Irradiated Lymphocytes Research Articles

Radioprotective effect of mefenamic acid against radiation-induced genotoxicity in human lymphocytes.

PurposeMefenamic acid (MEF) as a non-steroidal anti-inflammatory drug is used as a medication for relieving of pain and inflammation. Radiation-induced inflammation process is involved in DNA damage and cell death. In this study, the radioprotective effect of MEF was investigated against genotoxicity induced by ionizing radiation in human blood lymphocytes.Materials and MethodsPeripheral blood samples were collected from human volunteers and incubated with MEF at different concentrations (5, 10, 50, or 100 µM) for two hours. The whole blood was exposed to ionizing radiation at a dose 1.5 Gy. Lymphocytes were cultured with mitogenic stimulation to determine the micronuclei in cytokinesis blocked binucleated lymphocyte.ResultsA significant decreasing in the frequency of micronuclei was observed in human lymphocytes irradiated with MEF as compared to irradiated lymphocytes without MEF. The maximum decreasing in frequency of micronuclei was observed at 100 µM of MEF (38% decrease), providing maximal protection against ionizing radiation.ConclusionThe radioprotective effect of MEF is probably related to anti-inflammatory property of MEF on human lymphocytes.

Automation of the cytokinesis-block micronucleus cytome assay by laser scanning cytometry and its potential application in radiation biodosimetry.

Here we describe the adaptation of laser scanning cytometry (LSC) to measure micronuclei (MN) automatically in lymphocytes. MN frequencies were determined in irradiated human lymphocytes using the cytokinesis-block technique, and the results from LSC were compared with visual scoring results obtained from slides of cells stained using Fast Green and 4',6-diamidino-2-phenylindole (DAPI). This fluorescent approach allowed clear identification of binucleated cells and detection of MN. The dose responses measured visually and by LSC showed similar trends and correlated positively (r = 0.9689; P < 0.0001). High-content analysis was developed to further automatically score MN within mono-, tri- and tetra-nucleated cells and to determine the nuclear division index and nuclear circularity values. The high-throughput nature of LSC can provide unique advantages in future DNA damage diagnostics in experimental and epidemiological studies. Importantly, it allows for co-detection of other biomarkers of interest within a single lymphocyte, and further development of this capability is anticipated.

Tetramethylpyrazine protects lymphocytes from radiation-induced apoptosis through nuclear factor-κB

AimRadiation induces an important apoptosis response in irradiated organs. The objective of this study was to investigate the radioprotective effect of tetramethylpyrazine (TMP) on irradiated lymphocytes and discover the possible mechanism of protection. MethodLymphocytes were pretreated for 12 h with TMP (25–200 μmol·L−1) and then exposed to 4 Gy radiation. Cell apoptosis and the signaling pathway were analyzed. ResultsIrradiation increased cell death, DNA fragmentation, activated caspase activation and cytochrome c translocation, downregulated B-cell lymphoma 2 (Bcl-2) and up-regulated Bcl-2-associated X protein (Bax). Pretreated with TMP significantly reversed this tendency. Several anti-apoptotic characteristics of TMP, including the ability to increase cell viability, inhibit caspase-9 activation, and upregulate Bcl-2 and down-regulate Bax in 4Gy-irradiated lymphocytes were determined. Signal pathway analysis showed TMP could translate nuclear factor-κB (NF-κB) from cytosol into the nucleus. ConclusionThe results suggest that TMP had a radioprotective effect through the NF-κB pathway to inhibit apoptosis, and it may be an effective candidate for treating radiation diseases associated with cell apoptosis.

Edaravone protects human peripheral blood lymphocytes from γ-irradiation-induced apoptosis and DNA damage.

Radiation-induced cellular injury is attributed primarily to the harmful effects of free radicals, which play a key role in irradiation-induced apoptosis. In this study, we investigated the radioprotective efficacy of edaravone, a licensed clinical drug and a powerful free radical scavenger that has been tested against γ-irradiation-induced cellular damage in cultured human peripheral blood lymphocytes in studies of various diseases. Edaravone was pre-incubated with lymphocytes for 2h prior to γ-irradiation. It was found that pretreatment with edaravone increased cell viability and inhibited generation of γ-radiation-induced reactive oxygen species (ROS) in lymphocytes exposed to 3Gy γ-radiation. In addition, γ-radiation decreased antioxidant enzymatic activity, such as superoxide dismutase and glutathione peroxidase, as well as the level of reduced glutathione. Conversely, treatment with 100μM edaravone prior to irradiation improved antioxidant enzyme activity and increased reduced glutathione levels in irradiated lymphocytes. Importantly, we also report that edaravone reduced γ-irradiation-induced apoptosis through downregulation of Bax, upregulation of Bcl-2, and consequent reduction of the Bax:Bcl-2 ratio. The current study shows edaravone to be an effective radioprotector against γ-irradiation-induced cellular damage in lymphocytes in vitro. Finally, edaravone pretreatment significantly reduced DNA damage in γ-irradiated lymphocytes, as measured by comet assay (% tail DNA, tail length, tail moment, and olive tail moment) (p < 0.05). Thus, the current study indicates that edaravone offers protection from radiation-induced cytogenetic alterations.

Genotoxicity and cytotoxicity of sevoflurane in two human cell lines in vitro with ionizing radiation

To determine the in vitro toxicity of different concentrations of sevoflurane in cells exposed to X-ray. The genotoxic effects of sevofluorane were studied by means of the micronucleus test in cytokinesis-blocked cells of irradiated human lymphocytes. Subsequently, its cytotoxic effects on PNT2 (normal prostate) cells was determined using the cell viability test (MTT) and compared with those induced by different doses of X-rays. A dose- and time-dependent cytotoxic effect of sevofluorane on PNT2 cells was determined (p >0.001) and a dose-dependent genotoxic effect of sevofluorane was established (p >0.001). However, at volumes lower than 30 μL of sevofluorane at 100%, a non-toxic effect on PNT2 cells was shown. Sevofluorane demonstrates a genotoxic capacity as determined in vitro by micronucleus test in cytokinesis-blocked cells of irradiated human lymphocytes.

Cytogenetic characteristics of the radiation-induced bystander effect and its persistence in human blood lymphocytes

Cytogenetic characteristics of the radiation-induced chromosomal instability and its persistence in human peripheral blood lymphocytes due to the bystander effect from exposure to ionizing radiation in vitro and in vivo at low and high doses has been established using a coculturing of irradiated and nonirradiated human peripheral blood lymphocytes from persons of different sexes and G-banding metaphase chromosomes staining.

DNA-Damage Foci to Detect and Characterize DNA Repair Alterations in Children Treated for Pediatric Malignancies

PurposeIn children diagnosed with cancer, we evaluated the DNA damage foci approach to identify patients with double-strand break (DSB) repair deficiencies, who may overreact to DNA-damaging radio- and chemotherapy. In one patient with Fanconi anemia (FA) suffering relapsing squamous cell carcinomas of the oral cavity we also characterized the repair defect in biopsies of skin, mucosa and tumor.Methods and MaterialsIn children with histologically confirmed tumors or leukemias and healthy control-children DSB repair was investigated by counting γH2AX-, 53BP1- and pATM-foci in blood lymphocytes at defined time points after ex-vivo irradiation. This DSB repair capacity was correlated with treatment-related normal-tissue responses. For the FA patient the defective repair was also characterized in tissue biopsies by analyzing DNA damage response proteins by light and electron microscopy.ResultsBetween tumor-children and healthy control-children we observed significant differences in mean DSB repair capacity, suggesting that childhood cancer is based on genetic alterations affecting DNA repair. Only 1 out of 4 patients with grade-4 normal-tissue toxicities revealed an impaired DSB repair capacity. The defective DNA repair in FA patient was verified in irradiated blood lymphocytes as well as in non-irradiated mucosa and skin biopsies leading to an excessive accumulation of heterochromatin-associated DSBs in rapidly cycling cells.ConclusionsAnalyzing human tissues we show that DSB repair alterations predispose to cancer formation at younger ages and affect the susceptibility to normal-tissue toxicities. DNA damage foci analysis of blood and tissue samples allows one to detect and characterize DSB repair deficiencies and enables identification of patients at risk for high-grade toxicities. However, not all treatment-associated normal-tissue toxicities can be explained by DSB repair deficiencies.

DNA double-strand break repair and induction of apoptosis in ex vivo irradiated blood lymphocytes in relation to late normal tissue reactions following breast radiotherapy

This study aimed to test whether induction of apoptosis following ex vivo X-irradiation of unstimulated blood lymphocytes correlated with clinical radiosensitivity and DNA double-strand break (DSB) repair in breast radiotherapy patients and healthy volunteers. Using small molecule inhibitors, the relationship between DSB repair and radiation-induced apoptosis was examined. Sixteen breast cancer patients with minimal (controls, n = 8) or extremely marked late radiation-induced change (cases, n = 8) and eight healthy volunteers were selected. DSBs were quantified by γH2AX/53BP1 immunofluorescence, and apoptosis was measured using a fluorogenic inhibitor of caspases assay. Mean γH2AX/53BP1 focus levels 24 h after exposure to 4 Gy were higher in cases (12.7 foci per cell) than in controls (10.3 foci per cell, p = 0.002). In contrast, the mean apoptotic fraction 48 h after 8 Gy was comparable, 37.2 % in cases and 34.7 % in controls (p = 0.442). Residual focus and apoptosis levels were not correlated within individuals (Spearman's R = -0.0059, p = 0.785). However, cells treated with DNA-PK inhibitor Nu7441 had higher focus and apoptosis levels 48 h after 1 Gy compared to mock-treated cells, suggesting that apoptosis induction following irradiation is modulated by DSB repair. This effect required functional ATM since cells treated simultaneously with Nu7441 and the ATM inhibitor Ku55933 were resistant to apoptosis despite high levels of residual foci. One clinical case displayed an impaired DNA-PK-dependent end-joining cellular phenotype. In summary, clinical radiosensitivity may be associated with impaired DSB repair in some patients. Although pharmaceutical inhibition of ATM and DNA-PK affected apoptosis induction and DSB repair, no association was observed between apoptosis and residual focus levels in patients and volunteers.

Genetic factors in individual radiation sensitivity

Cancer risk and radiation sensitivity are often associated with alterations in DNA repair, cell cycle, or apoptotic pathways. Interindividual variability in mutagen or radiation sensitivity and in cancer susceptibility may also be traced back to polymorphisms of genes affecting e.g. DNA repair capacity. We studied possible associations between 70 polymorphisms of 12 DNA repair genes with basal and initial DNA damage and with repair thereof. We investigated DNA damage induced by ionizing radiation in lymphocytes isolated from 177 young lung cancer patients and 169 cancer-free controls. We also sought replication of our findings in an independent sample of 175 families (in total 798 individuals). DNA damage was assessed by the Olive tail moment (OTM) of the comet assay. DNA repair capacity (DRC) was determined for 10, 30 and, 60min of repair. Genes involved in the single-strand-repair pathway (SSR; like XRCC1 and MSH2) as well as genes involved in the double-strand-repair pathway (DSR; like RAD50, XRCC4, MRE11 and ATM) were found to be associated with DNA damage. The most significant association was observed for marker rs3213334 (p=0.005) of XRCC1 with basal DNA damage (B), in both cases and controls. A clear additive effect on the logarithm of OTM was identified for the marker rs1001581 of the same LD-block (p=0.039): BCC=-1.06 (95%-CI: -1.16 to -0.96), BCT=-1.02 (95%-CI: -1.11 to -0.93) and BTT=-0.85 (95%-CI: -1.01 to -0.68). In both cases and controls, we observed significantly higher DNA basal damage (p=0.007) for carriers of the genotype AA of marker rs2237060 of RAD50 (involved in DSR). However, this could not be replicated in the sample of families (p=0.781). An alteration to DRC after 30min of repair with respect to cases was observed as borderline significant for marker rs611646 of ATM (involved in DSR; p=0.055), but was the most significant finding in the sample of families (p=0.009). Our data indicate that gene variation impacts measurably on DNA damage and repair, suggesting at least a partial contribution to radiation sensitivity and lung cancer susceptibility.

Differential effects of p53 on bystander phenotypes induced by gamma ray and high LET heavy ion radiation

High LET particle irradiation has several potential advantages over γ-rays such as p53-independent response. The purpose of this work is to disclose the effect of p53 on the bystander effect induced by different LET irradiations and underlying mechanism. Lymphocyte cells of TK6 (wild type p53) and HMy2.CIR (mutated p53) were exposed to either low or high LET irradiation, then their mitochondrial dysfunction and ROS generation were detected. The micronuclei (MN) induction in HL-7702 hepatocytes co-cultured with irradiated lymphocytes was also measured. It was found that the mitochondrial dysfunction, p66Shc activation, and intracellular ROS were enhanced in TK6 but not in HMy2.CIR cells after γ-ray irradiation, but all of them were increased in both cell lines after carbon and iron irradiation. Consistently, the bystander effect of MN formation in HL-7702 cells was only triggered by γ-irradiated TK6 cells but not by γ-irradiated HMy2.CIR cells. But this bystander effect was induced by both lymphocyte cell lines after heavy ion irradiation. PFT-μ, an inhibitor of p53, only partly inhibited ROS generation and bystander effect induced by 30 keV/μm carbon-irradiated TK6 cells but failed to suppress the bystander effect induced by the TK6 cells irradiated with either 70 keV/μm carbon or 180 keV/μm iron. The mitochondrial inhibitors of rotenone and oligomycin eliminated heavy ion induced ROS generation in TK6 and HMy2.CIR cells and hence diminished the bystander effect on HL-7702 cells. These results clearly demonstrate that the bystander effect is p53-dependent for low LET irradiation, but it is p53-independent for high LET irradiation which may be because of p53-independent ROS generation due to mitochondrial dysfunction.

Allogeneic lymphocyte-licensed DCs expand T cells with improved antitumor activity and resistance to oxidative stress and immunosuppressive factors

Adoptive T-cell therapy of cancer is a treatment strategy where T cells are isolated, activated, in some cases engineered, and expanded ex vivo before being reinfused to the patient. The most commonly used T-cell expansion methods are either anti-CD3/CD28 antibody beads or the “rapid expansion protocol” (REP), which utilizes OKT-3, interleukin (IL)-2, and irradiated allogeneic feeder cells. However, REP-expanded or bead-expanded T cells are sensitive to the harsh tumor microenvironment and often short-lived after reinfusion. Here, we demonstrate that when irradiated and preactivated allosensitized allogeneic lymphocytes (ASALs) are used as helper cells to license OKT3-armed allogeneic mature dendritic cells (DCs), together they expand target T cells of high quality. The ASAL/DC combination yields an enriched Th1-polarizing cytokine environment (interferon (IFN)-γ, IL-12, IL-2) and optimal costimulatory signals for T-cell stimulation. When genetically engineered antitumor T cells were expanded by this coculture system, they showed better survival and cytotoxic efficacy under oxidative stress and immunosuppressive environment, as well as superior proliferative response during tumor cell killing compared to the REP protocol. Our result suggests a robust ex vivo method to expand T cells with improved quality for adoptive cancer immunotherapy.

Detection of DNA Strand Breaks in Gamma-Irradiated Lymphocytes Using Surface Plasmon Resonance

Surface plasmon resonance (SPR) is a sensitive, rapid, simple and low cost method for detection of biological molecules. In this study, SPR technology with alkaline phosphatase as a probe was utilized to measure DNA strand breaks induced by (60)Co gamma rays. The doses were from 0.01-10 Gy with a dose rate of 0.1 Gy/min. The results demonstrate that the SPR technology can be used to estimate strand breaks of calf thymus DNA. SPR signals of the calf thymus DNA samples increased with increasing gamma ray doses and the relationship of y = sqrt (3297x + 582.6) (r = 0.99) between the SPR signal and gamma dose was obtained. Estimation of DNA strand breaks in irradiated lymphocytes by SPR also demonstrated an increase in SPR signal with increasing dose and the exponential relationship of y = 169.43 × (1 - exp(-0.89x)) (r = 0.93) was obtained. The initial yield of the SPR signal is about 150.79 mdeg · Gy(-1) and compared to the sensitivity of 0.05 Gy achieved by the neutral single cell gel electrophoresis (SCGE), the SPR-based assay of DNA strand breaks was found to be more sensitive (0.02 Gy). We therefore propose that SPR technology with alkaline phosphatase as the probe is a sensitive, simple and quick method for detection of DNA strand breaks in gamma-irradiated lymphocytes.

Simultaneous analysis of p53 protein expression and cell proliferation in irradiated human lymphocytes by flow cytometry.

P53 protein has an intrinsic role in modulating the cellular response against DNA radioinduced damages and has been pointed out as an indirect indicator of individual radiosensitivity. The rate of cell proliferation is also a parameter that has been related to tissue sensitivity to radiation. However, this feature is yet understudied. In this context, the aim of this work was to employ Flow Cytometry (FC) for simultaneously assessing of p53 protein expression levels together with cellular proliferation rate of irradiated human lymphocytes. From in vitro irradiated human blood samples, mononuclear cells were isolated and labeled with Carboxylfluorescein Diacetate Succinimidyl Ester (CFSE) prior to phytohaemagglutinin (PHA) stimulation in culture for 96 hours. Cells were also labeled with anti-p53 monoclonal antibody PE-conjugated in order to analyze either proliferation rate or p53 expression levels by FC. It was verified a reduction in the proliferation rate of irradiated lymphocytes and, in parallel, a rise in the p53 expression levels, similar for quiescent and proliferating lymphocytes. The results emphasize the importance of the use of CFSE-stained lymphocytes in assays associated to proliferation rate and the use of this methodology in several studies, such as for evaluating individual radiosensitivity.

Rapid assessment of high-dose radiation exposures through scoring of cell-fusion-induced premature chromosome condensation and ring chromosomes

Analysis of premature chromosome condensation (PCC) mediated by fusion of G0-lymphocytes with mitotic CHO cells in combination with rapid visualization and quantification of rings (PCC-Rf) is proposed as an alternative technique for dose assessment of radiation-exposed individuals. Isolated lymphocytes or whole blood from six individuals were γ-irradiated with 5, 10, 15 and 20Gy at a dose rate of 0.5Gy/min. Following either 8- or 24-h post-exposure incubation of irradiated samples at 37°C, chromosome spreads were prepared by standard PCC cytogenetic procedures. The protocol for PCC fusion proved to be effective at doses as high as 20Gy, enabling the analysis of ring chromosomes and excess PCC fragments. The ring frequencies remained constant during the 8–24-h repair time; the pooled dose relationship between ring frequency (Y) and dose (D) was linear: Y=(0.088±0.005)×D. During the repair time, excess fragments decreased from 0.91 to 0.59 chromatid pieces per Gy, revealing the importance of information about the exact time of exposure for dose assessment on the basis of fragments. Compared with other cytogenetic assays to estimate radiation dose, the PCC-Rf method has the following benefits: a 48-h culture time is not required, allowing a much faster assessment of dose in comparison with conventional scoring of dicentrics and rings in assays for chemically-induced premature chromosome condensation (PCC-Rch), and it allows the analysis of heavily irradiated lymphocytes that are delayed or never reach mitosis, thus avoiding the problem of saturation at high doses. In conclusion, the use of the PCC fusion assay in conjunction with scoring of rings in G0-lymphocytes offers a suitable alternative for fast dose estimation following accidental exposure to high radiation doses.

Comparison of in vivo skin and in vitro blood lymphocyte models for the prediction of late normal tissue responses in breast radiotherapy (RT) patients.

11116 Background: Critical opinions for the lack of success of DNA double-strand break (DSB) repair as a predictive marker of normal tissue radiosensitivity include the argument that in vitro cellular responses correlate poorly with in vivo responses due to the modifying influence of tissue environment. In this study, we test the hypothesis that a DNA damage assay based on in vivo irradiated skin tissues better predicts clinical responses in human skin, as opposed to the same assay performed in ex vivo irradiated lymphocytes. Methods: DSB levels (24 h post-4 Gy) were quantified using γH2AX/53BP1 immunostaining in irradiated skin tissues and G0 lymphocytes of 35 breast RT patients. Patients were selected on the basis of late RT effects in their breast and individuals with marked or minimal effects were classified as cases and controls, respectively. Risk factors of late effects established from multivariate analyses of outcomes of two breast RT trials were also considered in patient selection. They were 1) total RT dose, 2) RT dosimetry, 3) tumour bed boost, 4) breast size, 5) surgical cavity, and 6) axillary treatment. Results: Clinical parameters were balanced in both patient groups. Residual foci levels in skin epidermis and dermis were comparable between cases (n = 20) and controls (n = 15). Mean foci per cell were 3.29 in cases, 2.80 in controls for dermal fibroblasts (p = 0.07); 3.28 in cases, 2.60 in controls for endothelial cells (p = 0.08); 2.87 in cases, 2.41 in controls for superficial keratinocytes (p = 0.45); 2.32 in cases, 2.35 in controls for basal keratinocytes (p = 0.27). Residual foci levels in lymphocytes were however significantly higher among cases (foci per cell = 12.1) compared to controls (foci per cell = 10.3, p = 0.01). Of the different cell types, only residual foci levels of dermal fibroblasts and lymphocytes correlated with clinical severity (R = 0.722, p &lt; 0.001; 0.593, p = 0.01, respectively). Interestingly, foci levels were not correlated between skin cells and lymphocytes of the same patients. Conclusions: DSB repair of ex vivo irradiated lymphocytes appears to be a better predictive marker of late effects to breast RT than DSB repair of in vivo irradiated skin.

Abstract 1792: MDM2 promoter variants and mRNA expression in irradiated lymphocytes.

Abstract Background. The ubiquitin ligase protein MDM2 plays an important role in cellular processes like growth arrest, senescence and apoptosis, and one of its main targets is the key cell cycle regulator p53. MDM2 is found overexpressed in several solid tumors, and its dysregulation is caused by gene amplification or by enhanced transcription or translation. The MDM2 gene has two main promoters; promoter P1 is constitutive while P2 is considered to be inducible in response to cellular stress. One polymorphism located in the MDM2 promoter P2 region, SNP309T&amp;gt;G (rs2279744), has been shown to enhance Sp1 transcription factor binding and is associated with increased cancer risk. Recently we reported a second polymorphism SNP285G&amp;gt;C (rs117039649) located 24 bp upstream of rs2279744. SNP285C resides on the SNP309G allele and was found to reduce Sp1 binding and lower the risk of ovarian, endometrial and breast cancer. A third SNP located in the same region, SNP344T&amp;gt;A (rs1196333) has been found not to be associated with cancer risk. In this study we examined the expression levels from the two MDM2 promoters among individuals harboring the different SNP variants (SNP309T/G, SNP285G/C and SNP344T/A) before and after irradiation. Materials and methods. Blood samples were collected from 219 healthy individuals. From this blood the buffy coat fraction was separated, divided into two and cultured. Prior to RNA extraction and subsequent cDNA synthesis, one half of the cells were irradiated with a dose equivalent to 3 Grey. Each sample was investigated for SNP285, SNP309 and SNP44 status by sequencing, and MDM2 expression was determined by qPCR using hydrolysis probes. All statistical analyses were calculated using SPSS and GraphPad Prism 5. Results. After stress (ionizing radiation) we observed, on average, a 8 and 60 fold increase in mRNA levels from promoter P1 and P2, respectively (p&amp;lt;0,0001). Statistical analysis of MDM2 mRNA expression from promoter P1 and P2, showed no significant difference between individuals harboring the different genotypes of SNP309 (TT/TG/GG), SNP285 (GG/GC/CC) or SNP344 (TT/TA/AA) before or after stress. Since the SNP285C allele counteracts the effect of SNP309G (reduces Sp1 binding), we analyzed the impact of SNP309G in the subgroup of individuals harboring SNP285 GG, but found no correlation to higher expression levels in this subgroup either. Conclusions. Despite their association with risk of several major solid tumor forms, we found no correlation between MDM2 promoter SNP285 or SNP309 with mRNA expression levels in WBC. Citation Format: Liv B. Gansmo, Stian Knappskog, Per E. Lonning. MDM2 promoter variants and mRNA expression in irradiated lymphocytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1792. doi:10.1158/1538-7445.AM2013-1792

Radioprotective effect of adenine on irradiation-induced apoptosis

AimTo investigate the radioprotective effect of adenine on irradiated lymphocytes and discover the possible mechanisms of protection. MethodsLymphocytes were pretreated for 12 h with adenine (0.001–0.1 μmol·L−1) and then exposed to 4 Gy radiation. Cell viability was observed by the MTS assay, apoptosis was detected by Annexin V-FITC/PI, DNA ladder, and caspase 3/7 activity. Caspase-9, Bax, and Bcl-2 gene expression was investigated by RT-PCR. ResultsIrradiation increased cell death and DNA fragmentation. Pretreatment with adenine significantly reversed this tendency. Furthermore, several anti-apoptotic characteristics of adenine were determined, including the ability to inhibit caspase 3/7, upregulate B-cell lymphoma (Bcl-2) and downregulate Bcl-2- associated X (Bax), capase-9 gene expression in 4 Gy-irradiated AHH-1 cells. ConclusionThe results suggest that adenine had a radioprotective effect to inhibit apoptosis in a concentration dependent manner.

Sunitinib (SU) plus extended courses of irradiated allogeneic lymphocytes (IAL) for patients with metastatic renal cell carcinoma (RCC).

464 Background: Treatment of patients with RCC using IAL from a haploidentical donor (sibling/parent), without prior conditioning of the recipient, induced tumor regressions in 3/13 patients with minimal toxicity (Strair J Clin Onc 2003:3785). Responses were not detected until after 2-3 courses, consistent with an immunologic mechanism. We hypothesize that combining the angiogenesis inhibitor SU with IAL will improve outcomes given the potential immune stimulatory benefit of VEGF inhibition and the benefit from preventing clinical progression prior to the development of an immune response in patients with rapid disease progression. Single agent SU has a median PFS of 11mos and response rate of 31%. Methods: Eligible pts with measurable metastatic clear cell RCC, no prior systemic therapy, adequate organ function, no CNS met, and a suitable donor (≥2/6 HLA A, B, DR match, CMV-, HIV-, HepB/C-) received SU 50mg/day in a 4 wk on/2wk off schedule with IAL administration commencing at the start of cycle number 2 of SU. IAL was given once every 8-16 weeks. The primary objective of the study is to determine the PFS of the combination of SU and IAL in 35 patients for 80% power to detect an increase in median PFS from 11 to 18 months. Results: A pre-planned futility analysis was performed. To date 9 pts (7M/2F), median age 63 (range 49-74) have been treated with 29 cycles of DLI. Median number of DLI/pt 2(1-7). 5 pts discontinued DLI due to disease progression, 4 due to loss of donor availability. Median time to disease progression was 41 weeks (range 12-180 weeks), with 4 pts on study for &gt;80 weeks. Response rate (CR+PR): 44% (1/9 CR, 3/9 PR, 2/9 SD, 3/9 PD). DLI was well tolerated with G2 fever in 1pt and G1 fever in 5 pts as the only DLI related AEs. SU related AE lead to SU dose reduction in 8/9 pts. Conclusions: Co-administration of SU and IAL is feasible and safe with no significant IAL related toxicity. Further study accrual is required to determine if SU plus IAL is superior to single agent SU. Clinical trial information: NCT00853125.

Low Amount Expression of Ku 70 Protein Related to Radiation-induced Chromosome Instability and Radiosensitivity in Normal Lymphocytes, and Leukemia and Lymphoma Cell Lines

Previous our experiment showed that B-lymphoid cell lines established from α ray- and γ-ray irradiated lymphocytes had higher chromosome instabilities through many passages than that from non-irradiated lymphocytes. Present protein study revealed that these B-cell lines had low amount of Ku 70 protein expression before and after second irradiation, longer localization of Ku 70 protein in nucleus, longer period of cell cycle delay at G1 or G2 phases and high activity of DNA-PK. The first αray- or γ-ray irradiation had an effect of persisting impaired DNA repair system in cells, which resulted in a decreased repair capacity after the second irradiation. Leukemia and lymphoma cell lines also showed similar characteristics after irradiation to those of the B-cell lines having high chromosome instability. These similarities are suggesting that characteristics on abnormal Ku 70 protein might persist long term and might be related to initiating leukemogenesis and lymphomagenesis as well as that such an ability of DNA repair protein might apply to prediction of radiosensitivity of leukemia or lymphoma cells.

Determination of Molecular Markers for BRCA1 and BRCA2 Heterozygosity Using Gene Expression Profiling

Approximately 5% of all breast cancers can be attributed to an inherited mutation in one of two cancer susceptibility genes, BRCA1 and BRCA2. We searched for genes that have the potential to distinguish healthy BRCA1 and BRCA2 mutation carriers from noncarriers based on differences in expression profiling. Using expression microarrays, we compared gene expression of irradiated lymphocytes from BRCA1 and BRCA2 mutation carriers versus control noncarriers. We identified 137 probe sets in BRCA1 carriers and 1,345 in BRCA2 carriers with differential gene expression. Gene Ontology analysis revealed that most of these genes relate to regulation pathways of DNA repair processes, cell-cycle regulation, and apoptosis. Real-time PCR was conducted on the 36 genes, which were most prominently differentially expressed in the microarray assay; 21 genes were shown to be significantly differentially expressed in BRCA1 and/or BRCA2 mutation carriers as compared with controls (P < 0.05). On the basis of a validation study with 40 mutation carriers and 17 noncarriers, a multiplex model that included six or more coincidental genes of 18 selected genes was constructed to predict the risk of carrying a mutation. The results using this model showed sensitivity 95% and specificity 88%. In summary, our study provides insight into the biologic effect of heterozygous mutations in BRCA1 and BRCA2 genes in response to ionizing irradiation-induced DNA damage. We also suggest a set of 18 genes that can serve as a prediction and screening tool for BRCA1 or BRCA2 mutational carriers by using easily obtained lymphocytes.