The influence of photodynamic therapy on apoptosis in human melanoma cell line

Melanoma is the most severe of all skin cancers as it may grow rapidly and metastasize. The application of photodynamic therapy (PDT) opens new perspectives in treatment of this cancer. Numerous studies suggest that the exposure of tumor cells to PDT can lead to cell death via two separate processes: apoptosis or necrosis. The aim of this study was to assess in vitro photodynamic therapy which induces apoptosis in the human Beidegr?m Melanoma (BM) cell line, using neutral comet assay. The cells were incubated with Photofrin II (15 microg/ml and 30 microg/ml) 4 h before and 3 h after irradiation for 5 or 10 min with the light intensity of 10 mW/cm2, using a lamp with red filter (632.8 nm). The percentage of apoptotic cells was significantly higher after PDT comparing to control cells. We observed 25% and 70% of apoptotic cells after shorter irradiation and treatment with 15 microg/ml and 30 microg/ml of Ph II, respectively. After longer irradiation, the respective values were 71.9% and 90%. The results suggest that induction of apoptosis is an important determinant of photodynamic sensitivity in the studied cell line and that some types of DNA damage are dependent on photosensitizer concentration and time of irradiation.     

ZnPcS2P2-based photodynamic therapy induces mitochondria-dependent apoptosis in K562 cells

Mitochondria play a key role in the regulation of apoptosis induced by numerous antitumor chemotherapeutic and other toxic agents. Photodynamic therapy (PDT) exerts significant cellular killing efficacy through either an apoptotic or necrotic cell death pathway. This study investigated the mechanism underlying the killing effects of a novel amphipathic photosensitizer [di-sulfonated di-phthalimidomethy lphthalocyanine zinc (ZnPcS2P2)]-mediated photodynamic therapy (ZnPcS2P2-PDT) on K562 cells. Apoptosis was evident in the post-PDT cells through the TdT-mediated dUTP nick end labeling (TUNEL) method and DNA fragmentation assay. After ZnPcS2P2-PDT, K562 cells underwent mitochondria-dependent apoptosis as evidenced by the release of cytochrome c from mitochondria into cytosol, accompanied by mitochondrial membrane potential (A~m) reduction, indicating the opening of the mitochondrial permeability transition pore (PTP). The activities of protease from the caspase family and caspase-3 were also significantly elevated. Furthermore, ZnPcS2P2-PDT down-regulated the expression of chimaeric Bcr-Abl oncoprotein, which is the molecular hallmark of chronic myelogenous leukemia (CML).     

Photodynamic therapy-induced death of HCT 116 cells: Apoptosis with or without Bax expression

Cell death following photodynamic therapy (PDT) with the photosensitizer Pc 4 involves the intrinsic pathway of apoptosis. To evaluate the importance of Bax in apoptosis after PDT, we compared the PDT responses of Bax-proficient (Bax^+/−) and Bax knock-out (BaxKO) HCT116 human colon cancer cells. PDT induced a slow apoptotic process in HCT Bax^+/− cells following a long delay in the activation of Bax and release of cytochrome c from mitochondria. Although cytochrome c was not released from mitochondria following PDT in BaxKO cells, an alternative mechanism of caspase-dependent apoptosis with extensive chromatin and DNA degradation was found in these cells. This alternative process was less efficient and slower than the normal apoptotic process observed in Bax^+/− cells. Early events upon PDT, such as the loss of mitochondrial membrane potential, photodamage to Bcl-2, and activation of p38 MAP kinase, were observed in both HCT116 cell lines. In spite of differences in the efficiency and mode of apoptosis induced by PDT in the Bax^+/− and BaxKO cells, they were found to be equally sensitive to killing by PDT, as determined by loss of clonogenicity. Thus, for Pc 4-PDT, the commitment to cell death occurs prior to and independent of Bax activation, but the process of cellular disassembly differs in Bax-expressing vs. non-expressing cells.     

Cell death via mitochondrial apoptotic pathway due to activation of Bax by lysosomal photodamage

Lysosomal photosensitizers have been used in photodynamic therapy. The combination of such photosensitizers and light causes lysosomal photodamage, inducing cell death. Lysosomal disruption can lead to apoptosis but its signaling pathways remain to be elucidated. In this study, N-aspartyl chlorin e6 (NPe6), an effective photosensitizer that preferentially accumulates in lysosomes, was used to study the mechanism of apoptosis caused by lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells (ASTC-a-1) after NPe6-photodynamic treatment (NPe6-PDT) was studied using real-time single-cell analysis. Our results demonstrated that NPe6-PDT induced rapid generation of reactive oxygen species (ROS). The photodynamically produced ROS caused a rapid destruction of lysosomes, leading to release of cathepsins, and the ROS scavengers vitamin C and NAC prevent the effects. Then the following spatiotemporal sequence of cellular events was observed during cell apoptosis: Bcl-2-associated X protein (Bax) activation, cytochrome c release, and caspase-9/-3 activation. Importantly, the activation of Bax proved to be a crucial event in this apoptotic machinery, because suppressing the endogenous Bax using siRNA could significantly inhibit cytochrome c release and caspase-9/-3 activation and protect the cell from death. In conclusion, this study demonstrates that PDT with lysosomal photosensitizer induces Bax activation and subsequently initiates the mitochondrial apoptotic pathway.     

Atg7 deficiency increases resistance of MCF-7 human breast cancer cells to photodynamic therapy

Photodynamic therapy (PDT) uses a photosensitizer, light, and oxygen to produce extensive oxidative damage to organelles housing the photosensitizer. Although PDT is an efficient trigger of apoptosis, it also induces autophagy in many kinds of cells. Autophagy can serve as both a cell survival and a cell death mechanism. Our previous study indicates that autophagy contributes to cell death after PDT, especially in apoptosis-deficient cells. Here, we provide further evidence to support the role of autophagy in cell killing after PDT. Autophagy was blocked by knockdown of one essential factor, LC3 or Atg7, in MCF-7 cells. The cells were exposed to a range of doses of PDT sensitized by the phthalocyanine Pc 4; steps in autophagy were monitored by western blotting for LC3-II and by fluorescence microscopy for the uptake of monodansylcadaverine or for the distribution of transfected GFP-LC3; and overall cell death was monitored by MTT assay and by clonogenic assay. We find that blocking autophagy increased the survival of MCF-7 cells after PDT and increased the shoulder on the dose-response curve. In response to Pc 4-PDT, Atg7-deficient MCF-7 cells remained capable of robust accumulation of LC3-II, but were defective in comparison to Atg7+ cells in the formation of autophagosomes. We conclude that apoptosis-deficient cells rely on autophagy for cell death after Pc 4-PDT and that the strong activation of LC3 maturation in response to PDT could occur even in cells with limited or no Atg7 expression.     

FADD null mouse embryonic fibroblasts undergo apoptosis after photosensitization with the silicon phthalocyanine Pc 4

Oxidative stress, such as photodynamic therapy with the silicon phthalocyanine Pc 4 (Pc 4-PDT), can induce apoptosis and tumor necrosis factor alpha (TNF) production. TNF receptors, as well as other death receptors, have been implicated in stress-induced apoptosis. To assess directly the role of FADD, a death receptor-associated protein, in induction of apoptosis post-Pc 4-PDT, embryonic fibroblasts from FADD knock out (k/o) and wild-type (wt) mice were used. Pc 4-PDT induced casp-3 activation and apoptosis in both cell types. In the presence of zVAD, a pancaspase inhibitor, Pc 4-PDT-induced apoptosis was abrogated in both cell lines. Fumonisin B1 (FB), an inhibitor of ceramide synthase, had no effect on apoptosis after Pc 4-PDT in either cell line. Similar to Pc 4-PDT, exogenous C6-ceramide bypassed FADD deficiency and induced zVAD-sensitive apoptosis. In contrast to Pc 4 photosensitization, TNF did not induce either apoptosis or ceramide accumulation in FADD k/o cells. In the absence of FADD deficiency, TNF-induced apoptosis was zVAD-sensitive and FB-insensitive. Induced ceramide levels remained elevated after cotreatment with TNF and zVAD in FADD wt cells. Taken together, these data provide genetic evidence for a lack of FADD requirement in Pc 4-PDT- or C6-ceramide-induced apoptosis. FB-sensitive ceramide production accompanies, but does not suffice, for apoptosis after Pc 4 photosensitization or TNF.     

Niemann-Pick human lymphoblasts are resistant to phthalocyanine 4-photodynamic therapy-induced apoptosis.

Stress-induced activation of sphingomyelinase (SMase) leading to generation of ceramide, a lipid mediator, has been associated with apoptosis in several malignant and nonmalignant cell lines. Photodynamic therapy (PDT), with the phthalocyanine photosensitizer Pc 4 [HOSiPcOSi(CH3)2(CH2)3N(CH3)2], is an oxidative stress associated with increased ceramide generation and subsequent induction of apoptosis in various cell types. We assessed the role of SMase in photocytotoxicity. Normal human lymphoblasts accumulated ceramide and underwent apoptosis after Pc 4-PDT. In contrast, Niemann-Pick disease (NPD) lymphoblasts, which are deficient in acid sphingomyelinase (ASMase) activity, failed to respond to Pc 4-PDT with ceramide accumulation and apoptosis, suggesting that ASMase may be a Pc 4-PDT target. NPD lymphoblasts were exposed to exogenous bacterial sphingomyelinase (bSMase) to test whether these defects are reversible. Treatment of NPD cells with bSMase itself led to elevated ceramide formation, which did not translate into induction of apoptosis. However, a combination of Pc 4-PDT + bSMase induced a significant apoptotic response. Thus, the combined treatment of Pc 4-PDT + bSMase, rather than bSMase alone, was required to restore apoptosis in NPD cells. These data support the hypothesis that SMase is a proapoptotic factor determining responsiveness of cells to Pc 4-PDT.     

A role for manganese superoxide dismutase in apoptosis after photosensitization

The oxidative stress triggered by photodynamic therapy (PDT) involves generation of cytotoxic reactive oxygen species, including superoxide radical, accumulation of de novo-generated ceramide, and induction of apoptosis. Since PDT with the photosensitizer phthalocyanine Pc 4 induces mitochondrial damage and the superoxide scavenger manganese superoxide dismutase (MnSOD) is localized to mitochondria, here we tested genetically the role of MnSOD in apoptosis and ceramide accumulation after photosensitization with Pc 4. Jurkat cells overexpressing wild-type MnSOD were protected from Pc 4-PDT-initiated apoptosis, but not from increased ceramide response to Pc 4-PDT. In Jurkat cells overexpressing mutant MnSOD, however, DEVDase activation and ceramide formation were promoted post-Pc 4-PDT. Similarly, in MnSOD-null cells, Pc 4-PDT-induced apoptosis, as well as ceramide accumulation, were enhanced compared to their normal counterparts. The data show that MnSOD affects sensitivity of cells to Pc 4-PDT-initiated apoptosis, and partly ceramide accumulation, suggesting that the processes are superoxide-mediated.     

A study on the photodynamic properties of chlorophyll derivatives using human hepatocellular carcinoma cells.

Photodynamic therapy (PDT) is an alternative anticancer treatment in which direct tumor-cell killing results from selective accumulation of photosensitizers in the tumor sites and phototoxicity occurs when light-activated photosensitizers transfer the energy to oxygen nearby to produce singlet oxygen. The objective of this study was to investigate the effects of PDT using chlorophyll derivatives such as pheophytin a (phe a), pheophytin b (phe b), pheophorbide a (pho a) and pheophorbide b (pho b) as the photosensitizers, and the 660 nm light-emitting diodes (LEDs) irradiation on human hepatocellular carcinoma cells (HuH-7). The drug concentration-dependent inhibition of HuH-7 cell viability was studied under LEDs irradiation (10 mW cm(-2)) at radiant exposure of 5.1 and 10.2 J cm(-2) by MTT assay. Significant inhibition of the survival of HuH-7 cells (<10%) was observed when an irradiation dose of 10.2 J cm(-2) combined with the concentration of 0.5 microg ml(-1) of phe a, 0.125 microg ml(-1) of pho a, 0.25 microg ml(-1) of phe b, and 0.125 microg ml(-1) of pho b were applied. The results from Annexin V-propidium iodide staining revealed that phe a, phe b, pho a and pho b could induce cell death in HuH-7 cells predominantly via a necrotic process. The results from immunoblot analyses exhibited that chlorophyll derivative-mediated PDT initiated cytochrome c release, caspase-9 and caspase-3 activation, followed by poly ADP-ribose polymerase (PARP) cleavage. Thus, apoptosis also occurred in HuH-7 cells after PDT treatment, and the execution of the apoptotic process may be initiated from the loss of mitochondrial function. Our findings demonstrate that both apoptosis and necrosis can be induced in HuH-7 cells after PDT using phe a, phe b, pho a and pho b and LEDs.     

Caspase-resistant vimentin suppresses apoptosis after photodynamic treatment with a silicon phthalocyanine in Jurkat cells

Oxidative stress, such as photodynamic therapy, is an apoptosis inducer. Apoptosis, as well as photosensitization, have been associated with disruption of the cytoskeletal network. The purpose of the present study was to assess the role of vimentin, a major cytoskeletal protein, in apoptosis after photodynamic treatment (PDT) with the silicon phthalocyanine Pc 4 in human Jurkat T cells. Here we show for the first time that photosensitization with Pc 4 initiates vimentin cleavage and that this event precedes poly(ADP-ribose) polymerase (PARP) degradation. Similar findings were obtained in the presence of C2-ceramide, an inducer of oxidative stress and apoptosis. In the presence of benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone, a pan-caspase inhibitor, Pc 4-PDT-induced vimentin and PARP cleavage were abolished. In Jurkat cells transfected with a caspase-resistant vimentin apoptosis was partly suppressed and delayed post-Pc 4-PDT. We suggest that the full-length vimentin confers resistance to nuclear apoptosis after PDT with Pc 4.     

藻红蛋白亚基光敏剂对小鼠移植瘤作用的超微结构研究

目的：从形态学角度探讨藻红蛋白(R-PE)β亚基光动力学抗肿瘤效果及其作用机理。方法：用不同密度的波长为496nm的氩离子激光对S180小鼠移植瘤进行β亚基光动力学治疗,并对治疗后的瘤体进行透射电镜的形态学观察。结果：用100μg／m1的β亚基,在200J/cm2激光照射剂量条件下治愈了瘤体直径为0．5cm-0．7cm大小的小鼠移植瘤,发现瘤组织中引起细胞死亡的途经有差异,被PDT抑制的肿瘤内部细胞表现出典型的凋亡细胞特征。结论：R-PE β亚基具较强的光动力学抗肿瘤效果,光动力治疗机理可能涉及肿瘤内部细胞死亡主要是凋亡途径而瘤周为坏死,且与血管系统破坏及白细胞参与的抗炎症反应相关。     

ZnPcS2P2在K562和HL-60细胞中的定位研究

目的:探讨ZnPcS2P2在K562细胞,HL-60细胞亚细胞结构中的精确定位,揭示光动力学疗法(photody-namic therapy,PDT)的作用机制。方法:将K562细胞,HL-60细胞与ZnPcS2P2共同孵育5 h。应用激光扫描共聚焦显微成像系统,选择特异性细胞器荧光探针(线粒体探针若丹明Rodanmine123、溶酶体探针LysoTrackerDND-26、内质网探针Dioc6(3)采用波形比较法对光敏剂进行亚细胞定位。结果:ZnPcS2P2在K562细胞,HL-60细胞中发出的荧光与负载的Rodanmine123、Lyso-TracKer DND-26、Dioc6(3)均有部分重叠,波形均有相似之处。ZnPc-S2P2在线粒体、溶酶体、内质网均有分布。结论:线粒体是ZnPcS2P2介导的PDT(ZnPcS2P2-PDT)光损伤的主要靶点,溶酶体、内质网也是ZnPcS2P2-PDT光损伤的靶点。     

Initiation of apoptosis and autophagy by photodynamic therapy

This study was designed to examine modes of cell death after photodynamic therapy (PDT). Murine leukemia L1210 cells and human prostate Bax-deficient DU-145 cells were examined after PDT-induced photodamage to the endoplasmic reticulum (ER). Previous studies indicated that this treatment resulted in a substantial loss of Bcl-2 function. Both apoptosis and autophagy occurred in L1210 cells after ER photodamage with the latter predominating after 24 hr. These processes were characterized by altered cellular morphology, chromatin condensation, loss of mitochondrial membrane potential and formation of vacuoles containing cytosolic components. Western blots demonstrated processing of LC3-I to LC3-II, a marker for autophagy. In DU145 cells, PDT initiated only autophagy. Phosphatidylinositol (PI) 3-kinase inhibitors suppressed autophagy in both cell lines as indicated by inhibition of vacuolization and LC3 processing. Inhibitors of apoptosis and/or autophagy were then used to delineate the contributions of the two pathways to the effects of PDT. Given the ability of autophagy to upregulate MHC-11 peptide presentation, autophagy may play a role in the ability of photodynamic therapy to stimulate immunologic recognition of target cells.     

Hypericin‐PDT‐induced rapid necrotic death in human squamous cell carcinoma cultures after multiple treatment

PDT (photodynamic therapy) has been used for the treatment of NMCC (non‐melanoma cutaneous cancer) particularly, human SCC (squamous cell carcinoma). However, the nature of the photosensitizer, the activation light source and the mode of cell death induced post‐PDT remains elusive. We tried to optimize PDT using the light‐activated (320–400 nm) St John's Wort‐derived compound, Hyp (hypericin). The work highlights the potential mode of cell death and the increased efficacy of the technique associated with multiple Hyp‐PDT treatment. SCC cells were exposed to different concentrations of Hyp and activated with light at 1 J/cm² for 1 or 2 days. Thereafter with the optimum dose of Hyp proliferation, ROS (reactive oxygen species), and apoptosis were analysed by XTT [2,3‐bis‐(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide] assay, FACS analysis and Fluorescent/Phase contrast microscopy was carried out for morphological studies. Hyp‐PDT produces more ROS after 1 day compared with 2 days and the mode of cell death is a necrotic caspase‐independent mechanism. We propose a novel ‘double‐hit/2‐day’ strategy to reduce the viability in SCC using Hyp‐based PDT as an adjunctive treatment modality.     

Photofrin--factor of photodynamic therapy induces apoptosis and necrosis

Photodynamic therapy (PDT) causes irreversible photodamage of tumor and other malignant tissues. The effect of reactive oxygen species generation in the presence of photofrin (HpD) was studied. The studies were performed on endothelial cell line from foetal aorta of calves and on normal fibroblasts cell line (3T3 -Balb) and also on malignant line (A431). The cells were grown in presence of photofrin at different time intervals. Time of interaction of photosensitiser with cells was very important. Short time of exposure of the cells to photofrin induced mostly apoptosis in normal cells and apoptotic or necrotic changes in malignant cells. Longer effect of these factors on cells provoked necrosis. The factors of PDT influence dynamic changes of SOD and CT activity. It was dependent on the intensity of factors. These results strongly suggest that HpD has an effect on generation of ROS, which are a signal for development of morphological changes (apoptosis or necrosis) in normal and malignant cells.     

Apoptotic and autophagic responses to Bcl-2 inhibition and photodamage.

Among the cellular responses to photodamage initiated by photodynamic therapy (PDT) are autophagy and apoptosis. While autophagy is a reversible process that can be both a survival and a death pathway, apoptosis is irreversible, leading only to cell death. In this study, we followed the fate of mouse leukemia L1210 cells after photodamage to the endoplasmic reticulum (ER) using a porphycene photosensitizer, where Bcl-2 was among the PDT targets. In wild-type cells, we observed a rapid wave of autophagy, presumed to represent the recycling of some damaged organelles, followed by apoptosis. Using shRNA technology, we created a Bax knockdown line (L1210/Bax(-)). In the latter cell line, we found a marked decrease in apoptosis after photodamage or pharmacologic inactivation of Bcl-2 function, but this did not affect PDT efficacy. Loss of viability was associated with a highly-vacuolated morphology consistent with autophagic cell death. Previous studies indicated pro-survival attributes of autophagy after low-dose PDT, suggesting that autophagy may be responsible for the 'shoulder' on the dose-response curve. It appears that attempts at extensive recycling of damaged organelles are associated with cell death, and that this phenomenon is amplified when apoptosis is suppressed.     

Effects of antrodia camphorata on viability, apoptosis, and [Ca2+]i in PC3 human prostate cancer cells

Antrodia camphorata (AC) has been used as a health supplement in Asia to control different cancers; however, the cellular mechanisms of its effects are unclear. The effect of AC on cultured human prostate cancer cells (PC3) has not been explored. This study examined the effect of AC on viability, apoptosis, mitogen-activated protein kinases (MAPKs) phosphorylation and Ca2+ handling in PC3 cells. AC at concentrations of 5-50 microg/ml did not affect cell viability, but at 100-200 microg/ml decreased viability and induced apoptosis in a concentration-dependent manner. AC at concentrations of 25-200 microg/ml did not alter basal [Ca2+]i, but at a concentration of 25 microg/ml decreased the [Ca2+]i increases induced by ATP, bradykinin, histamine and thapsigargin. ATP, bradykinin and histamine increased cell viability whereas thapsigargin decreased it. AC (25 microg/ml) pretreatment inhibited ATP-, bradykinin-, and histamine-induced enhancement on viability, but reversed thapsigargin-induced cytotoxicity. Immunoblotting showed that AC (200 microg/ml) did not induce the phosphorylation of ERK, JNK, and p38 MAPKs. Collectively, in PC3 cells, AC exerted multiple effects on viability and [Ca2+]i, caused apoptosis via pathways unrelated to [Ca2+]i signal and phosphorylation of ERK, JNK and p38 MAPKs.     

Design, synthesis, and in vitro photodynamic activities of benzochloroporphyrin derivatives as tumor photosensitizers

Novel benzochloroporphyrin derivatives (BCPDs) were designed, synthesized, and characterized. In vitro dark cytotoxicity and photodynamic efficacy of BCPDs were evaluated by MTT assay on human hepatoma BEL-7402 cells. The experimental results showed that BCPDs 15, 16, 17, and 18 have strong long wavelength absorptions around 670 nm and exhibit significantly lower dark cytotoxicity than BPDMA and possess potent photocytotoxicity, IC50 values 1.32 microg/mL for 15, 0.26 microg/mL for 16, 0.47 microg/mL for 17 of 0.27 microg/mL for 18, and 0.23 microg/mL for BPDMA. Among them, BCPDs 16 and 18 are more effective and promising PDT photosensitizers based on the studies with BEL-7402 cells and show nearly the same photodynamic efficacy as BPDMA. MG-P staining qualitative analysis also indicated that PDT with BCPDs 16 can induce apoptosis in BEL7402 cells.     

Low-dose radiation hypersensitivity in human tumor cell lines: effects of cell-cell contact and nutritional deprivation

The hyper-radiosensitivity at low doses recently observed in vitro in a number of cell lines is thought to have important implications for improving tumor radiotherapy. However, cell-cell contact and the cellular environment influence cellular radiosensitivity at higher doses, and they may alter hyper-radiosensitivity in vivo. To confirm this supposition, we investigated the effects of cell density, multiplicity and nutritional deprivation on low-dose hypersensitivity in vitro. Cell survival in the low-dose range (3 cGy to 2 Gy) was studied in cells of two human glioma (BMG-1 and U-87) and two human oral squamous carcinoma (PECA-4451 and PECA-4197) lines using a conventional macrocolony assay. The effects of cell density, multiplicity and nutritional deprivation on hyper-radiosensitivity/induced radioresistance were studied in cells of the BMG-1 cell line, which showed prominent hypersensitivity and induced radioresistance. The induction of growth inhibition, cell cycle delay, micronuclei and apoptosis was also studied at the hyper-radiosensitivity-inducing low doses. Hyper-radiosensitivity/induced radioresistance was evident in the cells of all four cell lines to varying extents, with maximum sensitivity at 10-30 cGy, followed by an increase in survival up to 50 cGy-1 Gy. Both the glioma cell lines had more prominent hyper-radiosensitivity than the two squamous carcinoma cell lines. Low doses inducing maximum hyper-radiosensitivity did not cause significant growth inhibition, micronucleation or apoptosis in BMG-1 cells, but a transient G(1)/S-phase block was evident. Irradiating and incubating BMG-1 cells at high density for 0 or 4 h before plating, as well as irradiating cells as microcolonies, reduced hyper-radiosensitivity significantly, indicating the role of cell-cell contact-mediated processes. Liquid holding of BMG-1 cells in HBSS + 1% serum during and after irradiation for 4 h significantly reduced hyper-radiosensitivity, suggesting that hyper-radiosensitivity may be due partly to active damage fixation processes at low doses. Therefore, our findings suggest that the damage-induced signaling mechanisms influenced by (or mediated through) cell-cell contact or the cellular environment, as well as the lesion fixation processes, play an important role in hyper-radiosensitivity. Further studies are required to determine the exact nature of the damage that triggers these responses as well as for evaluating the potential of low-dose therapy.