTOR inhibitior in human tongue squamous cell carcinoma.

e13585 Background: mTOR plays a critical role in malignant phenotype in many tumors. Everolimus [RAD001 (40-O-(2-hydroxyethyl)-rapamycin)] is a rapamycin rapalog that is being developed as an antitumor agent and is approved for various cancers. Recently, RAD001 has also been reported to increase the radiosensitization following for amplifying the autophagic pathway; however, it could promote an alternative mechanism that leads to apoptosis. In HNSCC, radiation therapy is a common form of cancer treatment, but locoregional recurrence and/or distant metastasis is the most cause of failure after RT and it results in HNSCC significant morbidity. Evidence from previous studies showed that RAD001 enhanced the cytotoxic effects of radiation in HNSCC cell lines. The molecular mechanism of radiosensitization by RAD001 in HNSCC has not cleared.Our aim is to evaluate the role of molecular mechanism of RAD001-mediated radiosensitization in HNSCC. Methods: Radiosensitivity effects of RAD001 were assays on human oral squamous cell carcinoma cell lines (SCC4 and DuFa). RAD001 (30 and 300 nM)and radiation (0~8 Gy) alone and in combination were evaluated for antitumor activity in the clonogenic assay. Next, we also evaluated the RAD001 on radiation-induced signal transduction by western blot analysis. Results: Here, we show that 300nM RAD001 plus 6 Gy cohort with significant HNSCC cell line growth suppression compared to 6 Gy alone (p=0.004) or 30n MRAD001 plus 6 Gy (p=0.008). RAD001 treatment effectively reduces mTOR downstream effector p-S6 protein compared with the vehicle or irradiated alone. RAD001 + 6 Gy significantly increases p53 and PARP cleavage, which facilitates apoptosis. Unexpectedly, autophagy pathway, such as beclin 1 and LC3II were not affected by RAD001. Addiationlly, we discover that Akt phosphorylation (Ser473) is up-regulated after RAD001/and irradiation of HNSCC cells. Conclusions: These findings suggest radiosensitization by the RAD001 involves the induction of apoptosis in HNSCC. Notwithstanding, Akt signaling is increased under RAD001 and it could attenuate anticancer efficacy instead, contribute to the development of resistance.

Towards Novel Radiosensitizing Agents: The Role of Cytosolic PLA2α in Combined Modality Cancer Therapy

The radioresistant nature of some tumors serves as an obstacle to curative therapy for several poor-prognosis malignancies. The radiosensitivity of a cancer is dependent not only on the intrinsic ability of tumor cells to recover from radiation-induced damage, but also the ability of stromal elements (e.g., vasculature) in the tumor microenvironment to survive and continue proliferating in the face of ionizing radiation. In this regard, it is important to understand the initial events activating radiation-induced signal transduction pathways. Among these events is the activation of cytosolic phospholipase A2 α and the subsequent production of the lipid second messengers. These events occur within minutes following exposure to ionizing radiation, and have been shown to enhance cell viability through a number of prosurvival signaling pathways. Furthermore, inhibition of cytosolic phospholipase A2 α has now been shown to reduce the viability of endothelial cells in culture after exposure to ionizing radiation, as well as slowing the growth of tumors in animal models of cancer.

SU11248 (Sunitinib) Sensitizes Pancreatic Cancer to the Cytotoxic Effects of Ionizing Radiation

SU11248 (sunitinib) is a small-molecule tyrosine kinase inhibitor which targets VEGFR and PDGFR isoforms. In the present study, the effects of SU11248 and ionizing radiation on pancreatic cancer were studied. For in vitro studies human pancreatic adenocarcinoma cells lines were treated with 1 microM SU11248 1 h before irradiation. Western blot analysis was used to determine the effect of SU11248 on radiation-induced signal transduction. To determine if SU11248 sensitized pancreatic cancer to the cytotoxic effects of ionizing radiation, a clonogenic survival assay was performed using 0-6 Gy. For in vivo assays, CAPAN-1 cells were injected into the hind limb of nude mice for tumor volume and proliferation studies. SU11248 attenuated radiation-induced phosphorylation of Akt and ERK at 0, 5, 15, and 30 min. Furthermore, SU11248 significantly reduced clonogenic survival after treatment with radiation (p < 0.05). In vivo studies revealed that SU11248 and radiation delayed tumor growth by 6 and 10 days, respectively, whereas combined treatment delayed tumor growth by 30 days. Combined treatment with SU11248 and radiation further attenuated Brdu incorporation by 75% (p = 0.001) compared to control. SU11248 (sunitinib) sensitized pancreatic cancer to the cytotoxic effects of radiation. This compound is promising for future clinical trials with chemoradiation in pancreatic cancer.

Current Concepts for the Combined Treatment Modality of Ionizing Radiation with Anticancer Agents

In current applied radiobiology, there exists a tremendous effort in basic and translational research to identify novel treatment modalities combining ionizing radiation with anticancer agents. This is mainly due to the highly improved molecular understanding of intrinsic radioresistance and the profiling of cellular stress responses to irradiation during recent years. Ionizing radiation not only damages DNA but also affects multiple cellular components that induce a multi-layered stress response. The treatment responses can be restricted to the individual cell level but might also be part of an intercellular stress communication network. Both DNA damage-induced signaling (which results in cell cycle arrest and induction of the DNA-repair machinery) and also ionizing radiation-induced signal transduction cascades, which are generated at cellular sites distant from and independent of DNA-damage, represent interesting targets for anticancer treatment modalities to sensitize for ionizing radiation. Due to the lack of molecular knowledge classic radiobiology assembled the cellular and tissue responses into four groups (4 R's of radiotherapy) which describe biological factors influencing the treatment response to fractionated radiotherapy. These classic 4 R's are Repair, Reassortment, Repopulation and Reoxygenation. With the tremendous progress in molecular oncology we now begin to understand theses factors on the molecular level. At the same time this classification may guide modern molecular radiobiologists to identify novel pharmaceuticals and antisignaling agents which can modulate the treatment response to irradiation. In this review we describe current approaches to sensitize tumor cells with novel anticancer agents along the lines of these 4 R's.

PKB/AKT is a molecular target to enhance radiation-induced cytotoxicity within tumor vascular endothelium

Signal transduction through the protein kinase B (PKB/AKT) pathway enhances cell viability within the vascular endothelium. To determine whether AKT signalling participates in the response of tumor vascular endothelium to radiation, we studied dominant negative genetic constructs and specific inhibitors of AKT in irradiated vascular endothelium. Radiation-induced signal transduction was studied by use of phospho-specific antibodies to AKT and its substrate glycogen synthase kinase 3β (GSK-3B). Akt was inhibited by overexpression of the mutant Akt dominant negative genetic construct within adenoviral vectors. Apoptosis was studied in transduced endothelial cells by analysis of caspase 3 cleavage and quantification of apoptotic nuclei. Endothelial cell migration was studied by use of Boyden chambers. Endothelial capillary tubule formation was studied in irradiated endothelial cells by use of Matrigel. The dorsal skin fold window was used to study vascular length density in LLC tumors implanted in mice. Immunohistochemistry was used to quantify apoptosis in tumor endothelium by use of TUNEL staining and endothelial-specific antibody staining. Irradiation of vascular endothelium resulted in phosphorylation of AKT and subsequent phosphorylation of GSK3B. Endothelial cells showed no caspase cleavage and no induction of apoptosis in response to 3 Gy. Overexpression of the Akt dominant negative genetic construct eliminated the apoptosis resistance phenotype in irradiated endothelial cells (p = 0.02). The dominant negative Akt also prevented migration and capillary tubule formation in irradiated endothelial cells within both Matrigel and animal models (p = 0.04). Because GSK3B attenuates apoptosis and mediates endothelial cell migration, we studied GSK3B inhibitors within endothelial cells treated with radiation and overexpression of Akt dominant negative genetic mutations. GSK-3B inhibitors attenuated migration and capillary tubule formation observed in irradiated endothelial cells (p = 0.05). Moreover, GSK-3B inhibitors reversed the induction of apoptosis in cells treated with Akt dominant negative and radiation. AKT inhibition by specific inhibitors enhanced radiation-induced apoptosis within vascular endothelium and significantly reduced clonogenic survival (p = 0.01). Therefore, AKT inhibiting compounds were used to study the role of this signalling pathway in vivo. Akt-specific inhibitors were administered to mice bearing irradiate flank tumors. Histologic sections of tumors also show enhanced apoptosis in tumor vascular endothelium (p = 0.05). The dorsal skin fold window analysis of tumor vasculature showed enhancement of destruction of tumor blood vessels when AKT inhibitors are administered prior to irradiation with 3 Gy. This was significantly enhanced as compared to tumor blood vessels treated with either AKT inhibitors alone or radiation alone (p = 0.05). Akt signalling therefore plays a central role in tumor vascular endothelial cell survival and capillary tubule formation following tumor irradiation. GSK3B phosphorylation can serve as a biomarker for pharmacodynamic studies of Akt inhibitors in planned clinical trials in the treatment of cancer.

Mechanisms of radiation-induced activation of the PI3K/Akt signal transduction pathway in vascular endothelium

Purpose/Objective: Receptor tyrosine kinases are key elements in initiation of signal transduction that results in proliferation and viability of cells. Previous studies have demonstrated that ionizing radiation activates the PI3K/Akt signal transduction pathway in tumor vascular endothelium. The purpose of the present study was to evaluate the role of receptor tyrosine kinases (RTKs) in this radiation-induced signal transduction in vascular endothelium.

Biological chemistry of reactive oxygen and nitrogen and radiation-induced signal transduction mechanisms

In the past few years, nuclear DNA damage-sensing mechanisms activated by ionizing radiation have been identified, including ATM/ATR and the DNA-dependent protein kinase. Less is known about sensing mechanisms for cytoplasmic ionization events and how these events influence nuclear processes. Several studies have demonstrated the importance of cytoplasmic signaling pathways in cytoprotection and mutagenesis. For cytoplasmic signaling, radiation-stimulated reactive oxygen species (ROS) and reactive nitrogen species (RNS) are essential activators of these pathways. This review summarizes recent studies on the chemistry of radiation-induced ROS/RNS generation and emphasizes interactions between ROS and RNS and the relative roles of cellular ROS/RNS generators as amplifiers of the initial ionization events. Cellular mechanisms for regulating ROS/RNS levels are discussed. The mechanisms by which cells sense ROS/RNS are examined in terms of how ROS/RNS modify protein structure and function, for example, interactions with metal-thiol clusters, protein tyrosine nitration, protein cysteine oxidation, S-thiolation and S-nitrosylation. We propose that radiation-induced ROS are the initiators and that nitric oxide (NO*) or derivatives are the effectors activating these signal transduction pathways. In responding to cellular ionization events, the cell converts an oxidative signal to a nitrosative one because ROS are too reactive and unspecific in their reactions for regulatory purposes and the cell is equipped to precisely modulate NO* levels.

Effect of Space Flight on the Frequency of Micronuclei and Expression of Stress-responsive Proteins in Cultured Mammalian Cells

Results of past space experiments suggest that the biological effect of space radiation could be enhanced under microgravity in some cases, especially in insects. To examine if such a synergistic effect of radiation and microgravity also exists in human cells, frequencies of chromosome instability and cellular levels of several stress-responsive proteins were analyzed in cultured human and rodent cells after space flight. Human (MCF7 and AT2KY), mouse (m5S) and hamster (SHE) cell lines were loaded on the Space Shuttle Discovery (STS-95 mission) and grown during a 9-day mission. After landing, the micronuclei resulting from abnormal nuclear division and accumulation of stress-responsive proteins such as p53 and mitogen-activated protein kinases (MAPKs), which are involved in radiation-induced signal transduction cascades, were analyzed. The frequencies of micronuclei in all the four mammalian cell strains tested were not significantly different between flight and ground control samples. Also, the cellular amounts of p53, p21 (WAF1/SDI1/CIP1) and activated (phosphorylated) forms of three distinct MAPKs in MCF7 and m5S cells of flight samples were similar to those of ground control samples. These results indicated that any effect of space radiation, microgravity, or combination of both were not detectable, at least under the present experimental conditions.

24] Radiation-induced signal transduction

Publisher Summary Cells depend for almost all their life reactions, from survival to apoptosis, from circadian rhythm to changes in behavior and differentiation, on extracellular cues. Much information has accumulated on the mechanisms of cell-cell communication and on the network of signaling inside the cell. It is now known the way hormones act and the way cytokines address the cellular program of gene expression and some of the components of the signal transduction network are known from the cell membrane to the cytoskeleton and to the nucleus. There are still many gaps in our understanding. One research field that may contribute to a better understanding of such pathways concerns cellular responses to light, to other types of radiation, and to non-natural chemicals. This chapter addresses some of the methodology that has been employed to study radiation-induced signaling and that led to the recognition of radiation-induced pathways and of some of the primary target molecules.

Defective regulation of Ca 2+/calmodulin-dependent protein kinase II in γ-irradiated ataxia telangiectasia fibroblasts

Recent indirect evidence suggests that a Ca 2+/calmodulin-dependent pathway, which may involve calmodulin-dependent protein kinase II (CaMKII), mediates the S-phase delay manifested by γ-ray-exposed human fibroblasts. This pathway is severely impaired in ataxia telangiectasia (A-T) cells [Mirzayans et al. (1995) Oncogene 11, 1597]. To extend these findings, we assayed CaMKII activity in irradiated normal and A-T fibroblasts. The radiation treatment induced the autonomous activity of the kinase in normal cells. In contrast, this activity was not elevated in either (i) normal cells pretreated with the selective CaMKII antagonist KN-62 or (ii) γ-irradiated A-T cells. Moreover, A-T fibroblasts, unlike normal cells, failed to mobilize intracellular Ca 2+ upon mitogenic stimulation. These findings identify a novel role for CaMKII in radiation-induced signal transduction and suggest its involvement in effecting the S-phase delay. The data also implicate ATM, the product of the gene responsible for A-T, as a key mediator of both intracellular Ca 2+ mobilization and CaMKII activation in response not only to genotoxic stress but also to physiological stimuli.

Histone H1 Dephosphorylation Is Mediated through a Radiation-induced Signal Transduction Pathway Dependent on ATM

Ionizing radiation is known to activate multiple signal transduction pathways, but the targets of these pathways are poorly understood. Phosphorylation of histone H1 is thought to have a role in chromatin condensation/decondensation, and we asked whether ionizing radiation (IR) would alter H1 phosphorylation. Our data demonstrate that low doses of IR result in a dramatic, but transient, dephosphorylation of H1 isoforms. The in vivo IR-induced dephosphorylation of H1 is completely blocked by wortmannin and is abrogated in ataxia telangiectasia cells. Furthermore, we measured radiation-induced inhibition of cyclin dependent kinase activity and activation of histone H1 phosphatase activity. Both activities were affected by radiation-induced signals in an ATM-dependent manner. Thus, the rapid IR-induced dephosphorylation of H1 involves a pathway including ATM and a wortmannin-sensitive step leading to both inhibition of cyclin-dependent kinase activities as well as activation of H1 phosphatase(s).

Radiation-induced signal transduction. Mechanisms and consequences

Over a dose range up to 50 Gy of low-LET (linear energy transfer) ionizing radiation and up to 5 k)/m2 UVB, mammalian cells convert molecular damage into productive response (mostly gain of function). By inactivation of negative regulatory components, such as protein tyrosine phosphatases as one mechanism discovered, the balance between restraining and stimulating influences is disturbed and an increase in signal flow results. Also DNA damage causing transcriptional arrest produces a signalling cascade of as yet unknown details. Such stimulation of the intracellular communication network can lead to apoptosis, elevated cell cycling and differentiation processes possibly including repair and recombination. The outcome likely depends on integration of all signals received which is as yet ill-understood. Although accurate determinations of low-dose inductions have not been achieved for technical reasons, the dose-response curves of induced signal transduction likely show threshold characteristics, in contrast to the direct consequences of DNA damage.

Mechanism of radiation-induced diacylglycerol production in primary cultured rat hepatocytes.

Protein kinase C (PKC) is known to be a key enzyme in radiation-induced signal transduction pathways. We have previously demonstrated that gamma-irradiation induces PKC activation and translocation from cytosol to membranes as a consequence of membrane lipid peroxidation in cultured rat hepatocytes (Int. J. Radiat. Biol. 70, 473-480, 1996). The present study was undertaken to investigate production of diacylglycerol, an endogenous activator of PKC, following gamma-irradiation of hepatocytes. Diacylglycerol content increased 3 min after irradiation, then decreased at 15 min and increased again at 30 min, indicating a biphasic pattern. This result implies participation of diacylglycerol in the radiation-induced activation of PKC in hepatocytes. In order to clarify the mechanism of the initial process of radiation-induced diacylglycerol production, the effects of reactive oxygens were investigated. Treatment of cells with hydroxyl radical, a major oxygen radical produced by radiation, induced diacylglycerol production without any change in the content of phosphatidylcholine, showing a peak at 1 min after treatment. No change in the diacylglycerol content was observed at that time by hydrogen peroxide treatment. Furthermore, the diacylglycerol production by hydroxyl radical was inhibited by pretreatment with neomycin sulfate, a phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor. These results suggest that radiation exerts PI-PLC activation through hydroxyl radical generation, followed by diacylglycerol production and PKC activation.

Radiation-sensitive tyrosine phosphorylation of cellular proteins: sensitive to changes in GSH content induced by pretreatment with n’acetyl-L-cysteine or L-buthionine-S,R-sulfoximine

Purpose: At relatively high concentrations, i.e., > 20 mM, N-acetyl-L-cysteine (NAC) scavenges reactive oxygen species produced by ionizing radiation in aqueous solution. Therefore, the ability of NAC to block signal transduction reactions in vivo, has lead to the suggestion that ROS are necessary for the normal propagation of these signals. In this paper we investigate the mechanism by which NAC alters signal transduction in whole cells. Results: Exposing CHO-K1 cells to ionizing radiation results in elevated pp59 fyn kinase activity. Moreover, we observe changes in the phosphotyrosine content of multiple cellular proteins, including one prominent phosphotyrosyl protein with a M r of 85 kDa. Both the radiation-induced changes in pp59 fyn kinase activity and the changes in phosphotyrosine content of pp85 were not affected by exposing K1 cells to NAC during the time of irradiation, suggesting that ROS generated extracellularly are not involved in the radiation-induced changes observed in phosphotyrosyl proteins. We also demonstrate that the cell membrane is an effective barrier against negatively charged NAC. Therefore, it seems unlikely that NAC’s ability to block signal transduction reactions is related to scavenging of ROS intracellularly. Chronic exposure, i.e., 1 h, to 20 mM NAC lead to a twofold elevation in GSH levels and resulted in a 17% decrease in the phosphotyrosine content of pp85 after exposure to 10 Gy. Moreover, pretreatment with L-buthionine-S,R-sulfoximine (BSO) decreased GSH levels and resulted in elevated phosphotyrosine levels in pp85 isolated from irradiated CHO-K1 cells. Conclusions: Since many signaling molecules contain redox sensitive cysteine residues that regulate enzyme activity, we suggest that the effects of NAC on radiation-induced signal transduction are due to its ability to alter the intracellular reducing environment, and not related to direct scavenging of ROS.

Radiation-Induced Signal Transduction and Stress Response

Radiation-induced DNA damage can induce death by apoptosis by activation of signal transduction pathways. One such pathway is the sphingomyelin/ceramide signal transduction pathway that is involved in initiation of stress-induced apoptosis in a variety of normal and neoplastic cells. This pathway is under regulation by the protein kinase C (PKC) pathway that constitutes an anti-apoptosis mechanism. DNA damage can also increase ceramide levels by activating the biosynthesis pathway, through the activation of the ceramide synthase enzyme. Both pathways could serve as potential targets for strategies that take advantage of signaling-based apoptosis to enhance cell killing in radiation therapy.

Platelet-derived growth factor (PDGF)-signaling mediates radiation-induced apoptosis in human prostate cancer cells with loss of p53 function

Platelet-derived growth factor (PDGF) signals a diversity of cellular responses in vitro, including cell proliferation, survival, transformation, and chemotaxis. PDGF functions as a “competence factor” to induce a set of early response genes expressed in G 1 including p21 WAF1 CIP1 , a functional mediator of the tumor suppressor gene p53 in G 1 S checkpoint. For PDGF-stimulated cells to progress beyond G 1 and transit the cell cycle completely, progression factors in serum such as insulin and IGF-1 are required. We have recently shown a novel role of PDGF in inducing apoptosis in growth-arrested murine fibroblasts. The PDGF-induced apoptosis is rescued by insulin, suggesting that G 1 S checkpoint is a critical determinant for PDGF-induced apoptosis. Because recent studies suggest that radiation-induced signal transduction pathways interact with growth factor-mediated signaling pathways, we have investigated whether activation of the PDGF-signaling facilitates the radiation-induced apoptosis in the absence of functional p53. For this study we have used the 125-IL cell line, a mutant p53-containing, highly metastatic, and hormone-unresponsive human prostate carcinoma cell line. PDGF signaling is constitutively activated by transfection with a p28 v-sis expression vector, which was previously shown to activate PDGF α- and β- receptors. Although the basal level of p21 WAF1 CIP1 expression and radiation-induced apoptosis were not detectable in control 125-IL cells as would be predicted in mutant p53-containing cells, activation of PDGF-signaling induced expression of p21 WAF1 CIP1 and radiation-induced apoptosis. Our study suggests that the level of “competence” growth factors including PDGF may be one of the critical determinants for radiation-induced apoptosis, especially in cells with loss of p53 function at the site of radiotherapy in vivo.

Radiation-Induced Autophosphorylation of Epidermal Growth Factor Receptor in Human Malignant Mammary and Squamous Epithelial Cells

In an effort to identify events initiating up-regulation of epidermal growth factor receptor after single and repeated radiation exposures, we investigated the role of epidermal growth factor receptor, a receptor protein tyrosine kinase, in radiation-induced signal transduction. Human malignant mammary, MCF-7, and squamous, A431, cells showed low baseline phospho-tyrosine levels of epidermal growth factor receptor, permitting reproducible dose-dependent stimulation of epidermal growth factor receptor autophosphorylation after exposure to epidermal growth factor. MCF-7 cells exhibited a mean 2.3-fold increase (95% confidence interval: 1.91, 2.65; P < 0.0001) in levels of epidermal growth factor phosphorylation in response to exposures of 2 Gy, which was substantially less than the epidermal growth factor receptor Y phosphorylation induced by epidermal growth factor. A quantitatively similar radiation response was seen in A431 cells. In the dose range of 1 to 4 Gy, no clear dose response was seen. There was a rapid induction of radiation-induced epidermal growth factor receptor Y phosphorylation, starting within 2 min, with maximum values between 0.5 and 5 min after radiation exposure followed by a slower decline to baseline levels after 20 min. The data presented identify the epidermal growth factor receptor protein tyrosine kinase associated with the plasma membrane as one target for ionizing radiation in the dose range used in radiotherapy.