The generation and characterization of a radiation-resistant model system to study radioresistance in human breast cancer cells.

To systematically study the selection of radioresistant cells in clinically advanced breast cancer, a model system was generated by treating MDA-MB231 breast cancer cells with fractionated gamma radiation. A clonogenic assay of the surviving cell populations showed that 2-6 Gy per fraction resulted in a rapid selection of radioresistant populations, within three to five fractions. Irradiation with additional fractions after this initial increase did not increase the radioresistance of the surviving population significantly. Doses of 0.5 and 8 Gy per fraction were not effective in selecting radioresistant cells. To further determine the cause of the changes in radiosensitivity, 15 clones were isolated from the cell populations treated with 40 or 60 Gy with 2 or 4 Gy per fraction, respectively, and were analyzed for radiosensitivity. The average D(10) for these clones was 6.75 +/- 0.36 Gy, which was higher than that for the parental cell population (D(10) = 6.0 +/- 0.2 Gy). The operation of cell cycle checkpoints and the doubling time were similar for both the nonirradiated parental population and the isolated radioresistant subclones. In contrast, a decrease in the apoptotic potential was correlated (r = 0.7, P < 0.01) with increased survival after irradiation, suggesting that apoptosis is an important factor in determining radioresistance under our experimental conditions. We also isolated several subclones from the nonirradiated parental cell population and analyzed them to determine their radiosensitivity after fractionated irradiation. Ten fractions of 4 Gy (40 Gy in total) did not result in a significant increase in the radioresistance of these subclones compared to the irradiated cell populations. The possible mechanisms of the increased radioresistance after fractionated irradiation are discussed.     

Evidence against enhancement of the radioresistance of Escherichia coli by cloned Deinococcus radiodurans DNA

Deinococcus radiodurans genomic DNA, introduced to Escherichia coli in cloning vectors, has been reported to produce radioresistant E. coli that can be selected by gamma irradiation. In this report prior results are reassessed experimentally, and additional studies are presented. Results to date suggest that the acquired radioresistance of E. coli selected by gamma irradiation does not stem from expression of stable plasmid-encoded D. radiodurans sequences, and that acquired radioresistance is not readily transmitted to naive (unirradiated) E. coli by transformation of plasmid recovered from the radioresistant isolates. Several interpretations are discussed.     

Recovery of ionizing-radiation damage after high doses of gamma ray in the hyperthermophilic archaeon Thermococcus gammatolerans

The recently discovered hyperthermophilic and radioresistant archaeon Thermococcus gammatolerans is of great interest to compare and contrast the impact of its physiology on radioresistance and its ability to repair damaged chromosomes after exposure to gamma irradiation with radioresistant bacteria. We showed that, in contrast to other organisms, cell survival was not modified by the cellular growth phase under optimal growth conditions but nutrient-limited conditions did affect the T. gammatolerans radioresistance. We determined the first kinetics of damaged DNA recovery in an archaeon after exposure to massive doses of gamma irradiation and compared the efficiency of chromosomal DNA repair according to the cellular growth phase, nutrient availability and culture conditions. Chromosomal DNA repair kinetics showed that stationary phase cells reconstitute disrupted chromosomes more rapidly than exponential phase cells. Our data also revealed that this radioresistant archaeon was proficient to reconstitute shattered chromosomes either slowly or rapidly without any loss of viability. These results suggest that rapid DNA repair is not required for the extreme radioresistance of T. gammatolerans. Angels Tapias and Christophe Leplat contributed equally to this work.     

Identification of differentially expressed genes contributing to radioresistance in lung cancer cells using microarray analysis

Radiotherapy has played a key role in the control of tumor growth in many cancer patients. It is usually difficult to determine what fraction of the tumor cell population is radioresistant after a course of radiotherapy. The response of tumor cells to radiation is believed to be accompanied by complex changes in the gene expression pattern. It may be possible to use these to sensitize radioresistant tumor cells and improve radiocurability. Based on the biological effects of ionizing radiation, in the present study, we developed one oligonucleotide microarray to analyze the expression of 143 genes in cells of two lung cancer cell lines with different radiosensitivities. Compared to NCI-H446 cells, expression of 18 genes significantly increased the basal levels in the radioresistant A549 cells, in which eight genes were up-regulated and 10 genes were down-regulated. In A549 cells irradiated with 5 Gy, 22 (19 up-regulated and three down-regulated) and 26 (eight up-regulated and 18 down-regulated) differentially expressed genes were found 6 and 24 h after irradiation, respectively. In NCI-H446 cells, the expression of 17 (nine up-regulated and eight down-regulated) and 18 (six up-regulated and 12 down-regulated) genes was altered 6 and 24 h after irradiation, respectively. RT-PCR was performed, and we found that MDM2, BCL2, PKCZ and PIM2 expression levels were increased in A549 cells and decreased in NCI-H446 cells after irradiation. Genes involved in DNA repair, such as XRCC5, ERCC5, ERCC1, RAD9A, ERCC4 and the gene encoding DNA-PK, were found to be increased to a higher level in A549 cells than in NCI-H446 cells. Antisense suppression of MDM2 resulted in increased radiosensitivity of A549 cells. Taken together, these results demonstrate the possibility that a group of genes involved in DNA repair, regulation of the cell cycle, cell proliferation and apoptosis is responsible for the different radioresistance of these two lung cancer cells. This list of genes may be useful in attempts to sensitize the radioresistant lung cancer cells.     

Alterations in DNA repair efficiency are involved in the radioresistance of esophageal adenocarcinoma

To study radioresistance in esophageal adenocarcinoma, we generated an isogenic cell line model by exposing OE33 esophageal adenocarcinoma cells to clinically relevant fractionated doses of radiation (cumulative dose 50 Gy). A clonogenic assay confirmed enhanced survival of the radioresistant OE33 subline (OE33 R). To our knowledge, we are the first to generate an isogenic model of radioresistance in esophageal adenocarcinoma. This model system was characterized in terms of growth, cell cycle distribution and checkpoint operation, apoptosis, reactive oxygen species generation and scavenging, and DNA damage. While similar properties were found for both the parental OE33 (OE33 P) cells and radioresistant OE33 R cells, OE33 R cells demonstrated greater repair of radiation-induced DNA damage. Our results suggest that the radioresistance of OE33 R cells is due at least in part to increased DNA repair.     

GS-nitroxide (JP4-039)-mediated radioprotection of human Fanconi anemia cell lines

Fanconi anemia (FA) is an inherited disorder characterized by defective DNA repair and cellular sensitivity to DNA crosslinking agents. Clinically, FA is associated with high risk for marrow failure, leukemia and head and neck squamous cell carcinoma (HNSCC). Radiosensitivity in FA patients compromises the use of total-body irradiation for hematopoietic stem cell transplantation and radiation therapy for HNSCC. A radioprotector for the surrounding tissue would therefore be very valuable during radiotherapy for HNSCC. Clonogenic radiation survival curves were determined for pre- or postirradiation treatment with the parent nitroxide Tempol or JP4-039 in cells of four FA patient-derived cell lines and two transgene-corrected subclonal lines. FancG(-/-) (PD326) and FancD2(-/-) (PD20F) patient lines were more sensitive to the DNA crosslinking agent mitomycin C (MMC) than their transgene-restored subclonal cell lines (both P < 0.0001). FancD2(-/-) cells were more radiosensitive than the transgene restored subclonal cell line (? = 2.0 ± 0.7 and 4.7 ± 2.2, respectively, P = 0.03). In contrast, FancG(-/-) cells were radioresistant relative to the transgene-restored subclonal cell line (? = 9.4 ± 1.5 and 2.2 ± 05, respectively, P = 0.001). DNA strand breaks measured by the comet assay correlated with radiosensitivity. Cell lines from a Fanc-C and Fanc-A patients showed radiosensitivity similar to that of Fanc-D2(-/-) cells. A fluorophore-tagged JP4-039 (BODIPY-FL) analog targeted the mitochondria of the cell lines. Preirradiation or postirradiation treatment with JP4-039 at a lower concentration than Tempol significantly increased the radioresistance and stabilized the antioxidant stores of all cell lines. Tempol increased the toxicity of MMC in FancD2(-/-) cells. These data provide support for the potential clinical use of JP4-039 for normal tissue radioprotection during chemoradiotherapy in FA patients.     

ITGB1 enhances the Radioresistance of human Non-small Cell Lung Cancer Cells by modulating the DNA damage response and YAP1-induced Epithelial-mesenchymal Transition

Objectives: Radiotherapy has played a limited role in the treatment of non-small cell lung cancer (NSCLC) due to the risk of tumour radioresistance. We previously established the radioresistant non-small cell lung cancer (NSCLC) cell line H460R. In this study, we identified differentially expressed genes between these radioresistant H460R cells and their radiosensitive parent line. We further evaluated the role of a differentially expressed gene, ITGB1, in NSCLC cell radioresistance and as a potential target for improving radiosensitivity.Materials and Methods: The radiosensitivity of NSCLC cells was evaluated by flow cytometry, colony formation assays, immunofluorescence, and Western blotting. Bioinformatics assay was used to identify the effect of ITGB1 and YAP1 expression in NSCLC tissues.Results: ITGB1 mRNA and protein expression levels were higher in H460R than in the parental H460 cells. We observed lower clonogenic survival and cell viability and a higher rate of apoptosis of ITGB1-knockdown A549 and H460R cells than of wild type cells post-irradiation. Transfection with an ITGB1 short hairpin (sh) RNA enhanced radiation-induced DNA damage and G2/M phase arrest. Moreover, ITGB1 induced epithelial-mesenchymal transition (EMT) of NSCLC cells. Silencing ITGB1 suppressed the expression and intracellular translocation of Yes-associated protein 1 (YAP1), a downstream effector of ITGB1.Conclusions: ITGB1 may induce radioresistance via affecting DNA repair and YAP1-induced EMT. Taken together, our data suggest that ITGB1 is an attractive therapeutic target to overcome NSCLC cell radioresistance.     

Multiplication of Bacteriophage P22 in Penicillin-Induced Spheroplasts of Salmonella typhimurium

The spheroplasts of Salmonella typhimurium (LT2) prepared by treatment with penicillin were capable of adsorbing phage P22 C(1). The normal multiplication of the phage took place, although the burst size was reduced to one-fourth of that in intact cells. Rate of incorporation of (14)C-thymidine into spheroplasts was increased severalfold on phage infection. Multiplication of C(+) also took place, but no lysogeny could be established in spheroplasts. Furthermore, spheroplasts prepared from cells lysogenized with wild-type phage, LT2 (C(+)), and a temperature-inducible C(2) mutant, LT2(tsC(2)), were not inducible. Unlike normal cells, both mitomycin C and actinomycin D interfered with the phage multiplication in spheroplasts. The spheroplast system offers great advantages in the study of the synthesis of nucleic acids and proteins in phage-infected LT2.     

Role of Rad52 in fractionated irradiation induced signaling in A549 lung adenocarcinoma cells

The effect of fractionated doses of γ-irradiation (2Gy per fraction over 5 days), as delivered in cancer radiotherapy, was compared with acute doses of 10 and 2Gy, in A549 cells. A549 cells were found to be relatively more radioresistant if the 10Gy dose was delivered as a fractionated regimen. Microarray analysis showed upregulation of DNA repair and cell cycle arrest genes in the cells exposed to fractionated irradiation. There was intense activation of DNA repair pathway-associated genes (DNA-PK, ATM, Rad52, MLH1 and BRCA1), efficient DNA repair and phospho-p53 was found to be translocated to the nucleus of A549 cells exposed to fractionated irradiation. MCF-7 cells responded differently in fractionated regimen. Silencing of the Rad52 gene in fractionated group of A549 cells made the cells radiosensitive. The above result indicated increased radioresistance in A549 cells due to the activation of Rad52 gene.     

Isolation and characterization of highly radioresistant malignant hamster fibroblasts that survive acute gamma irradiation with 20 Gy

To study the acquired radioresistance of tumor cells, a model system of two cell lines, Djungarian hamster fibroblasts (DH-TK-) and their radioresistant progeny, was established. The progeny of irradiated cells were isolated by treating the parental cell monolayer with a single dose of 20 Gy (PIC-20). The genetic and morphological features, clonogenic ability, radiosensitivity, cell growth kinetics, ability to grow in methylcellulose, and tumorigenicity of these cell lines were compared. The plating efficiency of PIC-20 cells exceeded that of DH-TK- cells. The progeny of irradiated cells were more radioresistant than parental cells. The average D0 for PIC-20 cells was 7.4 +/- 0.2 Gy, which is three times higher than that for parental cells (2.5 +/- 0.1 Gy). Progeny cell survival in methylcellulose after irradiation with a dose of 10 Gy was 15 times higher than that of DH-TK- cells. In contrast to parental cells, the progeny of irradiated cells showed fast and effective repopulation after irradiation with doses of 12.5 and 15 Gy. The tumor formation ability of irradiated progeny cells was higher than that of parental cells; after 15 Gy irradiation, PIC-20 cells produced tumors as large as unirradiated progeny of irradiated cells, whereas the tumor development of DH-TK- cells diminished by 70%. High radioresistance of progeny of irradiated cells was reproduced during the long period of cultivation (more than 80 passages). The stability of the radioresistant phenotype of PIC-20 cells allows us to investigate the possible mechanisms of acquired tumor radioresistance.     

X-irradiation of equine peripheral blood lymphocytes stimulated with phytohaemagglutinin in vitro.

Small lymphocytes were isolated from the peripheral blood of horses and incubated at 37 degrees C in Eagle's medium supplemented with 20 per cent foetal calf serum. The addition of phytohaemagglutinin (PHA) to the cultures resulted in: increased RNA and protein synthesis; the enlargement of the small lymphocyte into a lymphoblast-like cell; the initiation of DNA synthesis, and cell division. When survival was measured 24 hours after X-irradiation by means of phase-contrast microscopy, the lymphoblast-like cell was much more radio-resistant (D0 = 250 rad) than the small lymphocyte (D0 = 20 rad). This increase in radioresistance, however, was not observed until 12-24 hours after PHA treatment. To investigate which of the changes occurring during the transformation of the small lymphocyte was responsible for the increased resistance to irradiation, the percentage of cells surviving irradiation was compared with the percentage of cells incorporating significant amounts of 3HTdR, 3H-UR, or 3H-leucine at the time of irradiation. For this comparison, a dose of 100 rad was used because 100 rad killed essentially all of the small lymphocytes, but less than 35 percent of the cells which had become radioresistant from the PHA treatment. The results indicated that the increase in radioresistance was not associated with DNA synthesis, but instead correlated with the increase in RNA and protein synthesis which the cells had attained at the time of irradiation.     

Enhancement of autophagy is a potential modality for tumors refractory to radiotherapy

Radiotherapy is a well-established treatment for cancer. However, the existence of radioresistant cells is one of the major obstacles in radiotherapy. In order to understand the mechanism of cellular radioresistance and develop more effective radiotherapy, we have established clinically relevant radioresistant (CRR) cell lines, which continue to proliferate under daily exposure to 2 Gray (Gy) of X-rays for >30 days. X-ray irradiation significantly induced autophagic cells in parental cells, which was exiguous in CRR cells, suggesting that autophagic cell death is involved in cellular radiosensitivity. An autophagy inducer, rapamycin sensitized CRR cells to the level of parental cells and suppressed cell growth. An autophagy inhibitor, 3-methyladenine induced radioresistance of parental cells. Furthermore, inhibition of autophagy by knockdown of Beclin-1 made parental cells radioresistant to acute radiation. These suggest that the suppression of autophagic cell death but not apoptosis is mainly involved in cellular radioresistance. Therefore, the enhancement of autophagy may have a considerable impact on the treatment of radioresistant tumor.     

Genome-scale CRISPR-Cas9 knockout screening in nasopharyngeal carcinoma for radiosensitive and radioresistant genes

BackgroundGenome-scale CRISPR-Cas9 knockout screening may provide new insights into the mechanism underlying clinical radioresistance in nasopharyngeal carcinoma (NPC), which is remain largely unknown. Our objective was to screen the functional genes associated with radiosensitivity and radioresistance in NPC, laying a foundation for further research on its functional mechanismand.MethodsCRISPR-Cas9 library lentivirus screening in radiation-treated NPC cells was combined with second-generation sequence technology to identify functional genes, which were further validated in radioresistant NPC cells and patient tissues.ResultsEleven radiosensitive and radioresistant genes were screened. Among these genes, the expression of FBLN5, FAM3C, MUS81, and DNAJC17 were significantly lower and TOMM20, CDKN2AIP, SNX22, and SP1 were higher in the radioresistant NPC cells (C666-1R, 5-8FR) (p < 0.05). CALD1 was highly expressed in C666-1R. Furthermore, we found knockout of FBLN5, FAM3C, MUS81 and DNAJC17 promoted the proliferation of NPC cells, while CDKN2AIP and SP1 had the opposed results (p < 0.05). This result was verified in NPC patient tissues. Meanwhile, KEGG analysis showed that the Fanconi anemia pathway and the TGF-β signaling pathway possibly contributed to radiosensitivity or radioresistance in NPC.ConclusionsNine genes involved in the radiosensitivity or radioresistance of NPC: four genes for radiosensitivity (FBLN5, FAM3C, MUS81, and DNAJC17), two genes for radioresistance (CDKN2AIP, SP1), two potential radioresistant genes (TOMM20, SNX22), and a potential radiosensitive gene (CALD1). Genome-scale CRISPR-Cas9 knockout screening for radiosensitive and radioresistant genes in NPC may provide new insights into the mechanisms underlying clinical radioresistance to improve the efficacy of radiotherapy for NPC.     

Dying of Gamma-Irradiated Escherichia coli Studied by the Use of Prophage

Loss of the biological activity of deoxyribonucleic acid in gamma-irradiated Escherichia coli cells was studied. The study is based on two sets of experimental data: (i) post-irradiation heat inducibility of the cells whose chromosomes were "labeled" with the thermoinducible lambdacI857ind prophage, and (ii) post-irradiation capacity of nonlysogenic cells to promote growth of the unirradiated lambdacI857ind phage. The results show that, at the beginning of incubation after irradiation, the number of plaques formed upon heat induction of lysogenic cells was much higher than the viable cell count of the nonheated culture. This high resistance of the heat inducibility gradually decreased during post-irradiation incubation. Finally, after a period of 4 h, there was no difference in sensitivity between the heat inducibility and the colony-forming ability of gamma-irradiated cells. The capacity of gamma-irradiated bacteria to support growth of unirradiated lambdacI857ind is radioresistant; this resistance, in contrast to that of heat inducibility, is much less affected during post-irradiation incubation. A continuous decrease in radioresistance of heat inducibility without a corresponding decrease in radioresistance of the capacity suggests that functional failure of initially undamaged and/or repaired parts of the chromosome gradually develops after irradiation. From the fact that after 4 h all colony formers are capable of being induced by heat, whereas no chromosomal activity can be detected in nonviable cells, two conclusions may be drawn: (i) gamma-irradiated E. coli cells destined to die reach their biological end point within 4 h of post-irradiation incubation; (ii) in most cells, functional failure of the whole chromosome is the immediate cause of death.     

DNA protection mechanisms are not involved in the radioresistance of the hyperthermophilic archaea<Emphasis Type="Italic"> Pyrococcus abyssi</Emphasis> and<Emphasis Type="Italic"> P. furiosus</Emphasis>

Hyperthermophilic archaea of the genus Pyrococcus are resistant to gamma radiation, suggesting that efficient mechanisms for DNA repair exist in these organisms. To determine whether protective mechanisms might also be implicated in this radioresistance, we have estimated the linear density of DNA double-stranded breaks caused by gamma irradiation in the genomic DNA of two Pyrococcus species, using Escherichia coli and the radioresistant bacterium Deinococcus radiodurans as controls. The linear density of double-stranded breaks was essentially the same in all four microorganisms when irradiation was carried under similar anaerobic conditions, indicating that no specific DNA protection mechanisms exist in Pyrococcus species. Using one- and two-dimensional gel electrophoresis we compared the protein patterns from Pyrococcus abyssi and P. furiosus cells that had or had not been exposed to gamma rays. We did not detect any significant protein induction following DNA damage in either species.     

Minicell production and bacteriophage superinducibility of thymidine-requiring strains of Haemophilus influenzae.

Aminopterin- or trimethoprin-resistant thymidine-requiring strains of Haemophilus influenzae produce minicells, and the ratio of minicells to cells increases during the stationary phase of growth. Strain LB11, isolated after mutagenesis of a thymidine-requiring strain (Rd thd), produces more minicells than the parent strain. The mutations involved in high frequency minicell production have been transferred into the wild type (strain Rd) by transformation. The thymidine requirement in the resulting strain, MCl, is essential for minicell production, since spontaneous revertants of MCl to prototrophy do not produce minicells. The ratio of minicells to cells was increased more than 10(3)-fold by differential centrifugation. The minicells contain little or no deoxyribonucleic acid (DNA). Phage HPlcl apparently cannot attach to minicells. Competent cells of LB11 and its thymidine-requiring parent strain produce defective phage as a result of exposure to transforming DNA, whereas only LB11 produces many defective phage in response to the competence regime alone. Competent HP1c1 and S2 lysogens of MC1 and Rd thd are also superinducible by transforming DNA, but competent LB11 lysogens produced about the same amount of HP1c1 or S2 phage with or without exposure to transforming DNA possibly because of competition between the induced defective phage and Hp1c1 or S2 phage.     

Raman Spectroscopic Study of Radioresistant Oral Cancer Sublines Established by Fractionated Ionizing Radiation

Radiotherapy is an important treatment modality for oral cancer. However, development of radioresistance is a major hurdle in the efficacy of radiotherapy in oral cancer patients. Identifying predictors of radioresistance is a challenging task and has met with little success. The aim of the present study was to explore the differential spectral profiles of the established radioresistant sublines and parental oral cancer cell lines by Raman spectroscopy. We have established radioresistant sublines namely, 50Gy-UPCI:SCC029B and 70Gy-UPCI:SCC029B from its parental UPCI:SCC029B cell line, by using clinically admissible 2Gy fractionated ionizing radiation (FIR). The developed radioresistant character was validated by clonogenic cell survival assay and known radioresistance-related protein markers like Mcl-1, Bcl-2, Cox-2 and Survivin. Altered cellular morphology with significant increase (p<0.001) in the number of filopodia in radioresistant cells with respect to parental cells was observed. The Raman spectra of parental UPCI:SCC029B, 50Gy-UPCI:SCC029B and 70Gy-UPCI:SCC029B cells were acquired and spectral features indicate possible differences in biomolecules like proteins, lipids and nucleic acids. Principal component analysis (PCA) provided three clusters corresponding to radioresistant 50Gy, 70Gy-UPCI:SCC029B sublines and parental UPCI:SCC029B cell line with minor overlap, which suggest altered molecular profile acquired by the radioresistant cells due to multiple doses of irradiation. The findings of this study support the potential of Raman spectroscopy in prediction of radioresistance and possibly contribute to better prognosis of oral cancer.     

Bmi-1 confers adaptive radioresistance to KYSE-150R esophageal carcinoma cells

Radiotherapy (RT) is a major modality of cancer treatment. However, tumors often acquire radioresistance, which causes RT to fail. The exact mechanisms by which tumor cells subjected to fractionated irradiation (FIR) develop an adaptive radioresistance are largely unknown. Using the radioresistant KYSE-150R esophageal squamous cell carcinoma (ESCC) model, which was derived from KYSE-150 parental cells using FIR, the role of Bmi-1 in mediating the radioadaptive response of ESCC cells to RT was investigated. The results showed that the level of Bmi-1 expression was significantly higher in KYSE-150R cells than in the KYSE-150 parental cells. Bmi-1 depletion sensitized the KYSE-150R cells to RT mainly through the induction of apoptosis, partly through the induction of senescence. A clonogenic cell survival assay showed that Bmi-1 depletion significantly decreased the radiation survival fraction in KYSE-150R cells. Furthermore, Bmi-1 depletion increased the generation of reactive oxygen species (ROS) and the expression of oxidase genes (Lpo, Noxo1 and Alox15) in KYSE-150R cells exposed to irradiation. DNA repair capacities assessed by γ-H2AX foci formation were also impaired in the Bmi-1 down-regulated KYSE-150R cells. These results suggest that Bmi-1 plays an important role in tumor radioadaptive resistance under FIR and may be a potent molecular target for enhancing the efficacy of fractionated RT.