Oncogenic KRAS drives radioresistance through upregulation of NRF2-53BP1-mediated non-homologous end-joining repair.

KRAS-activating mutations are oncogenic drivers and are correlated with radioresistance of multiple cancers, including colorectal cancer, but the underlying precise molecular mechanisms remain elusive. Herein we model the radiosensitivity of isogenic HCT116 and SW48 colorectal cancer cell lines bearing wild-type or various mutant KRAS isoforms. We demonstrate that KRAS mutations indeed lead to radioresistance accompanied by reduced radiotherapy-induced mitotic catastrophe and an accelerated release from G2/M arrest. Moreover, KRAS mutations result in increased DNA damage response and upregulation of 53BP1 with associated increased non-homologous end-joining (NHEJ) repair. Remarkably, KRAS mutations lead to activation of NRF2 antioxidant signaling to increase 53BP1 gene transcription. Furthermore, genetic silencing or pharmacological inhibition of KRAS, NRF2 or 53BP1 attenuates KRAS mutation-induced radioresistance, especially in G1 phase cells. These findings reveal an important role for a KRAS-induced NRF2-53BP1 axis in the DNA repair and survival of KRAS-mutant tumor cells after radiotherapy, and indicate that targeting NRF2, 53BP1 or NHEJ may represent novel strategies to selectively abrogate KRAS mutation-mediated radioresistance.

Bach2 Deficiency Promotes Intestinal Epithelial Regeneration by Accelerating DNA Repair in Intestinal Stem Cells.

Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury, although the repair mechanisms are unclear. Here, we found that Bach2 deficiency promotes intestinal epithelial cell proliferation during homeostasis. Moreover, genetic inactivation of Bach2 in mouse intestinal epithelium facilitated crypt regeneration after irradiation, resulting in a reduction in mortality. RNA-sequencing analysis of isolated crypts revealed that Bach2 deficiency altered the expression of numerous genes, including those regulating double-strand break repair. Mechanistic characterizations indicated that Bach2 deletion facilitated DNA repair in intestinal crypt cells, as evidenced by faster resolution of γ-H2AX and 53BP1 foci in Bach2-/- crypt cells, compared with Bach2+/+ control. Together, our studies highlight that Bach2 deficiency promotes intestinal regeneration by accelerating DNA repair in intestinal stem cells after radiation damage.

Shift in G1-Checkpoint from ATM-Alone to a Cooperative ATM Plus ATR Regulation with Increasing Dose of Radiation.

The current view of the involvement of PI3-kinases in checkpoint responses after DNA damage is that ATM is the key regulator of G1-, S- or G2-phase checkpoints, that ATR is only partly involved in the regulation of S- and G2-phase checkpoints and that DNA-PKcs is not involved in checkpoint regulation. However, further analysis of the contributions of these kinases to checkpoint responses in cells exposed to ionizing radiation (IR) recently uncovered striking integrations and interplays among ATM, ATR and DNA-PKcs that adapt not only to the phase of the cell cycle in which cells are irradiated, but also to the load of DNA double-strand breaks (DSBs), presumably to optimize their processing. Specifically, we found that low IR doses in G2-phase cells activate a G2-checkpoint that is regulated by epistatically coupled ATM and ATR. Thus, inhibition of either kinase suppresses almost fully its activation. At high IR doses, the epistatic ATM/ATR coupling relaxes, yielding to a cooperative regulation. Thus, single-kinase inhibition suppresses partly, and only combined inhibition suppresses fully G2-checkpoint activation. Interestingly, DNA-PKcs integrates with ATM/ATR in G2-checkpoint control, but functions in its recovery in a dose-independent manner. Strikingly, irradiation during S-phase activates, independently of dose, an exclusively ATR-dependent G2 checkpoint. Here, ATM couples with DNA-PKcs to regulate checkpoint recovery. In the present work, we extend these studies and investigate organization and functions of these PI3-kinases in the activation of the G1 checkpoint in cells irradiated either in the G0 or G1 phase. We report that ATM is the sole regulator of the G1 checkpoint after exposure to low IR doses. At high IR doses, ATM remains dominant, but contributions from ATR also become detectable and are associated with limited ATM/ATR-dependent end resection at DSBs. Under these conditions, only combined ATM + ATR inhibition fully abrogates checkpoint and resection. Contributions of DNA-PKcs and CHK2 to the regulation of the G1 checkpoint are not obvious in these experiments and may be masked by the endpoint employed for checkpoint analysis and perturbations in normal progression through the cell cycle of cells exposed to DNA-PKcs inhibitors. The results broaden our understanding of organization throughout the cell cycle and adaptation with increasing IR dose of the ATM/ATR/DNA-PKcs module to regulate checkpoint responses. They emphasize notable similarities and distinct differences between G1-, G2- and S-phase checkpoint regulation that may guide DSB processing decisions.

Continuous Exposure to 1.7 GHz LTE Electromagnetic Fields Increases Intracellular Reactive Oxygen Species to Decrease Human Cell Proliferation and Induce Senescence.

Due to the rapid development of mobile phone technology, we are continuously exposed to 1.7 GHz LTE radio frequency electromagnetic fields (RF-EMFs), but their biological effects have not been clarified. Here, we investigated the non-thermal cellular effects of these RF-EMFs on human cells, including human adipose tissue-derived stem cells (ASCs), Huh7 and Hep3B liver cancer stem cells (CSCs), HeLa and SH-SY5Y cancer cells, and normal fibroblast IMR-90 cells. When continuously exposed to 1.7 GHz LTE RF-EMF for 72 h at 1 and 2 SAR, cell proliferation was consistently decreased in all the human cells. The anti-proliferative effect was higher at 2 SAR than 1 SAR and was less severe in ASCs. The exposure to RF-EMF for 72 h at 1 and 2 SAR did not induce DNA double strand breaks or apoptotic cell death, but did trigger a slight delay in the G1 to S cell cycle transition. Cell senescence was also clearly observed in ASC and Huh7 cells exposed to RF-EMF at 2 SAR for 72 h. Intracellular ROS increased in these cells and the treatment with an ROS scavenger recapitulated the anti-proliferative effect of RF-EMF. These observations strongly suggest that 1.7 GHz LTE RF-EMF decrease proliferation and increase senescence by increasing intracellular ROS in human cells.

Astragaloside IV ameliorates radiation-induced senescence via antioxidative mechanism.

OBJECTIVES: Ageing is a universal and gradual process of organ deterioration. Radiation induces oxidative stress in cells, which leads to genetic damage and affects cell growth, differentiation and senescence. Astragaloside (AS)-IV has antioxidative, anti-apoptotic and anti-inflammatory properties. METHODS: To study the protective mechanism of AS-IV on radiation-induced brain cell senescence, we constructed a radiation-induced brain cell ageing model, using biochemical indicators, senescence-associated galactosidase (SA-ß-gal) senescence staining, flow cytometry and Western blotting to analyse the AS-IV resistance mechanism to radiation-induced brain cell senescence. KEY FINDINGS: Radiation reduced superoxide dismutase (SOD) activity and expressions of cyclin-dependent kinase (CDK2), CDK4, cyclin E and transcription factor E2F1 proteins, and increased expressions of p21, p16, cyclin D and retinoblastoma (RB) proteins, malondialdehyde (MDA) activity, SA-ß-gal-positive cells and cells stagnating in G1 phase. After treatment with AS-IV, the level of oxidative stress in cells significantly decreased and expression of proteins related to the cell cycle and ageing significantly changed. In addition, SA-ß-gal-positive cells and cells arrested in G1 phase were significantly reduced. CONCLUSIONS: These data suggest that AS-IV can antagonize radiation-induced brain cells senescence; and its mechanism may be related to p53-p21 and p16-RB signalling pathways of ageing regulation.

Involvement of TGF-ß and ROS in G1 Cell Cycle Arrest Induced by Titanium Dioxide Nanoparticles Under UVA Irradiation in a 3D Spheroid Model.

BACKGROUND: As one of the most widely produced engineered nanomaterials, titanium dioxide nanoparticles (nano-TiO2) are used in biomedicine and healthcare products, and as implant scaffolds; therefore, the toxic mechanism of nano-TiO2 has been extensively investigated with a view to guiding application. Three-dimensional (3D) spheroid models can simplify the complex physiological environment and mimic the in vivo architecture of tissues, which is optimal for the assessment of nano-TiO2 toxicity under ultraviolet A (UVA) irradiation. METHODS AND RESULTS: In the present study, the toxicity of nano-TiO2 under UVA irradiation was investigated in 3D H22 spheroids cultured in fibrin gels. A significant reduction of approximately 25% in spheroid diameter was observed following treatment with 100 µg/mL nano-TiO2 under UVA irradiation after seven days of culture. Nano-TiO2 under UVA irradiation triggered the initiation of the TGF-ß/Smad signaling pathway, increasing the expression levels of TGF-ß1, Smad3, Cdkn1a, and Cdkn2b at both the mRNA and protein level, which resulted in cell cycle arrest in the G1 phase. In addition, nano-TiO2 under UVA irradiation also triggered the production of reactive oxygen species (ROS), which were shown to be involved in cell cycle regulation and the induction of TGF-ß1 expression. CONCLUSION: Nano-TiO2 under UVA irradiation induced cell cycle arrest in the G1 phase and the formation of smaller spheroids, which were associated with TGF-ß/Smad signaling pathway activation and ROS generation. These results reveal the toxic mechanism of nano-TiO2 under UVA irradiation, providing the possibility for 3D spheroid models to be used in nanotoxicology studies.

Genetic aberrations in iPSCs are introduced by a transient G1/S cell cycle checkpoint deficiency.

A number of point mutations have been identified in reprogrammed pluripotent stem cells such as iPSCs and ntESCs. The molecular basis for these mutations has remained elusive however, which is a considerable impediment to their potential medical application. Here we report a specific stage at which iPSC generation is not reduced in response to ionizing radiation, i.e. radio-resistance. Quite intriguingly, a G1/S cell cycle checkpoint deficiency occurs in a transient fashion at the initial stage of the genome reprogramming process. These cancer-like phenomena, i.e. a cell cycle checkpoint deficiency resulting in the accumulation of point mutations, suggest a common developmental pathway between iPSC generation and tumorigenesis. This notion is supported by the identification of specific cancer mutational signatures in these cells. We describe efficient generation of human integration-free iPSCs using erythroblast cells, which have only a small number of point mutations and INDELs, none of which are in coding regions.

Unlike reactivity of mono- and binuclear imine-copper(II) complexes toward melanoma cells via a tyrosinase-dependent mechanism.

The cytotoxicity of a dinuclear imine-copper (II) complex 2, and its analogous mononuclear complex 1, toward different melanoma cells, particularly human SKMEL-05 and SKMEL-147, was investigated. Complex 2, a tyrosinase mimic, showed much higher activity in comparison to complex 1, and its reactivity was verified to be remarkably activated by UVB-light, while the mononuclear compound showed a small or negligible effect. Further, a significant dependence on the melanin content in the tumor cells, both from intrinsic pigmentation or stimulated by irradiation, was observed in the case of complex 2. Similar tests with keratinocytes and melanocytes indicated a much lower sensitivity to both copper (II) complexes, even after exposition to UV light. Clonogenic assays attested that the fractions of melanoma cells survival were much lower under treatment with complex 2 compared to complex 1, both with or without previous irradiation of the cells. The process also involves generation of reactive oxygen species (ROS), as verified by EPR spectroscopy, and by using fluorescence indicators. Autophagic assays indicated a remarkable formation of cytoplasmic vacuoles in melanomas treated with complex 2, while this effect was not observed in similar treatment with complex 1. Monitoring of specific protein LC3 corroborated the simultaneous occurrence of autophagy. A balance interplay between different modes of cell death, apoptosis and autophagy, occurs when melanomas were treated with the dinuclear complex 2, in contrast to the mononuclear complex 1. These results pointed out to different mechanisms of action of such complexes, depending on its nuclearity.

Synergistic effect of phototherapy and chemotherapy on bladder cancer cells.

Drug resistance as an important barrier to cancer treatment, has a close relation with alteration of cancer metabolism. Therefore, in this study the synergistic effect of phototherapy and chemotherapy were investigated on the bladder cancer cells viability. The cytotoxicity effect of blue light irradiation was measured by the MTT assay. Glucose consumption, lactate and ammonium formation were analyzed in the blue LED-irradiated cancer cells culture. Also, the expression of some genes involved in apoptosis and epithelial-mesenchymal transition was assessed using real-time PCR in comparison with the control group. The analysis of the results indicated that blue light irradiation inhibited the cell viability in a dose-dependent manner. Blue light irradiation decreased the cell viability by 7% and 19% (p < .05) in 5637 cells at doses of 8.7 J/cm2 and 17.5 J/cm2 in comparison with the control group respectively. Glucose consumption, lactate and ammonium formation diminished in the blue LED-irradiated 5637 cells in both doses. The real time PCR results indicated that the expression of Bax increased in blue light-irradiated cells. In addition, the cell cycle analysis showed that blue light irradiation arrested the bladder cancer in the G1 phase. Also, the effect of combination therapy on cancer cells was investigated in presence of blue light irradiation and cisplatin. The obtained results of the MTT assay indicated that blue light irradiation enhance the cytotoxicity effect of cisplatin on bladder cancer cells.

Radiosensitization of esophageal carcinoma cells by knockdown of HMGB1 expression.

Radiotherapy (RT) is a traditional and important treatment for carcinoma of the esophagus along with surgery and chemotherapy. High mobility group box 1 (HMGB1) plays a crucial part in inhibiting the apoptosis of cancer cells after irradiation treatment. The present study, was designed to analyze the function of HMGB1 in esophageal cancer progression and elucidate the effects of HMGB1 on the radiosensitivity of human esophageal cancer cell lines. In the present study, an immunohistochemical evaluation of HMGB1 was performed on 77 biopsies, and the results revealed that HMGB1 overexpression was positively correlated with gross tumor volume (GTV), tumor­node­metastasis (TNM) stage, T classification, distant metastasis, and relapse and negatively correlated with patient survival rates, suggesting that HMGB1 acts as a key factor in the development of esophageal cancer. An shRNA targeting HMGB1 was designed for the knockdown of HMGB1 in ECA109 and TE13 cells, and the transfection efficiency of the shRNA was assessed using quantitative real­time reverse transcription polymerase chain reaction and western blot analysis. CCK­8 and clonogenic assays were used to analyze the effect of HMGB1 on the proliferation and radiosensitivity, respectively, of esophageal cancer cells in vitro. The influence of HMGB1 on radiation­induced changes in the migration, invasion, and cell cycle as well as apoptosis of tumor cells was examined by wound­healing and Transwell assays and flow cytometry, respectively. In addition, xenograft tumor models were constructed to observe the effect of HMGB1 on tumor growth in vivo. The results of the study in vitro revealed that the proliferation of the HMGB1­shRNA group decreased after irradiation, and the radiation treatment reduced the tumor volume of the xenograft model which was more marked in HMGB1­shRNA group. Moreover, HMGB1 was involved in the phosphorylation of H2AX after irradiation, and HMGB1 knockdown blocked the cell cycle in the G0/G1 phase and increased apoptosis. HMGB1 deficiency was also correlated with the upregulation of p16, Bax and caspase­9 and the downregulation of MMP­2, MMP­9, cyclin D1, CDK4, γH2AX and Bcl­2. These data indicated that the overexpression of HMGB1 prior to treatment was correlated with poor clinical outcome in esophageal carcinoma and that knockdown HMGB1 expression in human esophageal cancer cell lines increased their radiosensitivity by allowing the induction of apoptosis and G0/G1 arrest after exposure to radiation.

Radiation Sensitization of Basal Cell and Head and Neck Squamous Cell Carcinoma by the Hedgehog Pathway Inhibitor Vismodegib.

Vismodegib, an inhibitor of the Hedgehog signaling pathway, is an approved drug for monotherapy in locally advanced or metastatic basal cell carcinoma (BCC). Data on combined modality treatment by vismodegib and radiation therapy, however, are rare. In the present study, we examined the radiation sensitizing effects of vismodegib by analyzing viability, cell cycle distribution, cell death, DNA damage repair and clonogenic survival in three-dimensional cultures of a BCC and a head and neck squamous cell carcinoma (HNSCC) cell line. We found that vismodegib decreases expression of the Hedgehog target genes glioma-associated oncogene homologue (GLI1) and the inhibitor of apoptosis protein (IAP) Survivin in a cell line- and irradiation-dependent manner, most pronounced in squamous cell carcinoma (SCC) cells. Furthermore, vismodegib significantly reduced proliferation in both cell lines, while additional irradiation only slightly further impacted on viability. Analyses of cell cycle distribution and cell death induction indicated a G1 arrest in BCC and a G2 arrest in HNSCC cells and an increased fraction of cells in SubG1 phase following combined treatment. Moreover, a significant rise in the number of phosphorylated histone-2AX/p53-binding protein 1 (γH2AX/53BP1) foci in vismodegib- and radiation-treated cells was associated with a significant radiosensitization of both cell lines. In summary, these findings indicate that inhibition of the Hedgehog signaling pathway may increase cellular radiation response in BCC and HNSCC cells.

BRAF Inhibitors and Radiation Do Not Act Synergistically to Inhibit WT and V600E BRAF Human Melanoma.

BACKGROUND/AIM: Recent evidence suggests that melanoma patients treated with BRAF inhibitors experience radiosensitization with an increased frequency of side-effects. This could also imply increased effectiveness when treating melanoma. MATERIALS AND METHODS: To test whether the BRAF inhibitors dabrafenib and vemurafenib together with ionizing radiation more effectively inhibit melanoma cells, primary human melanoma tumor cell lines expressing wild-type (WT) or mutant V600E BRAF were analyzed by cell survival, cell death, and cell-cycle testing. RESULTS: All melanoma cell lines examined were radioresistant in these assays. BRAF inhibitor treatment alone suppressed cell survival more effectively than radiation in all the mutant V600E BRAF cell lines, and vemurafenib, but not dabrafenib, also inhibited cell survival in the WT BRAF cell lines at clinically relevant concentrations. However, when cells were treated with BRAF inhibitor followed by radiation, there was no increased effect on the suppression of cell survival. Vemurafenib induced more necrosis than radiation in most melanoma cell lines, irrespective of BRAF status, but this effect was not additive with the combination treatment. BRAF inhibitors and radiation had variable, but independent effects on the induction of cell-cycle arrest. CONCLUSION: These results suggest that BRAF inhibitors and ionizing radiation do not act synergistically to inhibit the growth of primary human melanoma cells.

Recruitment kinetics of the homologous recombination pathway in procyclic forms of Trypanosoma brucei after ionizing radiation treatment.

One of the most important mechanisms for repairing double-strand breaks (DSBs) in model eukaryotes is homologous recombination (HR). Although the genes involved in HR have been found in Trypanosoma brucei and studies have identified some of the proteins that participate in this HR pathway, the recruitment kinetics of the HR machinery onto DNA during DSB repair have not been clearly elucidated in this organism. Using immunofluorescence, protein DNA-bound assays, and DNA content analysis, we established the recruitment kinetics of the HR pathway in response to the DSBs generated by ionizing radiation (IR) in procyclic forms of T. brucei. These kinetics involved the phosphorylation of histone H2A and the sequential recruitment of the essential HR players Exo1, RPA, and Rad51. The process of DSB repair took approximately 5.5 hours. We found that DSBs led to a decline in the G2/M phase after IR treatment, concomitant with cell cycle arrest in the G1/S phase. This finding suggests that HR repairs DSBs faster than the other possible DSB repair processes that act during the G1/S transition. Taken together, these data suggest that the interplay between DNA damage detection and HR machinery recruitment is finely coordinated, allowing these parasites to repair DNA rapidly after DSBs during the late S/G2 proficient phases.

Translational control of a human CDKN1A mRNA splice variant regulates the fate of UVB-irradiated human keratinocytes.

In response to sublethal ultraviolet B (UVB) irradiation, human keratinocytes transiently block progression of the cell cycle to allow ample time for DNA repair and cell fate determination. These cellular activities are important for avoiding the initiation of carcinogenesis in skin. Central to these processes is the repression of initiation of mRNA translation through GCN2 phosphorylation of eIF2α (eIF2α-P). Concurrent with reduced global protein synthesis, eIF2α-P and the accompanying integrated stress response (ISR) selectively enhance translation of mRNAs involved in stress adaptation. In this study, we elucidated a mechanism for eIF2α-P cytoprotection in response to UVB in human keratinocytes. Loss of eIF2α-P induced by UVB diminished G1 arrest, DNA repair, and cellular senescence coincident with enhanced cell death in human keratinocytes. Genome-wide analysis of translation revealed that the mechanism for these critical adaptive responses by eIF2α-P involved induced expression of CDKN1A encoding the p21 (CIP1/WAF1) protein. We further show that human CDKN1A mRNA splice variant 4 is preferentially translated following stress-induced eIF2α-P by a mechanism mediated in part by upstream ORFs situated in the 5'-leader of CDKN1A mRNA. We conclude that eIF2α-P is cytoprotective in response to UVB by a mechanism featuring translation of a specific splice variant of CDKN1A that facilitates G1 arrest and subsequent DNA repair.

Novel focal adhesion kinase 1 inhibitor sensitizes lung cancer cells to radiation in a p53-independent manner.

Focal adhesion kinase 1 (FAK1) is known to promote tumor progression and metastasis by controlling cell movement, invasion, survival and the epithelial-to-mesenchymal transition in the tumor microenvironment. As recent reports imply that FAK1 is highly associated with tumor cell development and malignancy, the inhibition of FAK1 activity could be an effective therapeutic approach for inhibiting the growth and metastasis of tumor cells. In this study, we aimed to determine the effect of a novel synthetic FAK1 inhibitor 2-[2-(2-methoxy-4-morpholin-4-yl-phenylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-N-methyl-benzamide, (MPAP) on lung cancer cells. MPAP suppressed cancer cell proliferation and the phosphorylation of FAK1. Combined treatment with MPAP and irradiation (IR) showed enhanced suppression of cancer cell proliferation in wild-type p53 cells and more intense suppression in p53-null cells. In addition, the combination treatment effectively induced G1 cell cycle arrest in a p53-independent manner. In an in vivo tumor xenograft mouse model, treatment with both MPAP and IR reduced tumor growth more than the treatment with IR or MPAP alone. Overall, these data demonstrate that the radiosensitizing effect of MPAP is mediated by the regulation of retinoblastoma protein (RB) phosphorylation in a p53-independent manner.

Up-Regulated ATF4 Expression Increases Cell Sensitivity to Apoptosis in Response to Radiation.

BACKGROUND/AIMS: Activating transcription factor 4 (ATF4) is a member of the activating transcription factor family which regulates the expression of genes involved in amino acid metabolism, redox homeostasis and ER stress responses. ATF4 is also over-expressed in human solid tumors, although its effect on responsiveness to radiation is largely unexplored. METHODS: Real-time PCR was used to detect ATF4 mRNA levels in cells treated with different doses of 60Coγ radiation. Cell viability was assayed using a cell counting kit. The cell cycle was analyzed using flow cytometry, and cell apoptosis was assayed using Annexin V-PI double labeling. Small interfering RNA (siRNA) against ATF4 was transfected into ECV304 cells using Lipofectamine 2000. An ATF4 over-expression plasmid (p-ATF4-CGN) was transfected into HEK293 cells that endogenously expressed low levels of ATF4. The levels of intracellular reactive oxygen species (ROS) were measured using CM-H2DCFDA as a probe. RESULTS: ATF4 mRNA and protein expression levels were higher after radiation and increased in a dose- and time-dependent manner in AHH1 lymphoblast cells (P < 0.05). An increase in ATF4 levels was also observed after radiation in primary murine spleen cells, human endothelial ECV304 cells, human liver LO2 cells, breast cancer MCF7 cells, and human hepatocellular carcinoma HEPG2 cells. No change was observed in human embryonic kidney 293 (HEK293) cells. Over-expressing ATF4 in HEK293 cells inhibited cell proliferation, increased cell apoptosis and significantly increased the proportion of cells in G1 phase. Conversely, when ATF4 expression was knocked down using siRNA in ECV304 cells, it protected the cells from radiation-induced apoptosis. These findings suggest that ATF4 may play a role in radiation-induced cell killing by inhibiting cell proliferation and promoting cell apoptosis. CONCLUSIONS: In this study, we found that radiation up-regulated the expression of ATF4. We used ATF4 knockdown and over-expression systems to show that ATF4 may play a role in radiation-induced cellular apoptosis.

GADD45α is involved in the apoptosis of lymphocytes induced by riboflavin and ultraviolet light.

BACKGROUND: Riboflavin plus ultraviolet (UV) pathogen reduction technology (RF-PRT) is an effective method for inactivating the residual white blood cells (WBCs) in blood components. The RF-PRT system for platelets is known to activate many signaling pathways, including p38 and NF-κB. Nevertheless, proteomic studies in WBCs after riboflavin plus UV treatment requires further analysis. STUDY DESIGN AND METHODS: ABO/D-matched lymphocytes were pooled, split, and treated with RF-PRT or UV light or left untreated. After treatment, cell apoptosis was measured. In addition, cell proliferation and the cycle distribution were evaluated upon stimulation with phytohemagglutinin. The changes in the protein expression levels of growth arrest and DNA damage-inducible (GADD)45α, p38, and c-Jun N-terminal kinase (JNK) were determined by Western blotting. The effect of GADD45α, p38, and JNK on apoptosis was assessed. RESULTS: RF-PRT significantly inhibited proliferation and induced G1 arrest in lymphocytes. Furthermore, the percentage of apoptotic cells was increased in RF-PRT-treated lymphocytes compared to UV-treated cells or untreated cells, associated with the up regulation of GADD45α expression. Consistent with these observations, the inhibition of GADD45α expression partially counteracted the effects of riboflavin plus UV treatment. The p38 and JNK signaling pathways were activated by GADD45α in RF-PRT-treated lymphocytes. CONCLUSIONS: These data revealed that RF-PRT effectively inhibited proliferation and induced apoptosis of lymphocytes by promoting GADD45α expression, which subsequently activates p38 and JNK signaling pathways.

Matrilin-2 regulates proliferation, apoptosis and cell cycle during radiation-induced injury in HPAEpiC cell.

Radiation pulmonary injury is related to the accumulation of extracellular matrix proteins in the alveolar interstitial space. Matrilin-2 as a component of extracellular filamentous networks, present higher level in the lung tissue from irradiated mice and irradiated pulmonary epithelial cell line, HPAEpiC cells. Knockdown of endogenous matrilin-2 prevents the apoptosis of HPAEpiC cell induced by the irradiation injury. Consistently, over-expression of matrilin-2 reduced the proliferation and induced apoptosis of HPAEpiC cells. Matrilin-2 promotes the expression of p21 via increasing the transcriptional activity of p53, by which induces the G1 phase arresting in HPAEpiC cells. In summary, matrilin-2, increased by irradiation, reduced the proliferation and induces apoptosis of pulmonary epithelial cells via p53/p21 pathway.

p27Kip1 Is Required to Mediate a G1 Cell Cycle Arrest Downstream of ATM following Genotoxic Stress.

The DNA damage response (DDR) is a coordinated signaling network that ensures the maintenance of genome stability under DNA damaging stress. In response to DNA lesions, activation of the DDR leads to the establishment of cell cycle checkpoints that delay cell-cycle progression and allow repair of the defects. The tumor suppressor p27Kip1 is a cyclin-CDK inhibitor that plays an important role in regulating quiescence in a variety of tissues. Several studies have suggested that p27Kip1 also plays a role in the maintenance of genomic integrity. Here we demonstrate that p27Kip1 is essential for the establishment of a G1 checkpoint arrest after DNA damage. We also uncovered that ATM phosphorylates p27Kip1 on a previously uncharacterized residue (Ser-140), which leads to its stabilization after induction of DNA double-strand breaks. Inhibition of this stabilization by replacing endogenous p27Kip1 with a Ser-140 phospho-mutant (S140A) significantly sensitized cells to IR treatments. Our findings reveal a novel role for p27Kip1 in the DNA damage response pathway and suggest that part of its tumor suppressing functions relies in its ability to mediate a G1 arrest after the induction of DNA double strand breaks.

Thioridazine Sensitizes Esophageal Carcinoma Cell Lines to Radiotherapy-Induced Apoptosis In Vitro and In Vivo.

BACKGROUND Radiotherapy is one of the primary treatments for esophageal squamous cell carcinoma (ESCC). Identification of novel radio-sensitizing agents will improve the therapeutic outcome of radiotherapy. This study aimed to determine the radio-sensitizing effect of the antipsychotic agent thioridazine in ESCC and explored the underlying mechanisms. MATERIAL AND METHODS ECA-109 and TE-1 ESCC cells were treated with thioridazine and radiotherapy alone and in combination. Cell survival was measured by MTT assay. Cell cycle and apoptosis were monitored by flow cytometry. Western blot analysis was used to analyze the expression of phospho-PI3K, phosphor-AKT, phospho-mTOR, Caspase-3, Caspase-9, Bax, Bcl-2, Bal-xl, Bak, and p53. The xenograft mouse model was used to study the in vivo anticancer effect of thioridazine and irradiation. RESULTS Combined treatment with thioridazine and irradiation significantly reduced viability of ESCC cells compared with thioridazine or irradiation treatment alone. Thioridazine and irradiation treatment induced G0/G1 phases cell cycle arrest through down-regulation of CDK4 and cyclinD1. In addition, thioridazine and irradiation treatment induced apoptosis through up-regulation of cleaved capase-3 and 9, as well as an increase in the expression of Bax and Bak and a decrease in the expression of Bcl-2 and Bcl-xl. Furthermore, thioridazine and irradiation treatment inhibited the PI3K-AKT-mTOR pathway and up-regulated the expression of p53. In xenograft mice, thioridazine and irradiation reduced ESCC tumor growth. CONCLUSIONS Thioridazine sensitizes ESCC cells to radiotherapy. Thioridazine may play a role in ESCC radiation therapy as a promising radiosensitizer.