Normal Human Lymphoblastoid Cells Research Articles

Carbon ion and proton beam irradiation of a normal human TK6 lymphoblastoid cell line within a magnetic field of 1.0 tesla.

BackgroundConsidering the increasing simultaneous application of magnetic resonance imaging (MRI) for more precise photon radiotherapy, it will be likely for particle radiotherapy to adopt MRI for future image guiding. It will then be imperative to evaluate the potential biological effects of a magnetic field (MF) on particle irradiation. This study explores such effects on the highly radiosensitive TK6 lymphoblastoid human cell line.MethodsThe following three parameters were measured after irradiation with either carbon ion or proton beams using spread out Bragg peaks and applying different doses within a perpendicular 1.0 T MF: (1) cell survival fraction (14 days postirradiation), (2) treatment-specific apoptosis, which was determined through the measurement of population in the sub-G1 phase, and (3) cell cycle progression by means of flow cytometry. These were compared to the same parameters measured without an MF.ResultsThe clonogenic assay in both treatment groups showed almost identical survival curves with overlapping error bars. The calculated α values with and without an MF were 2.18 (σ=0.245) and 2.17 (σ=0.234) for carbon ions and 1.08 (σ=0.138) and 1.13 (σ=0.0679) for protons, respectively. Similarly, the treatment-specific apoptosis and cell cycle progression showed almost identical curves with overlapping error bars. A two-sample, unpooled t-test analysis was implemented for comparison of all mean values and showed p-values >0.05.ConclusionNo statistically significant difference in biological response of the TK6 cells was observed when they were irradiated using spreadout Bragg peaks within a perpendicular 1.0 T MF as compared to those, which received the same dose without the MF. This should serve as another supporting piece of evidence toward the implementation of MRI in particle radiotherapy, though further research is necessary.

The influence of a magnetic field on photon beam radiotherapy in a normal human TK6 lymphoblastoid cell line

BackgroundThe implementation of magnetic resonance imaging (MRI) guided radiotherapy (RT) continues to increase. Very limited in-vitro data on the interaction of ionizing radiation and magnetic fields (MF) have been published. In these experiments we focused on the radiation response in a MF of the TK6 human lymphoblastoid cells which are known to be highly radiosensitive due to efficient radiation-induced apoptosis.MethodsClonogenicity was determined 12–14 days after irradiation with 1–4 Gy 6 MV photons with or without a 1.0 Tesla MF. Furthermore, alterations in cell cycle distribution and rates of radiation induced apoptosis (FACS analysis of cells with sub-G1 DNA content) were analyzed.ResultsClonogenic survival showed an exponential dose-dependence, and the radiation sensitivity parameter (α = 1.57/Gy) was in accordance with earlier reports. Upon comparing the clonogenic survival between the two groups, identical results within error bars were obtained. The survival fractions at 2 Gy were 9% (without MF) and 8.5% (with MF), respectively.ConclusionA 1.0 Tesla MF does not affect the clonogenicity of TK6 cells irradiated with 1–4 Gy 6MV photons. This supports the use of MRI guided RT, however ongoing research on the interaction of MF and radiotherapy is warranted.

Effects of low oxygen levels on G2-specific cytogenetic low-dose hyper-radiosensitivity in irradiated human cells.

Low-dose hyper-radiosensitivity (HRS) has been reported in normal human lymphoblastoid cell lines for exposures at ≤ 20 cGy, but the cytogenetic effects of oxygen (O2 ) levels in tissue culture medium on HRS have not been evaluated. We asked whether HRS was lost in G2-irradiated cells grown in atmospheres of 2.5% or 5% O2 , compared to responses by cells cultured in ambient O2 (21%). The results indicate a loss of HRS when cells are cultured and irradiated either in 2.5% or 5% O2 . We then evaluated whether low O2 levels either before or after exposure were responsible for the loss of HRS. For cells irradiated in 5% O2 , subsequent immediate re-oxygenation to ambient O2 levels restored the HRS effect, while cells cultured and irradiated at ambient O2 levels and then transferred to 5% O2 exhibited little or no HRS, indicating that ambient O2 levels after, but not before, radiation substantially affect the amounts of cytogenetic damage. HRS was not observed when cells were irradiated in G1. At doses of 40-400 cGy there was significantly less cytogenetic damage when cells were recovering from radiation at low O2 levels than at ambient O2 levels. Here we provide the first cytogenetic evidence for the loss of HRS at low O2 levels in G2-irradiated cells; these results suggest that at low O2 levels for all doses evaluated there is either less damage to DNA, perhaps because of lower amounts of reactive oxygen species, or that DNA damage repair pathways are activated more efficiently.

Cytogenetic characterization of low-dose hyper-radiosensitivity in Cobalt-60 irradiated human lymphoblastoid cells

The dose-effect relationships of cells exposed to ionizing radiation are frequently described by linear quadratic (LQ) models over an extended dose range. However, many mammalian cell lines, when acutely irradiated in G2 at doses ≤0.3Gy, show hyper-radiosensitivity (HRS) as measured by reduced clonogenic cell survival, thereby indicating greater cell lethality than is predicted by extrapolation from high-dose responses. We therefore hypothesized that the cytogenetic response in G2 cells to low doses would also be steeper than predicted by LQ extrapolation from high doses. We tested our hypothesis by exposing four normal human lymphoblastoid cell lines to 0–400cGy of Cobalt-60 gamma radiation. The cytokinesis block micronucleus assay was used to determine the frequencies of micronuclei and nucleoplasmic bridges. To characterize the dependence of the cytogenetic damage on dose, univariate and multivariate regression analyses were used to compare the responses in the low- (HRS) and high-dose response regions. Our data indicate that the slope of the response for all four cell lines at ≤20cGy during G2 is greater than predicted by an LQ extrapolation from the high-dose responses for both micronuclei and bridges. These results suggest that the biological consequences of low-dose exposures could be underestimated and may not provide accurate risk assessments following such exposures.

Neutron exposures in human cells: bystander effect and relative biological effectiveness.

Bystander effects have been observed repeatedly in mammalian cells following photon and alpha particle irradiation. However, few studies have been performed to investigate bystander effects arising from neutron irradiation. Here we asked whether neutrons also induce a bystander effect in two normal human lymphoblastoid cell lines. These cells were exposed to fast neutrons produced by targeting a near-monoenergetic 50.5 MeV proton beam at a Be target (17 MeV average neutron energy), and irradiated-cell conditioned media (ICCM) was transferred to unirradiated cells. The cytokinesis-block micronucleus assay was used to quantify genetic damage in radiation-naïve cells exposed to ICCM from cultures that received 0 (control), 0.5, 1, 1.5, 2, 3 or 4 Gy neutrons. Cells grown in ICCM from irradiated cells showed no significant increase in the frequencies of micronuclei or nucleoplasmic bridges compared to cells grown in ICCM from sham irradiated cells for either cell line. However, the neutron beam has a photon dose-contamination of 5%, which may modulate a neutron-induced bystander effect. To determine whether these low doses of contaminating photons can induce a bystander effect, cells were irradiated with cobalt-60 at doses equivalent to the percent contamination for each neutron dose. No significant increase in the frequencies of micronuclei or bridges was observed at these doses of photons for either cell line when cultured in ICCM. As expected, high doses of photons induced a clear bystander effect in both cell lines for micronuclei and bridges (p<0.0001). These data indicate that neutrons do not induce a bystander effect in these cells. Finally, neutrons had a relative biological effectiveness of 2.0±0.13 for micronuclei and 5.8±2.9 for bridges compared to cobalt-60. These results may be relevant to radiation therapy with fast neutrons and for regulatory agencies setting standards for neutron radiation protection and safety.

Effect of 0.1 Gy X-ray irradiation on gene expression profiles in normal human lymphoblastoid cells

To investigate the effect of 0.1 Gy X-ray irradiation on the gene expression profiles in normal human lymphoblastoid cells using gene microarray and to explore the possible mechanism of the biological effect of low-dose irradiation. The NimbleGen 12×135 K microarray corresponding to 45033 genes was used to analyze the gene expression profiles in AHH-1 cells cultured for 6 h and 20 h after 0.1 Gy X-ray irradiation. A gene was identified as the differentially expressed gene if the ratio between its expression levels in irradiation group and control group was higher than 2 or lower than 0.5. RT-PCR and real-time PCR were used to confirm some differentially expressed genes. There were 760 up-regulated genes and 1222 down-regulated genes in the cells at 6 h after 0.1 Gy X-ray irradiation, while there were 463 up-regulated genes and 753 down-regulated genes at 20 h after 0.1 Gy X-ray irradiation; there were 92 differentially expressed genes in common. The expression of GADD45A, CDKN2A, and Cx43 measured using gene microarray was confirmed by RT-PCR and real-time PCR. Low-dose irradiation can affect the expression of many functional genes, which provides a basis for the research on the mechanism of radiation damage.

Bioenergetic and Antiapoptotic Properties of Mitochondria from Cultured Human Prostate Cancer Cell Lines PC-3, DU145 and LNCaP

The purpose of this work was to reveal the metabolic features of mitochondria that might be essential for inhibition of apoptotic potential in prostate cancer cells. We studied mitochondria isolated from normal prostate epithelial cells (PrEC), metastatic prostate cancer cell lines LNCaP, PC-3, DU145; and non-prostate cancer cells - human fibrosarcoma HT1080 cells; and normal human lymphoblastoid cells. PrEC cells contained 2 to 4 times less mitochondria per gram of cells than the three PC cell lines. Respiratory activities of PrEC cell mitochondria were 5-20-fold lower than PC mitochondria, depending on substrates and the metabolic state, due to lower content and lower activity of the respiratory enzyme complexes. Mitochondria from the three metastatic prostate cancer cell lines revealed several features that are distinctive only to these cells: low affinity of Complex I for NADH, 20-30 mV higher electrical membrane potential (ΔΨ). Unprotected with cyclosporine A (CsA) the PC-3 mitochondria required 4 times more Ca2+ to open the permeability transition pore (mPTP) when compared with the PrEC mitochondria, and they did not undergo swelling even in the presence of alamethicin, a large pore forming antibiotic. In the presence of CsA, the PC-3 mitochondria did not open spontaneously the mPTP. We conclude that the low apoptotic potential of the metastatic PC cells may arise from inhibition of the Ca2+-dependent permeability transition due to a very high ΔΨ and higher capacity to sequester Ca2+. We suggest that due to the high ΔΨ, mitochondrial metabolism of the metastatic prostate cancer cells is predominantly based on utilization of glutamate and glutamine, which may promote development of cachexia.

Relationships among micronuclei, nucleoplasmic bridges and nuclear buds within individual cells in the cytokinesis-block micronucleus assay

Micronuclei have been used extensively in studies as an easily evaluated indicator of DNA damage but little is known about their association with other types of damage such as nucleoplasmic bridges and nuclear buds. Here, radiation-induced clastogenic events were evaluated via the cytokinesis-block micronucleus assay in two normal human lymphoblastoid cell lines exposed to neutrons or γ-radiation. DNA damage induced by the chemical agents mitomycin C and phleomycin was also evaluated in two normal and two mitochondrial mutant human lymphoblastoid cell lines. In addition to micronuclei, nucleoplasmic bridges and nuclear buds were enumerated by recording the coincident presence of these end points within individual cells, and the associations among these three end points were evaluated for all treatment conditions. The common odds ratios for micronuclei and nucleoplasmic bridges were found to be significantly larger than unity, indicating that the presence of one or more micronuclei in a cell imposes a significant risk of having one or more nucleoplasmic bridges in that same cell, and vice versa. The strength of this association did not change significantly with radiation dose or concentration of the chemical clastogens. Common odds ratios for association between micronuclei and buds, and between bridges and buds were also found to be significantly higher than unity. However, associations between micronuclei and buds could not be calculated for some treatments due to heterogeneity in the odds ratios and hence may depend on chemical clastogen concentration or radiation dose. This study provides evidence of how paired analyses among genetic end points in the cytokinesis-block micronucleus assay can provide information concerning abnormalities of cell division and possibly about structural chromosomal rearrangements induced by clastogens.

Abstract 2121: Nonerythroid alpha spectrin prevents telomere fragility after DNA interstrand crosslink damage

Abstract Telomere dysfunction and increased telomere fragility are important contributing factors to chromosome instability. We have previously demonstrated that the structural protein, nonerythroid -spectrin(IISp), is critical for maintenance of chromosome stability after DNA interstrand crosslink (ICL) damage. However, whether IISp plays an important role in maintaining telomere stability after DNA damage is an extremely important question and the goal of the present study. IISp in normal human lymphoblastoid cells was depleted by siRNA and cells treated with an ICL agent, mitomycin C (MMC). Cells were analyzed using immunofluorescence and in situ hybridization (IF-FISH) and probed with anti–spectrin and a telomeric Cy3-conjugated-PNA probe or antibodies against two telomeric proteins, TRF1 or TRF2. Metaphase spreads of these cells were also examined and stained with the PNA probe and scored for telomeric abnormalities. In cells damaged with MMC but not in undamaged cells, a portion of IISp (50%) was found to co-localize with the PNA probe in the telomeres along with TRF1 and TRF2, indicating that IISp localizes to telomeres after ICL damage. To assess whether there was increased telomere fragility after ICL damage, chromosome ends were examined for loss of telomeric signal. After treatment with MMC, 28% of the chromatid ends showed a loss of telomeric signal compared to 2% in nondamaged cells. However, after depletion of IISp, 74% of the chromatid ends in MMC damaged cells displayed loss of telomeric signal compared to 18% in the nondamaged cells. This indicates that there was increased telomere fragility in MMC treated cells after knockdown of IISp. In addition to an increase in fragile telomeres in these cells, the chromosomes in the IISp siRNA treated cells damaged with MMC showed a significant increase in sister chromatid end fusions compared to the undamaged cells, which is indicative of aberrant telomere structure.These results on telomere fragility after loss of IISp are similar to those obtained using cells from the cancer prone genetic disease, Fanconi anemia complementation group A (FA-A), in which we have shown that there is a deficiency in IISp levels which correlates with a defect in ability to repair DNA ICLs. Examination of FA-A lymphoblastoid cells showed that there is an increase in fragile telomeres after MMC treatment: 76% of the chromatid ends showed loss of telomeric signal after MMC damage compared to 24% in undamaged cells. When levels of IISp were restored to normal in the FA-A cells by knocking down µ-calpain, a protease that cleaves IISp, the percentage of chromatid ends showing loss of telomeric signal (26%) after MMC was similar to that in normal cells. These studies collectively show that IISp plays an important and central role in maintenance of telomere stability after DNA ICL damage and suggests that it is an important factor in telomere function and chromosome stability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2121. doi:1538-7445.AM2012-2121

Oligonucleotide immobilization using 10-(carbomethoxy)decyl-dimethylchlorosilane for mRNA isolation and cDNA synthesis on a microfluidic chip

Fabrication of microfluidic systems capable of extracting and isolating nucleic acids from biological samples and preparing them for downstream applications within the same device is of interest as on-chip sample preparation reduces the time and effort expended on multi-step benchtop procedures. A microfluidic chip capable of cell lysis, nucleic acid extraction and immobilization has been developed in our laboratory. This report focuses on substrate development and chip integration of the nucleic acid immobilization platform. The immobilization region was initially developed using poly-adenylated mRNA released from normal human lymphoblastoid cells. After immobilization, the mRNA remains functional for amplification by the reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) gene expression analyses. Detailed surface functionalization, chemical modification, and covalent oligonucleotide immobilization methods are described. Vapor and liquid deposition techniques for Pyrex functionalization by 10-(carbomethoxy)decyl-dimethylchlorosilane (CMDCS) were developed and an optimal method was determined for microfluidic chip application. CMDCS substrate depositions and modification were characterized by atomic force microscopy and contact angle goniometry, respectively. Oligonucleotide immobilizations and mRNA bindings were determined by fluorescence microscopy. Functional, selective platform integration within the microfluidic chip was confirmed by standard RT-PCR and qPCR systems.

Immunostaining of modified histones defines high-level features of the human metaphase epigenome

BackgroundImmunolabeling of metaphase chromosome spreads can map components of the human epigenome at the single cell level. Previously, there has been no systematic attempt to explore the potential of this approach for epigenomic mapping and thereby to complement approaches based on chromatin immunoprecipitation (ChIP) and sequencing technologies.ResultsBy immunostaining and immunofluorescence microscopy, we have defined the distribution of selected histone modifications across metaphase chromosomes from normal human lymphoblastoid cells and constructed immunostained karyotypes. Histone modifications H3K9ac, H3K27ac and H3K4me3 are all located in the same set of sharply defined immunofluorescent bands, corresponding to 10- to 50-Mb genomic segments. Primary fibroblasts gave broadly the same banding pattern. Bands co-localize with regions relatively rich in genes and CpG islands. Staining intensity usually correlates with gene/CpG island content, but occasional exceptions suggest that other factors, such as transcription or SINE density, also contribute. H3K27me3, a mark associated with gene silencing, defines a set of bands that only occasionally overlap with gene-rich regions. Comparison of metaphase bands with histone modification levels across the interphase genome (ENCODE, ChIP-seq) shows a close correspondence for H3K4me3 and H3K27ac, but major differences for H3K27me3.ConclusionsAt metaphase the human genome is packaged as chromatin in which combinations of histone modifications distinguish distinct regions along the euchromatic chromosome arms. These regions reflect the high-level interphase distributions of some histone modifications, and may be involved in heritability of epigenetic states, but we also find evidence for extensive remodeling of the epigenome at mitosis.

Identifying regulatory relationships among genomic loci, biological pathways, and disease

Elucidating genetic factors of complex diseases is one of the most important challenges in biomedical research. Recently, a genetical genomics approach of mapping genotype to transcripts has been used in complex disease analysis. This approach treats messenger ribonucleic acid (mRNA) expression as a quantitative trait and identifies putative regulatory loci for the expression of an individual gene. However, the single-gene approach could not detect single nucleotide polymorphisms (SNP's) associated with the concerted activity of multiple genes. Since complex diseases result from the interactions of multiple genes, it is important to consider associations between genotype and multiple gene expression. We developed the differential allelic co-expression (DACE) that identifies regulatory loci that affect the inter-correlation structure of multiple genes or a gene set. We applied DACE to two benchmark datasets: the normal human lymphoblastoid cell dataset and the glioblastoma dataset. These datasets consist of both SNPs and mRNA expression profiles for each sample. When analyzing the lymphoblastoid cell dataset, principal component analysis (PCA) was compared with the DACE test. While PCA identified associations found by single-gene analysis, the DACE test detected associations not identified by the single-gene approach. Using the DACE test, seven putative regulatory loci of immune-related pathways were identified in lymphoblastoid cells after controlling for family-wise error rate. In the glioblastoma dataset, DACE identified 4582 SNPs associated with six pathways. In 231 of the 4582 SNPs, patient survival length was correlated significantly with the SNP genotype. This finding suggests that our integrative approach may provide a biological explanation for the putative relationship between sequence level variation and disease outcome, via expression of a functional pathway. The DACE test shows promise for finding regulatory relationships between a genomic locus and sets of genes which may be related to disease outcome.

Mitochondrial Gene Expression Changes in Normal and Mitochondrial Mutant Cells after Exposure to Ionizing Radiation

Mitochondrial DNA (mtDNA) contains 13 genes that encode proteins of the oxidative phosphorylation complex that are involved in ATP generation. Leber's optic atrophy and Leigh's syndrome are diseases that are caused by point mutations in the mitochondrial genome and that have phenotypes associated with energy deprivation. We hypothesized that energy deficiency from mitochondrial mutations in these cells leads to radiation hypersensitivity. Here we compared mitochondrial gene expression for the 13 mitochondrial protein-coding genes in two mitochondrial mutant cell lines, GM13740 (Leigh's syndrome) and GM10744 (Leber's optic atrophy) and a normal human lymphoblastoid cell line (GM15036) after X irradiation (0-4 Gy) 0 to 24 h postirradiation. Changes in gene expression were compared with cellular radiosensitivity. Statistically significant differences between Leigh's syndrome and normal cells were found in mitochondrial gene expression for all radiation doses and times that were commensurate with changes in radiation sensitivity. The data suggest that Leigh's syndrome cells have an impaired ability to repair radiation-induced DNA damage that results in radiation hypersensitivity. This may be attributable to mitochondrial dysfunction from reductions in mitochondrial gene expression and ATP generation, since Leigh's optic atrophy cells exhibit a mutation in the ATPase6 gene, which is an important component of Complex V of ATP synthase. In contrast, the mutation of the Leber's cells conferred radioresistance, which might be attributed to the mutation in the ND4 gene in the mitochondrial genome. The altered sensitivity of mitochondrial mutant cells to ionizing radiation can lead to decreased DNA repair, which may put individuals with mtDNA mutations at greater risk for cancer and other diseases.

Bystander effects induced by chemicals and ionizing radiation: evaluation of changes in gene expression of downstream MAPK targets

Radiation-induced bystander effects have been evaluated extensively, including the involvement of the mitogen-activated protein kinase (MAPK) pathways. However, few studies have examined the ability of chemicals to induce bystander effects, and the molecular mechanisms involved in chemical bystander effects have not been investigated. We have previously demonstrated the ability of mitomycin C (MMC) and phleomycin (PHL) to induce bystander effects in normal human lymphoblastoid cells. Here, we demonstrate changes in the expression of MAPK target genes following bystander exposure to MMC or PHL or ionizing radiation. The expression changes of 18 genes, which code for proteins that are downstream targets of MAPK proteins, were evaluated at various time points following direct or bystander exposure to MMC, PHL and ionizing radiation. The 18 genes were analysed as groups belonging to one of the seven possible combinations of the three MAPK pathways. We observed statistically significant changes in expression of several genes following exposure to each agent. However, when the expression changes were analysed in the bystander cells alone, significant increases in expression of MAPK target genes were observed for MMC- and radiation-induced bystander effects but not for PHL. PHL is an acknowledged radiomimetic agent; however, in the present study, PHL responses did not resemble those of radiation. These results provide evidence for bystander-induced changes in MAPK proteins and downstream targets and suggest that the bystander effects are a part of a general stress response.

Involvement of MAPK proteins in bystander effects induced by chemicals and ionizing radiation

Many studies have examined bystander effects induced by ionizing radiation, however few have evaluated the ability of chemicals to induce similar effects. We previously reported the ability of two chemicals, mitomycin C (MMC) and phleomycin (PHL) to induce bystander effects in normal human lymphoblastoid cell lines. The focus of the current study was to determine the involvement of the MAPK proteins in bystander effects induced by physical and chemical DNA damaging agents and to evaluate the effects of MAPK inhibition on bystander-induced caspase 3/7 activation. The phosphorylation levels of the MAPK proteins ERK1/2, JNK, and p38, were measured from 1 to 24h following direct or bystander exposure to MMC, PHL or radiation. We observed transient phosphorylation, at early time points, of all 3 proteins in bystander cells. We also evaluated the effect of MAPK inhibition on bystander-induced caspase 3/7 activity to determine the role of MAPK proteins in bystander-induced apoptosis. We observed bystander-induced activation of caspase 3/7 in bystander cells. Inhibition of MAPK proteins resulted in a decrease in caspase 3/7 activity at the early time points, and the caspase activity increased (in the case of ERK inhibition) or returned to basal levels (in the case of JNK or p38 inhibition) between 12 and 24h. PHL is considered to be a radiomimetic agent, however in the present study PHL behaved more like a chemical and not like radiation in terms of MAPK phosphorylation. These results point to the involvement of MAPK proteins in the bystander effect induced by radiation and chemicals and provide additional evidence that this response is not limited to radiation but is a generalized stress response in cells.

Chemical induction of the bystander effect in normal human lymphoblastoid cells

Many studies investigating the bystander effect have used ionizing radiation to evaluate this phenomenon, whereas very few have determined whether genotoxic chemicals are also capable of inducing this effect. Here, we show that two such chemicals, mitomycin C, a bifunctional alkylating agent and phleomycin, a glycopeptide antibiotic of the bleomycin family, cause normal human B lymphoblastoid cells to produce media soluble factors that induce a bystander effect in unexposed cells. Ionizing radiation was used in parallel experiments to verify the existence of the bystander effect in these cells. Micronuclei in Cytochalasin B-blocked binucleated cells were used as the endpoint. Conditioned media obtained from cells exposed to mitomycin C induced a 1.5–3 fold increase, while conditioned media from phleomycin induced a 1.5–4 fold increase, and conditioned media from irradiated cells induced a 2–8 fold increase in micronuclei. We conclude that the bystander effect is not restricted to ionizing radiation, suggesting it may be a part of a general cellular stress response.

Mitochondrial mutant cells are hypersensitive to ionizing radiation, phleomycin and mitomycin C

Mitochondrial DNA (mtDNA) is an important contributor to the ATP-generating oxidative phosphorylation complex. Single nucleotide mutations in mitochondrial genes involved in ATP synthesis result in a broad range of diseases. Leber optic atrophy and Leigh's syndrome are two such diseases arising from point mutations in the mitochondrial genome. Here, ionizing radiation, phleomycin and mitomycin C (MMC) were used to induce structural chromosomal aberrations in Leber's and Leigh's cells to investigate how these mitochondrial mutations affect the cell's DNA repair processes. Because of the energy deprivation that results from mitochondrial mutations, we hypothesized that these mutant cells would demonstrate hypersensitivity when exposed to oxidative and genotoxic stress and we also expected that these cells would not be able to repair nuclear DNA damage as efficiently as normal cells. As a consequence, these mutant cells are expected to show increased levels of DNA damage, longer cell cycle delays and increased levels of cell death. Following acute radiation exposure these mutant cells showed an increase in the number of chromosomal aberrations and decreased mitotic indices when compared with normal human lymphoblastoid cells with wild-type mtDNA. When exposed to phleomycin or MMC, the mitochondrial mutant cells again showed hypersensitivity and decreased mitotic indices compared to normal cells. These results suggest that Leber's and Leigh's cells have an impaired ability to cope with oxidative and genotoxic stress. These observations may help explain the role of ATP generation in understanding the enhanced sensitivity of mitochondrial mutant cells to cancer therapeutic agents and to adverse environmental exposure, suggesting that individuals with mtDNA mutations may be at a greater risk for cancer and other diseases that result from an accumulation of nuclear DNA damage.

Evaluation and validation of housekeeping genes in response to ionizing radiation and chemical exposure for normalizing RNA expression in real-time PCR

Gene expression changes are used with increasing frequency to assess the effects of exposure to environmental agents. Housekeeping (Hk) genes are essential in these analyses as internal controls for normalizing expression levels evaluated with Real-Time PCR (RT-PCR). Ideal Hk genes are constitutively expressed, do not respond to external stimuli and exhibit little or no sample-to-sample or run-to-run variation. Previous studies indicate that some commonly used Hk genes including glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin have differential expression in various cell lines. Here we examine the expression of 11 Hk genes in four normal human lymphoblastoid cell lines and one T-cell leukemia (Jurkat) cell line following exposure to graded doses of ionizing radiation or to varying ratio concentrations of phytohemagglutinin (PHA) and phorbol myristate acetate (PMA). PHA and PMA are known to have synergistic effects on the expression of some genes and have very different effects from those of radiation. There has been no systematic study performed to ascertain the best control genes for radiation and/or PHA/PMA exposures in lymphoblastoid cells. Using a two-step reverse-transcriptase RT-PCR protocol we show that following radiation doses ranging from 0 to 400 cGy, 18S rRNA, acidic ribosomal protein, beta-actin, cyclophilin, GAPDH, phosphoglycerokinase, beta-2 microglobulin (B2M), beta-glucuronidase, hypoxanthine phosphoribosyltransferase and transferrin receptor showed no significant variation in expression in normal lymphoblastoid cells. In contrast, only 18S rRNA levels were unchanged in Jurkat cells. After PHA/PMA treatment of the same normal cell lines, B2M showed no significant variation and 18S rRNA, GAPDH and transcription binding protein (TBP) were minimally responsive, whereas in Jurkat cells all these genes were unresponsive. While our results suggest that the utility of a particular Hk gene should be determined for each experimental condition, 18S rRNA and B2M appear to be excellent candidates for use as internal controls in RT-PCR in human lymphoblastoid cells because they have the most constant levels of expression across cell lines following exposure to ionizing radiation as well as to PHA/PMA.

828. Gene Transfer of Superoxide-Dismutase Genes and Catalase Confers Radioprotection on Normal Human Primary Lung Fibroblasts and Lymphoblastoid Cells

828. Gene Transfer of Superoxide-Dismutase Genes and Catalase Confers Radioprotection on Normal Human Primary Lung Fibroblasts and Lymphoblastoid Cells

Role of Apoptosis in Low-Dose Hyper-radiosensitivity

Little is known about the mode of cell killing associated with low-dose hyper-radiosensitivity, the radiation response that describes the enhanced sensitivity of cells to small doses of ionizing radiation. Using a technique that measures the activation of caspase 3, we have established a relationship between apoptosis detected 24 h after low-dose radiation exposure and low-dose hyper-radiosensitivity in four mammalian cell lines (T98G, U373, MR4 and 3.7 cells) and two normal human lymphoblastoid cell lines. The existence of low-dose hyper-radiosensitivity in clonogenic survival experiments was found to be associated with an elevated level of apoptosis after low-dose exposures, corroborating earlier observations (Enns et al., Mol. Cancer Res. 2, 557-566, 2004). We also show that enriching populations of MR4 and V79 cells with G(1)-phase cells, to minimize the numbers of G(2)-phase cells, abolished the enhanced low-dose apoptosis. These cell-cycle enrichment experiments strengthen the reported association between low-dose hyper-sensitivity and the radioresponse of G(2)-phase cells. These data are consistent with our current hypothesis to explain low-dose hyper-radiosensitivity, namely that the enhanced sensitivity of cells to low doses of ionizing radiation reflects the failure of ATM-dependent repair processes to fully arrest the progression of damaged G(2)-phase cells harboring unrepaired DNA breaks entering mitosis.