The ATM inhibitor KU55933 sensitizes radioresistant bladder cancer cells with DAB2IP gene defect

Abstract

Purpose: Our preliminary results showed that differentially expressed in ovarian cancer-2/disabled homolog 2 (DOC-2/DAB2) interactive protein (DAB2IP), a putative tumor suppressor gene, is down-regulated in bladder cancer (BCa) with aggressive phenotypes. In this study, we investigated how DAB2IP knockdown influenced BCa cell response to ionizing radiation (IR) and discussed possible ways to enhance cell radiosensitivity.

Methods and materials: The small interfering RNA (siRNA) system was implemented to inhibit endogenous DAB2IP expression in two human BCa cell lines, T24 and 5637. Cell sensitivity to IR alone or combined treatment was measured by a colony formation assay (CFA). Western blot was used to determine the phosphorylation levels of ataxia-telangiectasia mutated (ATM), catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) and related DNA damage repair (DDR) proteins. Immunofluorescence as well as a flow cytometry assay were employed to detect DNA double-strand break (DSB) repair and cell cycle distribution, respectively.

Results: DAB2IP-knockdown of BCa cells (i.e., siDAB2IP) exhibit increased clonogenic survival in response to IR compared with control cells (i.e., siCON) expressing an endogenous level of DAB2IP. The mechanism in siDAB2IP cells could be explained by elevated ATM expression and activation, increased S phase cell distribution as well as faster DSB repair kinetics. 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (KU55933) significantly sensitized siDAB2IP cells to IR due to inhibition of the phosphorylation of ATM and its downstream targets following IR and slower DSB repair kinetics.

Conclusions: Loss of DAB2IP expression in BCa cells signifies their radioresistance. KU55933, which suppresses ATM phosphorylation upon irradiation, could be applied in the radiotherapy of BCa patients with a DAB2IP gene defect.     

Telomere shortening and ionizing radiation: A possible role in vascular dysfunction?

Abstract

Purpose: In recent years, growing epidemiological evidence has linked ionizing radiation exposure to cardiovascular atherosclerotic disease. However, there are still major gaps in the knowledge of the molecular mechanisms of radiation-induced vascular disease, especially for low-dose levels. Telomeres, repetitive DNA sequences of (TTAGGG)_n located at the ends of eukaryotic chromosomes, play a role in regulating vascular aging, and shorter leukocyte telomere length has been demonstrated to predict cardiovascular disease and mortality. There is also evidence supporting the crucial role of telomeres in the formation of chromosome and chromatid aberrations induced by ionizing radiation.

Conclusions: The purpose of the present paper is to review the recent advances in the biological mechanisms determining telomere length erosion after ionizing radiation exposure as well as to examine the hypothesis that telomere shortening may be the crucial mediator leading to detrimental vascular effects after ionizing radiation exposure.     

ASPM influences DNA double-strand break repair and represents a potential target for radiotherapy

Abstract

Purpose: In a previous study using HiCEP (High coverage expression profiling), we demonstrated that ASPM (abnormal spindle-like microcephaly-associated) or the most common-type microcephaly (MCPH5) gene was selectively down-regulated by IR (ionizing radiation). The roles of ASPM on radiosensitivity, however, have never been studied.

Materials and methods: Using glioblastoma cell lines and normal human fibroblasts, we investigated how IR sensitivity (survived fraction, DNA repair and chromosome aberration) was affected by the reduction of ASPM by specific siRNA (small interfering RNA).

Results: Down-regulation of ASPM by siRNA enhanced radiosensitivity in three human cell lines examined. Constant-field gel electrophoreses and γ-H2AX (phosphorylated form of Histone H2A variant H2AX) foci analysis showed that ASPM-specific siRNA impaired DNA double-strand breaks (DSB) in irradiated cells. Elevated levels of abnormal chromosomes were also observed following ASPM siRNA. In addition IR-sensitization by ASPM knockdown was not enhanced in DNA-PK (DNA-dependent protein kinase) deficient glioblastoma cells suggesting that ASPM impacts upon a DNA-PK-dependent pathway.

Conclusions: Our results show for the first time that ASPM is required for efficient non-homologous end-joining in mammalian cells. In clinical applications, ASPM could be a novel target for combination therapy with radiation as well as a useful biomarker for tumor prognosis as ever described.     

Hypothermia modulates the DNA damage response to ionizing radiation in human peripheral blood lymphocytes

Purpose: Low temperature at exposure has been shown to act in a radioprotective manner at the level of cytogenetic damage. It was suggested to be due to an effective transformation of DNA damage to chromosomal damage at low temperature. The purpose of the study was to analyze the kinetics of aberration formation during the first hours after exposing human peripheral blood lymphocytes to ionizing radiation at 0.8?°C and 37?°C.

Materials and methods: To this end, we applied the technique of premature chromosome condensation. In addition, DNA damage response was analyzed by measuring the levels of phosphorylated DNA damage responsive proteins ATM, DNA-PK and p53 and mRNA levels of the radiation-responsive genes BBC3, FDXR, GADD45A, XPC, MDM2 and CDKN1A.

Results: A consistently lower frequency of chromosomal breaks was observed in cells exposed at 0.8?°C as compared to 37?°C already after 30?minutes postexposure. This effect was accompanied by elevated levels of phosphorylated ATM and DNA-PK proteins and a reduced immediate level of phosphorylated p53 and of the responsive genes.

Conclusions: Low temperature at exposure appears to promote DNA repair leading to reduced transformation of DNA damage to chromosomal aberrations.     

Role of various DNA repair pathways in chromosomal inversion formation in CHO mutants

Abstract

Purpose: In an effort to better understand the formation of chromosomal inversions, we investigated the role of various DNA repair pathways, including the non-homologous end joining (NHEJ), homologous recombination (HR), and Fanconi Anemia (FA) repair pathways for the formation of radiation induced chromosomal inversions.

Materials and methods: CHO10B2 wild type, CHO DNA repair-deficient, and CHO DNA repair-deficient corrected mutant cells were synchronized into G1 phase and exposed to gamma-rays. First post-irradiation metaphase cells were analyzed for chromosomal inversions by a differential chromatid staining technique involving a single cycle pre-irradiation ethynyl-uridine treatment and statistic calculations.

Results: It was observed that inhibition of the NHEJ pathway resulted in an overall decrease in the number of radiation-induced inversions, roughly a 50% decrease when compared to the CHO wild type. Interestingly, inhibition of the FA pathway resulted in an increase in both the number of spontaneous inversions and the number of radiation-induced inversions observed after exposure to 2 Gy of ionizing radiation. It was observed that FA-deficient cells contained roughly 330% (1.24 inversions per cell) more spontaneous inversions and 20% (0.4 inversions per cell) more radiation-induced inversions than the wild-type CHO cell lines. The HR mutants, defective in Rad51 foci, showed similar number of spontaneous and radiation-induced inversion as the wild-type cells. Gene complementation resulted in both spontaneous and radiation-induced inversions resembling the CHO wild-type cells.

Conclusions: We have concluded that the NHEJ repair pathway contributes to the formation of radiation-induced inversions. Additionally, through an unknown molecular mechanism it appears that the FA signal pathway prevents the formation of both spontaneous and radiation induced inversions.     

Radioresistance in a tumour cell line correlates with radiation inducible Ku 70/80 end-binding activity

Purpose: The aims of the present study were to better understand the role of Ku 80, which is involved in double-strand break repair in mammalian cells in the mechanism of radiation resistance and to verify the possibility of increasing cell radiosensitivity by targeted inhibition of Ku autoantigen 80 (Ku 80).

Materials and methods: Western blot and electrophoretic mobility shift assay (EMSA) were performed on the human bladder carcinoma cell line RT112 (radioresistant) and on the human colorectal carcinoma cell line SW48 (radiosensitive) to assess the expression levels of DNA-dependent protein kinase (DNA-PK) components and the DNA-binding activity of the Ku 70/80 heterodimer after exposure to radiation, respectively. Ku 80 silencing was carried out with the use of small interfering RNA (siRNA).

Results: Greater differences in the DNA-binding activity of Ku 70/80 and Ku 80 phosphorylation level were observed in RT112 as compared to SW48 after X-ray treatment. There is no correlation between Ku expression and DNA-binding activity at lower doses. A significant increase in nuclear Ku 80 expression was observed one hour after the exposure, only at the higher doses, while the DNA-PK catalytic subunits (DNA-PKcs) and Ku 70 levels did not change significantly. Inhibition of Ku 80 expression by siRNA induced radiosensitivity in the RT112 cell line.

Conclusions: Our data demonstrate that in a bladder tumour cell line up-regulation of Ku end-binding activity without any marked change in Ku expression underlie radiation resistance.     

Cerenkov Ultraviolet Radiation (137Cs γ-rays) and Direct Excitation (137Cs γ-rays and 50 kVp X-rays) Produce Photoreactivable Damage in Escherichia Coli

Summary

The mechanism of formation of photoreactivable damage in deoxyribonucleic acid (DNA) by ionizing radiation in a dark repair deficient strain of Escherichia coli (uvr A recA) has been investigated. By altering the ratio of damage produced directly (by ionization) to that formed indirectly (by Cerenkov ultraviolet (U.V.) radiation) by ¹³⁷Cs γ-rays, it has been demonstrated that the major portion of the photoreactivable damage is produced by Cerenkov U.V. radiation. The amount of photoreactivable damage produced by 50kVp X-rays, which cannot generate Cerenkov radiation, is similar to that component of photoreactivable damage produced by ¹³⁷Cs γ-rays that is not attributed to Cerenkov radiation. It is suggested that the second mechanism of formation of photoreactivable damage in DNA by ionizing radiation is the direct excitation of DNA. The possible role of Cerenkov U.V. radiation in ionizing radiation mutagenesis is discussed.     

Dotting the eyes: mouse strain dependency of the lens epithelium to low dose radiation-induced DNA damage

Purpose: Epidemiological evidence regarding the radiosensitivity of the lens of the eye and radiation cataract development has led to changes in the EU Basic Safety Standards for protection of the lens against ionizing radiation. However, mechanistic details of lens radiation response pathways and their significance for cataractogenesis remain unclear. Radiation-induced DNA damage and the potential impairment of repair pathways within the lens epithelium, a cell monolayer that covers the anterior hemisphere of the lens, are likely to be involved.

Materials and Methods: In this work, the lens epithelium has been analyzed for its DNA double-strand break (DSB) repair response to ionizing radiation. The responses of epithelial cells located at the anterior pole (central region) have been compared to at the very periphery of the monolayer (germinative and transitional zones). Described here are the different responses in the two regions and across four strains (C57BL/6, 129S2, BALB/c and CBA/Ca) over a low dose (0–25 mGy) in-vivo whole body X-irradiation range up to 24?hours post exposure.

Results: DNA damage and repair as visualized through 53BP1 staining was present across the lens epithelium, although repair kinetics appeared non-uniform. Epithelial cells in the central region have significantly more 53BP1 foci. The sensitivities of different mouse strains have also been compared.

Conclusions: 129S2 and BALB/c showed higher levels of DNA damage, with BALB/c showing significantly less inter-individual variability and appearing to be a more robust model for future DNA damage and repair studies. As a result of this study, BALB/c was identified as a suitable radiosensitive lens strain to detect and quantify early low dose ionizing radiation DNA damage effects in the mouse eye lens specifically, as an indicator of cataract formation.     

Evaluation of the radiosensitizing potency of chemotherapeutic agents in prostate cancer cells

Purpose: Despite recent advances in the treatment of metastatic prostate cancer, survival rates are low and treatment options are limited to chemotherapy and hormonal therapy. Although ionizing radiation is used to treat localized and metastatic prostate cancer, the most efficient use of radiotherapy is yet to be defined. Our purpose was to determine in vitro the potential benefit to be gained by combining radiation treatment with cytotoxic drugs.

Materials and methods: Inhibitors of DNA repair and heat shock protein 90 and an inducer of oxidative stress were evaluated in combination with X-radiation for their capacity to reduce clonogenic survival and delay the growth of multicellular tumor spheroids.

Results: Inhibitors of the PARP DNA repair pathway, olaparib and rucaparib, and the HSP90 inhibitor 17-DMAG, enhanced the clonogenic cell kill and spheroid growth delay induced by X-radiation. However, the oxidative stress-inducing drug elesclomol failed to potentiate the effects of X-radiation. PARP inhibitors arrested cells in the G₂/M phase when administered as single agents or in combination with radiation, whereas elesclomol and 17-DMAG did not affect radiation-induced cell cycle modulation.

Conclusion: These results indicate that radiotherapy of prostate cancer may be optimized by combination with inhibitors of PARP or HSP90, but not elesclomol.     

DNA Repair in Radiation Sensitive Mutants of Mammalian Cells: Possible Involvement of DNA Topoisomerases

Summary

Mutants are invaluable in the study of DNA repair processes. The past 10 years have seen a rapid proliferation of papers describing the isolation of mammalian cell mutants exhibiting DNA repair abnormalities. A variety of DNA-damaging agents, including radiation, alkylating agents and bleomycin, have been used to select mutants. This mini-review will concentrate on radiation (particularly ionizing radiation)-sensitive mutants, whether selected directly on the basis of radiation sensitivity or subsequently found to be cross-sensitive to radiation. Commonly observed DNA repair defects are associated with sensitivity to radiation. For UV-sensitive mutants a defect in the incision step of excision repair is frequently seen. For ionizing radiation-sensitive mutants, the common feature is a defect in the repair of DNA strand breaks. This may take the form of a reduced rate of strand-break rejoining or of a lowering in the fidelity of rejoining. Recent work suggests that the DNA topoisomerases may participate in the repair of DNA strand breaks and that strand breaks induced by both topoisomerase inhibitory drugs and radiation may be repaired by common pathways.     

Radiation-induced molecular changes in rat mammary tissue: Possible implications for radiation-induced carcinogenesis

Purpose:?Ionizing radiation is a potent mammary gland carcinogen, yet the exact molecular etiology of radiation-induced breast cancer remains unknown.

Materials and methods:?Our study utilized a rat model of breast carcinogenesis to analyse the molecular and epigenetic changes induced in mammary gland tissue upon exposure to ionizing radiation (IR). Using a methylation-sensitive cytosine extension assay we studied the IR-induced changes in DNA methylation. In parallel, we analysed the expression of proteins involved in DNA methylation, DNA repair and cell proliferation control. Molecular changes were related to cellular proliferation and apoptosis.

Results:?We found that IR led to a loss of genomic cytosine methylation in the exposed mammary tissue. Global DNA hypomethylation was paralleled by reduction in the levels of maintenance (DNMT1) and de novo (DNMT3a and 3b) DNA methyltransferases and methyl-binding protein MeCP2. The observed DNA hypomethylation was linked, at least in part, to activation of DNA repair processes. Concurrently, we observed increased levels of phosphorylated extracellular signal-regulated kinase (p-ERK1/2), phosphorylated AKT kinase (p-AKT), cyclin D1 and proliferating cells nuclear antigen (PCNA) proteins, suggesting IR alters intra-cellular signaling and cell cycle control mechanisms in mammary tissue. We also noted a significant induction of apoptosis in the exposed tissue 6 hours after irradiation. The observed apoptosis levels were paralleled by the slight elevation of cellular proliferation.

Conclusions:?We have demonstrated that a single exposure to 5 Gy of X rays leads to noticeable epigenetic changes in the rat mammary gland that occurred in the context of activation of DNA damage repair and alterations in the pro-survival growth-stimulatory cellular signaling pathways. The possible cellular repercussions of the observed changes in relationship to breast carcinogenesis are discussed.     

Molecular efficacy of radio- and chemotherapy sequences from direct DNA damage measurements

Purpose: To investigate the molecular aspects of the synergy between ionizing radiation and platinum (Pt) chemotherapeutic agents in cancer treatment with chemoradiation therapy (CRT) by measuring damages induced by low-energy electrons (LEE) to DNA bound to cisplatin. LEE are produced abundantly by any type of ionizing radiation and cisplatin represents a typical Pt-chemotherapeutic agents.

Materials and methods: Our strategy involves two parallel administrations of cisplatin and irradiation with a 4.6 and 9.6?eV electron fluence of 1.1?×?10¹²: (1) LEE bombardment of supercoiled DNA and its subsequent reaction with cisplatin; (2) the reaction of DNA with cisplatin followed by LEE irradiation. The damage yields for the loss of supercoiled (LS), single-strand breaks (SSB) and double-strand breaks (DSB) were obtained from gel electrophoresis analysis. Base modifications were revealed by treating the samples with Escherichia coli base excision repair endonuclease (Nth and Fpg).

Results: The yields were deduced from the respective time–response for the reaction of DNA with cisplatin. The results show that binding cisplatin to DNA followed by LEE irradiation, consistently yields more DNA damages than the reverse order. In comparison to non-treated DNA, administration (2) results in an increase of LS and SSB of 1.4–3.3 folds and of DSB by more than an order of magnitude. Furthermore, after enzyme treatment, the yields of DSB rise by factors of 5.3–15.4, indicating a large increase of clustered damages, which should at least partially translate into an increase of lethal damages in cancer cells during the CRT.

Conclusions: Our results demonstrate that a strong synergy between radiation and cisplatin can only be achieved at the molecular level, if the drug is present at the time of irradiation. Furthermore, this work confirms the LEE mechanism previously proposed to explain the synergy between radiation and Pt drugs in CRT. It involves chemical sensitization of DNA prior to irradiation, to facilitate strand breaks and clustered damages induced by the highly reactive LEE.     

Bioavailable serum estradiol may alter radiation risk of postmenopausal breast cancer: a nested case-control study

Purpose: Ionizing radiation and high levels of circulating estradiol are known breast cancer carcinogens. We investigated the risk of first primary postmenopausal breast cancer in relation to the combined effects of whole-body ionizing radiation exposure and prediagnostic levels of postmenopausal sex hormones, particularly bioavailable estradiol (bE₂).

Materials and methods: A nested case-control study of 57 incident breast cancer cases matched with 110 controls among atomic bomb survivors. Joint effects of breast radiation dose and circulating levels of sex hormones were assessed using binary regression and path analysis.

Results and conclusion: Radiation exposure, higher levels of bE₂, testosterone and progesterone, and established reproductive risk factors were positively associated with postmenopausal breast cancer risk. A test for mediation of the effect of radiation via bE₂ level suggested a small (14%) but significant mediation (p?=?0.004). The estimated interaction between radiation and bE₂ was large but not significant (interaction?=?3.86; p?=?0.32). There is accumulating evidence that ionizing radiation not only damages DNA but also alters other organ systems. While caution is needed, some portion of the radiation risk of postmenopausal breast cancer appeared to be mediated through bE₂ levels, which may be evidence for cancer risks due to both direct and indirect effects of radiation.     

Cellular Responses to Ionizing Radiation: Effects of Interrupting DNA Repair with Chemical Agents

Summary

This review is concerned with the influence of different classes of chemical agents on cellular repair of DNA damage induced by ionizing radiation. Single-strand break rejoining is little affected by inhibitors of DNA synthesis; however, such inhibitors do lead to a persistence of double-strand breaks in the DNA, and this correlates with an enhancement of chromosome aberrations and cell killing. Experiments with antagonists of topoisomerase II suggest an intriguing role for this DNA unwinding enzyme in double-strand break repair. Interference with poly(ADP-ribose) synthesis, by means of the inhibitor 3-aminobenzamide, does not have a clear-cut effect on recovery from ionizing radiation damage. Various substances (for example, caffeine and trypsin) affect DNA repair via a modulation of the cell cycle, altering the time available to the cell for repairing potentially lethal DNA damage before such damage is ‘fixed’ by the process of DNA replication. Finally, disturbing cellular energy metabolism, and depressing the level of ATP, can inhibit the repair of radiation damage.     

Bowman–Birk protease inhibitor activates DNA‐dependent protein kinase and reduces formation of radiation‐induced dicentric chromosomes

Purpose: To test a stimulatory effect of the radioprotector Bowman Birk protease inhibitor (BBI) upon DNA repair processes.

Materials and methods: An effect of BBI upon DNA repair was investigated by quantification of radiation‐induced dicentric chromosomes. Sensitivity to ionizing radiation was determined by clonogenic survival assay. Quantification of activity of the DNA‐dependent kinase was performed by immunoprecipitation and phosphorylation of a TP53‐derived peptide.

Results: The formation of radiation‐induced dicentric chromosomes was reduced significantly after pretreatment of cells with BBI. By using a cell line with an inducible expression of a mutated TP53, it was shown that the BBI‐mediated reduction of dicentric chromosome formation depended on the presence of wild‐type TP53. To get further insights into the molecular mode of action of BBI, activity of the DNA‐dependent protein kinase (DNA‐PK) was quantified. BBI treatment resulted in a stimulation of basal (DNA‐PK) activity. In SCID mouse fibroblasts deficient in DNA‐PK activity, BBI failed to reduce the amount of radiation‐induced dicentric chromosomes and the radioprotective effect was absent. Likewise, cells expressing mt.TP53 did not show radioprotection by BBI.

Conclusions: It was observed that BBI exerts its radioprotective effect by a reduction of incorrect DNA repair, resulting in a reduced amount of dicentric chromosomes. This effect on the fidelity of DNA repair is TP53 dependent and correlated with induction of DNA‐PK activity.     

Radiation-induced Potentially Lethal Damage: DNA Lesions Susceptible to Fixation

Summary

The various postirradiation incubation conditions reported to uncover potentially lethal damage (PLD) induced by ionizing radiation are outlined and critically discussed. The process of damage fixation is the most characteristic determinant in distinguishing between PLD and other forms of damage (lethal or non-lethal). The results compiled indicate the induction of two forms of PLD (termed alpha-and beta-PLD). Evidence is presented that repair and fixation of alpha-PLD may underlie the variation in radiosensitivity observed through the cycle. Beta-PLD appears to be sensitive only to postirradiation treatment in anisotonic salt solutions. Results obtained at the DNA and chromosome level, under conditions allowing repair or causing fixation of PLD, are reviewed and combined together to devise a qualitative model that outlines a possible sequence of events from damage fixation at the DNA level, to damage fixation at the chromosome level and, ultimately, to cell death. It is suggested that damage uncovered at the cellular level as potentially lethal, comprises DNA dsb (single, pairs or groups) and that fixation is mediated by forces transmitted to the double helix through alteration (local or general) in chromatin conformation. Changes in chromatin conformation are caused either as a result of the cell's progression through the cycle or in response to a postirradiation treatment. The fixation process leads to the induction of chromosome aberrations. The validity of the concept of PLD in in vivo systems is shown, and the possible importance of PLD repair in radiation therapy is reviewed. The concept of PLD is compared to the concept of sublethal damage, and the possibility that similar molecular lesions underlie both types of damage is discussed.     

Differing effects of breast cancer 1, early onset (BRCA1) and ataxia‐telangiectasia mutated (ATM) mutations on cellular responses to ionizing radiation

Purpose: The ataxia‐telangiectasia mutated (ATM) gene encodes a protein kinase, the activation of which is an early event in the cellular response to ionizing radiation. One of the many substrates of ATM is BRCA1 (breast cancer 1, early onset gene), which has been associated with susceptibility to breast and ovarian cancer, and has been implicated in DNA repair processes. Various cellular responses to radiation were analysed in cells with mutations in ATM or BRCA1 in an attempt to clarify which effects of ATM can be mediated through BRCA1.

Materials and methods: The response to radiation of cells with mutations in ATM or BRCA1 was examined, as were BRCA1‐mutant tumour cells transfected with an exogenous wild‐type BRCA1 allele. Assays included cell‐survival curves, studies of potentially lethal damage repair, measurement of chromosomal aberrations and of G1 arrest, and Western blot analysis of lysates of irradiated cells to determine the phosphorylation of the product of the human Mdm2 gene (HDM2).

Results: Both ATM and BRCA1 mutations were associated with sensitivity to ionizing radiation, deficient repair of potentially lethal damage and markedly increased chromosomal aberrations. A BRCA1‐mutated tumour cell line HCC1937, like ATM mutant cells, did not exhibit a normal G1 arrest but, unlike ATM mutant cells, did exhibit phosphorylation of HDM2. Expression of wild‐type BRCA1 in HCC1937 cells partially restored radioresistance, restored repair of potentially lethal damage and markedly reduced radiation‐induced chromosomal aberrations. G1 arrest, however, was not restored by expression of BRCA1.

Conclusions: The results are consistent with a model in which ATM phosphorylation of BRCA1 regulates DNA repair functions, particularly those involved in potentially lethal damage repair and chromosomal integrity, but not other aspects of the cellular response to radiation such as G1 cell cycle arrest. To the authors' knowledge, this is the first demonstration of the ability of exogenously expressed BRCA1 to restore the ability to perform potentially lethal damage repair and maintain chromosomal integrity in irradiated cells.     

FGF2 mediates DNA repair in epidermoid carcinoma cells exposed to ionizing radiation

Abstract

Purpose: Fibroblast growth factor 2 (FGF2) is a well-known survival factor. However, its role in DNA repair is poorly documented. The present study was designed to investigate in epidermoid carcinoma cells the potential role of FGF2 in DNA repair.

Materials and methods: The side population (SP) with cancer stem cell-like properties and the main population (MP) were isolated from human A431 squamous carcinoma cells. Radiation-induced DNA damage and repair were assessed using the alkaline comet assay. FGF2 expression was quantified by enzyme linked immunosorbent assay (ELISA).

Results: SP cells exhibited rapid repair of radiation induced DNA damage and a high constitutive level of nuclear FGF2. Blocking FGF2 signaling abrogated the rapid DNA repair. In contrast, in MP cells, a slower repair of damage was associated with low basal expression of FGF2. Moreover, the addition of exogenous FGF2 accelerated DNA repair in MP cells. When irradiated, SP cells secreted FGF2, whereas MP cells did not.

Conclusions: FGF2 was found to mediate DNA repair in epidermoid carcinoma cells. We postulate that carcinoma stem cells would be intrinsically primed to rapidly repair DNA damage by a high constitutive level of nuclear FGF2. In contrast, the main population with a low FGF2 content exhibits a lower repair rate which can be increased by exogenous FGF2.     

Radiation-induced DNA Damage and Its Repair

Summary

Application of modern methods of organic chemistry and recombinant DNA technologies has provided new insights in the field of DNA radiation damage and its repair. An overview of the chemical nature of the lesions inflicted on DNA by ionizing radiation is presented. The structures of 29 different DNA modified base or sugar residues are shown in comprehensive formation schemes.

A fraction of radiation-induced modified bases is spontaneously released from the DNA chain during irradiation. Another part remains attached to the DNA chain backbone and for its characterization mild formic acid or enzymatic hydrolysis have been used. Starting from the chemical formulae of the altered base residues, the specific repair enzymes and their modes of action are discussed. Various glycosylases and endonucleases have been purified to homogeneity, and in some cases the gene which encodes the protein cloned. Using methods derived from Maxam and Gilbert sequencing procedures and DNA fragment ³²P-labelled at one end, it has been shown that the alkali—labile sites in DNA induced by radiation are strongly dependent on the DNA base sequence. Enzymatic methods have been used to analyse the DNA base defects produced by gamma-irradiation of cells under in vivo conditions. Structures of modified bases were the same as those observed when DNA was irradiated in aqueous solution.