Bystander effects induced by diffusing mediators after photodynamic stress.

The bystander effect, whereby cells that are not traversed by ionizing radiation exhibit various responses when in proximity to irradiated cells, is well documented in the field of radiation biology, Here we demonstrate that considerable bystander responses are also observed after photodynamic stress using the membrane-localizing dye deuteroporphyrin (DP). Using cells of a WTK1 human lymphoblastoid cell line in suspension and a transwell insert system that precludes contact between targeted and bystander cells, we have shown that the bystander signaling is mediated by diffusing species. The extranuclear localization of the photosensitizer used suggests that primary DNA damage is not the trigger for initiating these bystander responses, which include elevated oxidative stress, DNA damage (micronucleus formation), mutagenesis and decreased clonogenic survival. In addition, oxidative stress in the bystander population was reduced by the presence of the membrane antioxidant vitamin E in the targeted cells, suggesting that lipid peroxidation may play a key role in mediating these bystander effects. The fluence responses for these bystander effects are non-linear, with larger effects seen at lower fluences and toxicity to the target cell population. Hence, when considering outcomes of photodynamic action in cells and tissue, bystander effects may be significant, especially at sublethal fluences.

Ionizing radiation-induced bystander mutagenesis and adaptation: quantitative and temporal aspects.

This work explores several quantitative aspects of radiation-induced bystander mutagenesis in WTK1 human lymphoblast cells. Gamma-irradiation of cells was used to generate conditioned medium containing bystander signals, and that medium was transferred onto naïve recipient cells. Kinetic studies revealed that it required up to 1h to generate sufficient signal to induce the maximal level of mutations at the thymidine kinase locus in the bystander cells receiving the conditioned medium. Furthermore, it required at least 1h of exposure to the signal in the bystander cells to induce mutations. Bystander signal was fairly stable in the medium, requiring 12-24h to diminish. Medium that contained bystander signal was rendered ineffective by a 4-fold dilution; in contrast a greater than 20-fold decrease in the cell number irradiated to generate a bystander signal was needed to eliminate bystander-induced mutagenesis. This suggested some sort of feedback inhibition by bystander signal that prevented the signaling cells from releasing more signal. Finally, an ionizing radiation-induced adaptive response was shown to be effective in reducing bystander mutagenesis; in addition, low levels of exposure to bystander signal in the transferred medium induced adaptation that was effective in reducing mutations induced by subsequent gamma-ray exposures.

Deficiencies of double-strand break repair factors and effects on mutagenesis in directly gamma-irradiated and medium-mediated bystander human lymphoblastoid cells.

Using RNA interference techniques to knock down key proteins in two major double-strand break (DSB) repair pathways (DNA-PKcs for nonhomologous end joining, NHEJ, and Rad54 for homologous recombination, HR), we investigated the influence of DSB repair factors on radiation mutagenesis at the autosomal thymidine kinase (TK) locus both in directly irradiated cells and in unirradiated bystander cells. We also examined the role of p53 (TP53) in these processes by using cells of three human lymphoblastoid cell lines from the same donor but with differing p53 status (TK6 is p53 wild-type, NH32 is p53 null, and WTK1 is p53 mutant). Our results indicated that p53 status did not affect either the production of radiation bystander mutagenic signals or the response to these signals. In directly irradiated cells, knockdown of DNA-PKcs led to an increased mutant fraction in WTK1 cells and decreased mutant fractions in TK6 and NH32 cells. In contrast, knockdown of DNA-PKcs led to increased mutagenesis in bystander cells regardless of p53 status. In directly irradiated cells, knockdown of Rad54 led to increased induced mutant fractions in WTK1 and NH32 cells, but the knockdown did not affect mutagenesis in p53 wild-type TK6 cells. In all cell lines, Rad54 knockdown had no effect on the magnitude of bystander mutagenesis. Studies with extracellular catalase confirmed the involvement of H2O2 in bystander signaling. Our results demonstrate that DSB repair factors have different roles in mediating mutagenesis in irradiated and bystander cells.

Partial deficiency of DNA-PKcs increases ionizing radiation-induced mutagenesis and telomere instability in human cells.

The correct repair of DNA double-strand breaks (DSBs) is essential to maintaining the integrity of the genome. Misrepair of DSBs is detrimental to cells and organisms, leading to gene mutation, chromosomal aberration, and cancer development. Nonhomologous end-joining (NHEJ) is one of the principal rejoining processes in most higher eukaryotic cells. NHEJ is facilitated by DNA-dependent protein kinase (DNA-PK), which is composed of a catalytic subunit, DNA-PKcs, and the heterodimeric DNA binding regulatory complex Ku70/86. Null mutation of DNA-PKcs leads to immunodeficiency, chromosomal aberration, gene mutation, telomeric end-capping failure, and cancer predisposition in animals and cells. However, it is unknown whether partial deficiency of DNA-PKcs as might occur in a fraction of the population (e.g., heterozygotes), influences cellular function. Using small interfering RNA (siRNA) transfection, we established partial deficiency of DNA-PKcs in human cells, ranging from 4 to 85% of control levels. Our results reveal for the first time, that partial deficiency of DNA-PKcs leads to increased ionizing radiation (IR)-induced mutagenesis, cell killing, and telomere dysfunction. Radiation mutagenesis was increased inversely with DNA-PKcs protein level, with the most pronounced effect being observed in cells with protein levels below 50% of controls. A small but statistically significant increase in IR-induced cell killing was observed as DNA-PKcs levels decreased, over the entire range of protein levels. Frequencies of IR-induced telomere-DSB fusion was increased at levels of DNA-PKcs as low as approximately 50%, similar to what would be expected in heterozygous individuals. Taken together, our results suggest that even partial deficiency of DNA repair proteins may represent a considerable risk to genomic stability.

Induction and loss of a TP53-dependent radioadaptive response in the human lymphoblastoid cell model TK6 and its abrogation by BCL2 over-expression.

PURPOSE: To characterize the radioadaptive response in the human lymphoblastoid cell model TK6, and determine: (i) Whether repeated low dose exposures are more effective than single acute exposures in inducing resistance, (ii) the time-course for induction and loss of resistance following chronic exposures, and (iii) the effect of TP53 deletion or BCL2 over-expression on the induction of an adaptive response. MATERIALS AND METHODS: TK6, a human B-lymphoblastoid cell line, TK6-BCL2, a TK6 line that over-expresses BCL2 and is resistant to radiation-induced apoptosis, and NH32, a TP53 knockout of TK6 that is also resistant to apoptosis were studied. Cells were exposed to chronic, daily doses of 10 cGy given over 1 -21 days before being challenged with 1 -5 Gy exposures. Cell survival and chromatid break induction following high dose challenge were used to evaluate adaptive radiation responses. RESULTS: Exposure to 10 cGy gamma rays induced resistance to killing and chromosome break induction in TK6 cells, but not in either TK6-BCL2 or NH32 cells. Resistance in TK6 was observed 4 h after exposure, and cells remained resistant for about 48 h. Maximal resistance was induced by a single 10 cGy dose. Repeated 10 cGy exposures had no additional effect on radiation sensitivity, except to maintain the induced radioresistance. CONCLUSION: An adaptive response is maximally and rapidly induced by a single low dose exposure in TK6 cells, and it has a limited lifespan. Induction of an adaptive response in TK6 cells can be abrogated by either TP53 loss or BCL2 over-expression. The characteristics of induced resistance in TK6 cells suggest that alterations in TP53-dependent apoptotic responses may be one mechanism for resistance.

NBS1 knockdown by small interfering RNA increases ionizing radiation mutagenesis and telomere association in human cells.

Hypomorphic mutations which lead to decreased function of the NBS1 gene are responsible for Nijmegen breakage syndrome, a rare autosomal recessive hereditary disorder that imparts an increased predisposition to development of malignancy. The NBS1 protein is a component of the MRE11/RAD50/NBS1 complex that plays a critical role in cellular responses to DNA damage and the maintenance of chromosomal integrity. Using small interfering RNA transfection, we have knocked down NBS1 protein levels and analyzed relevant phenotypes in two closely related human lymphoblastoid cell lines with different p53 status, namely wild-type TK6 and mutated WTK1. Both TK6 and WTK1 cells showed an increased level of ionizing radiation-induced mutation at the TK and HPRT loci, impaired phosphorylation of H2AX (gamma-H2AX), and impaired activation of the cell cycle checkpoint regulating kinase, Chk2. In TK6 cells, ionizing radiation-induced accumulation of p53/p21 and apoptosis were reduced. There was a differential response to ionizing radiation-induced cell killing between TK6 and WTK1 cells after NBS1 knockdown; TK6 cells were more resistant to killing, whereas WTK1 cells were more sensitive. NBS1 deficiency also resulted in a significant increase in telomere association that was independent of radiation exposure and p53 status. Our results provide the first experimental evidence that NBS1 deficiency in human cells leads to hypermutability and telomere associations, phenotypes that may contribute to the cancer predisposition seen among patients with this disease.

Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation.

Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

Suppression of DNA-PK by RNAi has different quantitative effects on telomere dysfunction and mutagenesis in human lymphoblasts treated with gamma rays or HZE particles.

Basic to virtually all relevant biological effects of ionizing radiation is the underlying damage produced in DNA and the subsequent cellular processing of such damage. The damage can be qualitatively different for different kinds of radiations, and the genetics of the biological systems exposed can greatly affect damage processing and ultimate outcome--the biological effect of concern. The accurate repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity and function. Incorrect repair of such lesions results in chromosomal rearrangements and mutations that can lead to cancer and heritable defects in the progeny of irradiated parents. We have focused on the consequent phenotypic effects of faulty repair by examining connections between cellular radiosensitivity phenotypes relevant for carcinogenesis after exposure to ionizing radiation, and deficiencies in various components of the non-homologous end-joining (NHEJ) system. Here we produced deficiencies of individual components of the DNA-dependent protein kinase (DNA-PK) holoenzyme (Ku86 and the catalytic subunit, DNA-PKcs), both singly and in combination, using RNA interference (RNAi) in human lymphoblastoid cell lines. Exposure of cells exhibiting reduced protein expression to either gamma rays or 1 GeV/nucleon iron particles demonstrated differential effects on telomere dysfunction and mutation frequency as well as differential effects between radiation qualities.

Telomeres and Telomerase in the Radiation Response: Implications for Instability, Reprograming, and Carcinogenesis.

Telomeres are nucleoprotein complexes comprised of tandem arrays of repetitive DNA sequence that serve to protect chromosomal termini from inappropriate degradation, as well as to prevent these natural DNA ends from being recognized as broken DNA (double-strand breaks) and triggering of inappropriate DNA damage responses. Preservation of telomere length requires telomerase, the specialized reverse transcriptase capable of maintaining telomere length via template-mediated addition of telomeric repeats onto the ends of newly synthesized chromosomes. Loss of either end-capping function or telomere length maintenance has been associated with genomic instability or senescence in a variety of settings; therefore, telomeres and telomerase have well-established connections to cancer and aging. It has long been recognized that oxidative stress promotes shortening of telomeres, and that telomerase activity is a radiation-inducible function. However, the effects of ionizing radiation (IR) exposure on telomeres per se are much less well understood and appreciated. To gain a deeper understanding of the roles, telomeres and telomerase play in the response of human cells to IRs of different qualities, we tracked changes in telomeric end-capping function, telomere length, and telomerase activity in panels of mammary epithelial and hematopoietic cell lines exposed to low linear energy transfer (LET) gamma(γ)-rays or high LET, high charge, high energy (HZE) particles, delivered either acutely or at low dose rates. In addition to demonstrating that dysfunctional telomeres contribute to IR-induced mutation frequencies and genome instability, we reveal non-canonical roles for telomerase, in that telomerase activity was required for IR-induced enrichment of mammary epithelial putative stem/progenitor cell populations, a finding also suggestive of cellular reprograming. Taken together, the results reported here establish the critical importance of telomeres and telomerase in the radiation response and, as such, have compelling implications not only for accelerated tumor repopulation following radiation therapy but also for carcinogenic potential following low dose exposures as well, including those of relevance to spaceflight-associated galactic cosmic radiations.

Radiation quality and mutagenesis in human lymphoblastoid cells.

An interesting problem associated with studying the effects of low doses of high atomic number and energy (HZE) particles, as found in space, is that not all cells will necessarily be similarly traversed during exposure, a scenario that greatly complicates the measurement of end points that require time to develop, gene-locus mutation being a perfect example. The standard protocol for measuring mutations at the heterozygous thymidine kinase locus in human lymphoblastoid cells involves waiting three days after treatment for newly induced mutants to fully express, at which time cells are then plated in the presence of the selective agent, and mutants are counted three weeks later. This approach is acceptable as long as all cells are uniformly affected, as is the case with low-linear energy transfer (LET) ionizing radiation. However, for HZE particles some fraction of cells may not be traversed or perhaps would receive fewer than the average number of "hits", and they would continue to grow at or closer to the normal rate, thus outpacing cells that received more damage. As a result, at three days post-treatment, more heavily damaged cells will have been "diluted" by the less damaged ones, and thus the measured mutant frequency (MF) will underestimate actual mutant frequency. We therefore developed a modified approach for measuring mutation that eliminates this problem and demonstrates that the mutagenicity of 1 GeV/n Fe ions are underestimated by a factor of two when using the standard MF protocol. Furthermore, we determined the mutagenic effects of a variety of heavy ions, all of which induced mutations in a linear fashion. We found that the maximal yield of mutations (i.e., highest relative biological efficiency) was about 7.5 times higher at an LET of 70 keV/µ (400 MeV/n Si) than for gamma rays. Nontargeted mutagenicity after treatment with ionizing radiation was also investigated. For each particular ion/energy examined and in agreement with many previous studies, there was no clear evidence of a dose response for bystander mutagenesis, i.e., the MF plateaued. Interestingly, the magnitudes of the bystander MFs induced by different ion/energy combinations did vary, with bystander MFs ranging from 0.8 to 2.2× higher than the background. Furthermore, the nontargeted MFs appeared to reflect a mirror image of that for direct mutagenesis.

Regulatory roles of tankyrase 1 at telomeres and in DNA repair: suppression of T-SCE and stabilization of DNA-PKcs.

Intrigued by the dynamics of the seemingly contradictory yet integrated cellular responses to the requisites of preserving telomere integrity while also efficiently repairing damaged DNA, we investigated roles of the telomere associated poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) tankyrase 1 in both telomere function and the DNA damage response following exposure to ionizing radiation. Tankyrase 1 siRNA knockdown in human cells significantly elevated recombination specifically within telomeres, a phenotype with the potential of accelerating cellular senescence. Additionally, depletion of tankyrase 1 resulted in concomitant and rapid reduction of the nonhomologous end-joining protein DNA-PKcs, while Ku86 and ATM protein levels remained unchanged; DNA-PKcs mRNA levels were also unaffected. We found that the requirement of tankyrase 1 for DNA-PKcs protein stability reflects the necessity of its PARP enzymatic activity. We also demonstrated that depletion of tankyrase 1 resulted in proteasome-mediated DNA-PKcs degradation, explaining the associated defective damage response observed; i.e., increased sensitivity to ionizing radiation-induced cell killing, mutagenesis, chromosome aberration and telomere fusion. We provide the first evidence for regulation of DNA-PKcs by tankyrase 1 PARP activity and taken together, identify roles of tankyrase 1 with implications not only for DNA repair and telomere biology, but also for cancer and aging.

Distinct signaling pathways after higher or lower doses of radiation in three closely related human lymphoblast cell lines.

PURPOSE: The tumor suppressor p53 plays an essential role in cellular responses to DNA damage caused by ionizing radiation; therefore, this study aims to further explore the role that p53 plays at different doses of radiation. MATERIALS AND METHODS: The global cellular responses to higher-dose (10 Gy) and lower dose (iso-survival dose, i.e., the respective D0 levels) radiation were analyzed using microarrays in three human lymphoblast cell lines with different p53 status: TK6 (wild-type p53), NH32 (p53-null), and WTK1 (mutant p53). Total RNAs were extracted from cells harvested at 0, 1, 3, 6, 9, and 24 h after higher and lower dose radiation exposures. Template-based clustering, hierarchical clustering, and principle component analysis were applied to examine the transcriptional profiles. RESULTS: Differential expression profiles between 10 Gy and iso-survival radiation in cells with different p53 status were observed. Moreover, distinct gene expression patterns were exhibited among these three cells after 10 Gy radiation treatment, but similar transcriptional responses were observed in TK6 and NH32 cells treated with iso-survival radiation. CONCLUSIONS: After 10 Gy radiation exposure, the p53 signaling pathway played an important role in TK6, whereas the NFkB signaling pathway appeared to replace the role of p53 in WTK1. In contrast, after iso-survival radiation treatment, E2F4 seemed to play a dominant role independent of p53 status. This study dissected the impacts of p53, NFkB and E2F4 in response to higher or lower doses of gamma-irradiation.

Real-time imaging of novel spatial and temporal responses to photodynamic stress.

Cells subjected to various forms of stress have been shown to induce bystander responses in nontargeted cells, thus extending the stress response to a larger population. However, the mechanism(s) of bystander responses remains to be clearly identified, particularly for photodynamic stress. Oxidative stress and cell viability were studied on the spatial and temporal levels after photodynamic targeting of a subpopulation of EMT6 murine mammary cancer cells in a multiwell plate by computerized time-lapse fluorescence microscopy. In the targeted population a dose-dependent loss of cell viability was observed in accordance with increased oxidative stress. This was accompanied by increased oxidative stress in bystander populations but on different time scales, reaching a maximum more rapidly in targeted cells. Treatment with extracellular catalase, or the NADPH oxidase inhibitor diphenyleneiodinium, decreased production of reactive oxygen species (ROS) in both populations. These effects are ascribed to photodynamic activation of NADPH-oxidase in the targeted cells, resulting in a rapid burst of ROS formation with hydrogen peroxide acting as the signaling molecule responsible for initiation of these photodynamic bystander responses. The consequences of increased oxidative stress in bystander cells should be considered in the overall framework of photodynamic stress.

Wild-type p53 reduces radiation hypermutability in p53-mutated human lymphoblast cells.

Many studies have shown that an alteration of p53 affects various cellular responses to DNA damage after treatment with ionizing radiation. The human lymphoblast cell WTK1, which contains a mutant p53 (ile237), is 10-fold hypermutable at the thymidine kinase (tk) locus compared with TK6 cells, which are from the same donor but contain wild-type p53. These results implied that the specific p53 mutation found in WTK1 may actively contribute to mutagenesis in a gain of function manner. To further investigate this, the present experiments involved transfecting WTK1 cells with a wild-type p53 vector; this restored p53 activity in WTK1 cells, as evidenced by radiation-induced expression of p21. We compared radiosensitivity, as measured both by clonogenic survival and the induction of apoptosis, as well as mutant fractions (MFs) at the tk locus. WTK1 cells expressing wild-type p53 were more sensitive to gamma-ray-induced toxicity as measured by either clonogenic survival or apoptosis. The mutation assays revealed that both the spontaneous and gamma-ray-induced MFs were significantly decreased in WTK1 cells expressing wild-type p53; the MFs were similar to those observed in p53-null NH32 cells, also derived from the same donor. These results indicate that wild-type p53 can reduce the apparent gain-of-function hypermutable effects of a particular p53 gene mutation and thereby help maintain genomic stability.

Applications of RNA interference for studies in DNA damage processing, genome stability, mutagenesis, and cancer.

RNA interference, discovered only five years ago, is an important system for the control of gene expression. It is also quickly becoming a valuable experimental tool as it allows investigators to knock down the level of expression of specific genes. In this paper, we review some applications of this technology for studies in DNA damage processing, genome stability, mutagenesis, and cancer.

Baseline levels of chromosome instability in the human lymphoblastoid cell TK6.

Induced genomic instability in the human B lymphoblastoid cell line TK6 manifests itself as increases in end-to-end chromosome fusions and non-reciprocal chromosome translocations. It is not associated with elevated frequencies of specific locus mutations or other cytogenetic alterations. Previous studies on a limited number of cells and end-points suggested that induced instability in TK6 mirrors spontaneous instability in terms of the types of alterations observed. In the present study we expanded on our previous analysis to include more cells and more end-points in order to derive a more precise measure of spontaneous instability in TK6 cells. The frequency of normal growth rate thymidine kinase mutants (TK(-/-)), measured in 44 independently isolated clones, was 2.73 +/- 0.78 x 10(-6)/cell, while that for slow growth mutants was 2.39 +/- 0.52 x 10(-6)/cell. These are similar to the frequencies observed for HPRT mutants in primary human cells. There was wide variation in chromatid break frequencies, but the average break frequency, at 0.04+/-0.01 breaks/cell, was only slightly higher than that reported for primary human cells. In contrast, the dicentric frequency of 0.006/cell was more than 10-fold higher for TK6 cells than that reported for normal primary human cells. Furthermore, the dicentrics in TK6 cells are unusual in that they are the result of end-to-end chromosome fusions. TK6 cells also show much higher levels of non-reciprocal chromosome translocations than are usually observed in primary human cells. The results suggest an inherent instability in TK6 cells that differs from what is observed in primary cells in that it affects the frequency of end-to-end chromosome fusions and non-reciprocal chromosome translocations, but not TK gene mutations or other cytogenetic alterations.