[Show abstract][Hide abstract] ABSTRACT: The human vasculature includes a vast network of microcapillaries networking the body and is a major target for non-carcinogenic effects of space radiation in the body. The brain microvascular system is crucial to healthy functioning of the brain and its pathology is not only a primary event in a range of neurodegenerative diseases but also an important influencing factor in many others. The vasculature is maintained by angiogenesis regenerating vessels as they are needed, this is particularly relevant if the blood–brain barrier is damaged by agents such as space radiation, thereby creating the need for angiogenic regeneration. The resulting lack of vasculature due to the inhibition of re-growth of vessels can, in turn, lead to a negative feedback loop and further pathologies.
Using three-dimensional human vessel cultures with human umbilical vein and brain microvascular endothelial cells, we have developed assays that determine at what stage angiogenesis is inhibited by ionizing radiation. The relative biological effect of high linear energy transfer (LET) 1 GeV Fe ions compared with low LET 1 GeV protons is only one for developing vessels but greater than four for mature vessels. This action of low LET protons on developing vessels was surprisingly effective (50% inhibition with 40 cGy exposure) and together with the effect of high LET ions may represent a significant hazard in the space environment.
The morphology of developing vessels 5 days after exposure showed significant differences that suggest distinct mechanisms of inhibition. Cells exposed to protons failed to make connections with other cells. Conversely, cells exposed to Fe ions extended cellular processes and made connections to other cells but did not develop a central lumen. The microtubule and actin cytoskeletons showed differences indicating that motility at the extending tips of endothelial cells is inhibited by protons but not Fe ions. Actin-rich protrusive structures that contain bundled and dynamic microtubules showed a 65% decrease when exposed to high-energy protons but not with the same dose of high-energy Fe ions. Since protein kinase C (PKC) has long been known to stimulate angiogenesis, we hypothesized that rescue of the capillary phenotype after proton exposure would be possible by stimulating PKC before irradiation. One-day-old vessel cultures were treated with 30 and 60 nM phorbol ester (PMA) 15 min before irradiation. Stimulation of PKC restored capillary formation in proton-treated cultures but not in Fe ion-treated cultures. More specifically, stimulation of PKC by PMA was able to restore the tip motility that was inhibited by low LET ions [
Further studies with various charged particles showed that low LET ion particles (Proton and He ions) with an LET lower or equal to 1 keV/μm inhibit vasculogenesis in the same way as protons. Higher LET charged particles (Silicon 1GeV, Oxygen 250 MeV and 1 GeV and Carbon 290 MeV and 1 GeV) with an LET ≥8 keV/μm inhibit vasculogenesis in the same way as Fe ions.
In conclusion, we have shown that low and high LET ions inhibit the formation of brain capillaries by different mechanisms. For low LET ions, inhibition involves regulation of PKC-dependent motile tips leading to a failure of cellular processes to migrate through the matrix and meet up with other processes. For high LET ions, the cells fail to complete angiogenesis by not migrating and forming tubular structures. This complexity of response opens up possibilities of greater control over angiogenesis and the resulting pathologies during coincident exposure or therapy. For exposure in space, knowledge of these mechanisms will enable more precise risk assessment and mitigation strategies. For radiotherapy, treatment could be manipulated to utilize the radiation effectively.
Journal of Radiation Research 03/2014; 55 Suppl 1(Suppl 1):i44-i45. DOI:10.1093/jrr/rrt172 · 1.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Charged particle radiation is known to be more biologically effective than photon radiation. One example of this is the inhibition of the formation of human blood vessels. This effect is an important factor influencing human health and is relevant to space travel as well as to cancer radiotherapy. We have previously shown that ion particles with a high energy deposition, or linear energy transfer (LET) are more than four times more effective at disrupting mature vessel tissue models than particles with a lower LET. For vasculogenesis however, the relative biological effectiveness between particles is the same. This unexpected result prompted us to investigate whether the inhibition of vasculogenesis was occurring by distinct mechanisms.
Using 3-Dimensional human vessel models, we developed assays that determine at what stage angiogenesis is inhibited. Vessel morphology, the presence of motile tip structures, and changes in the matrix architecture were assessed. To confirm that the mechanisms are distinct, stimulation of Protein Kinase C (PKC) with phorbol ester (PMA) was employed to selectively restore vessel formation in cultures where early motile tip activity was inhibited.
Endothelial cells in 3-D culture exposed to low LET protons failed to make connections with other cells but eventually developed a central lumen. Conversely, cells exposed to high LET Fe charged particles extended cellular processes and made connections to other cells but did not develop a central lumen. The microtubule and actin cytoskeletons indicated that motility at the extending tips of endothelial cells is inhibited by low LET but not high LET particles. Actin-rich protrusive structures that contain bundled microtubules showed a 65% decrease when exposed to low LET particles but not high LET particles, with commensurate changes in the matrix architecture. Stimulation of PKC with PMA restored tip motility and capillary formation in low but not high LET particle treated cultures.
Low LET charged particles inhibit the early stages of vasculogenesis when tip cells have motile protrusive structures and are creating pioneer guidance tunnels through the matrix. High LET charged particles do not affect the early stages of vasculogenesis but they do affect the later stages when the endothelial cells migrate to form tubes.
[Show abstract][Hide abstract] ABSTRACT: While gene expression studies have proved extremely important in understanding cellular processes, it is becoming more apparent that there may be differences in individual cells that are missed by studying the population as a whole. We have developed a qRT-PCR protocol that allows us to assay multiple gene products in small samples, starting at 100 cells and going down to a single cell, and have used it to study radiation responses at the single-cell level. Since the accuracy of qRT-PCR depends greatly on the choice of "housekeeping" genes used for normalization, initial studies concentrated on determining the optimal panel of such genes. Using an endogenous control array, it was found that for IMR90 cells, common housekeeping genes tend to fall into one of two categories-those that are relatively stably expressed regardless of the number of cells in the sample, e.g., B2M, PPIA, and GAPDH, and those that are more variable (again regardless of the size of the population), e.g., YWHAZ, 18S, TBP, and HPRT1. Further, expression levels in commonly studied radiation-response genes, such as ATF3, CDKN1A, GADD45A, and MDM2, were assayed in 100, 10, and single-cell samples. It is here that the value of single-cell analyses becomes apparent. It was observed that the expression of some genes such as FGF2 and MDM2 was relatively constant over all irradiated cells, while that of others such as FAS was considerably more variable. It was clear that almost all cells respond to ionizing radiation but the individual responses were considerably varied. The analyses of single cells indicate that responses in individual cells are not uniform and suggest that responses observed in populations are not indicative of identical patterns in all cells. This in turn points to the value of single-cell analyses.
[Show abstract][Hide abstract] ABSTRACT: To analyze the effect of different radiation qualities on the kinetics of p53 Binding Protein 1 (53BP1) formation and decline in human three-dimensional (3-D) vessel models.
Two-dimensional (2-D) and 3-D cultures of human umbilical vein cells were exposed to 80 cGy of Gamma radiation and high-energy protons and Fe ions. 53BP1 antibodies were used for foci visualization via immunocytochemistry. Computer analysis was used to determine the number and the size of foci up to 48 hours after irradiation.
DNA foci kinetics in 2-D and 3-D human vessel cultures show that foci formation and removal were the same in each type of culture. After 48 h, the number of foci induced by high-energy protons and gamma rays reduced to almost control levels while high linear energy transfer (LET) Fe particles produced more persistent damage.
The kinetics of radiation-induced 53BP1 foci in 3-D vessel models is essentially the same as in 2-D monolayers. Since the basal level of spontaneous foci is low in these differentiated non-proliferating cultures, the persistence of radiation-induced 53BP1 foci is detected longer than previously noted. Furthermore, analysis of foci sizes revealed that abnormal radiation-induced foci can persist even when foci frequencies are close to basal levels. The detection of these latent abnormalities could be useful for a more sensitive dosimetry.
International Journal of Radiation Biology 03/2012; 88(6):493-500. DOI:10.3109/09553002.2012.679382 · 1.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Exposure to ionizing radiation may induce a heritable genomic instability phenotype that results in a persisting and enhanced genetic and functional change among the progeny of irradiated cells. Since radiation-induced bystander effects have been demonstrated with a variety of biological end points under both in vitro and in vivo conditions, this raises the question whether cytoplasmic irradiation or the radiation-induced bystander effect can also lead to delayed genomic instability. In the present study, we used the Radiological Research Accelerator Facility charged-particle microbeam for precise nuclear or cytoplasmic irradiation. The progeny of irradiated and the bystander human hamster hybrid (A(L)) cells were analyzed using multicolor banding (mBAND) to examine persistent chromosomal changes. Our results showed that the numbers of metaphase cells involving changes of human chromosome 11 (including rearrangement, deletion and duplication) were significantly higher than that of the control in the progeny of both nuclear and cytoplasmic targeted cells. These chromosomal changes could also be detected among the progeny of bystander cells. mBAND analyses of clonal isolates from nuclear and cytoplasm irradiations as well as the bystander cell group showed that chromosomal unstable clones were generated. Analyses of clonal stability after long-term culture indicated no significant change in the number of unstable clones for the duration of culture in each irradiated group. These results suggest that genomic instability that is manifested after ionizing radiation exposure is not dependent on direct damage to the cell nucleus.
Radiation Research 11/2011; 177(1):25-34. DOI:10.2307/41408643 · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Little is known about the effects of space radiation on the human body. There are a number of potential chronic and acute effects, and one major target for noncarcinogenic effects is the human vasculature. Cellular stress, inflammatory response, and other radiation effects on endothelial cells may affect vascular function. This study was aimed at understanding the effects of space ionizing radiation on the formation and maintenance of capillary-like blood vessels. We used a 3D human vessel model created with human endothelial cells in a gel matrix to assess the effects of low-LET protons and high-LET iron ions. Iron ions were more damaging and caused significant reduction in the length of intact vessels in both developing and mature vessels at a dose of 80 cGy. Protons had no effect on mature vessels up to a dose of 3.2 Gy but did inhibit vessel formation at 80 cGy. Comparison with γ radiation showed that photons had even less effect, although, as with protons, developing vessels were more sensitive. Apoptosis assays showed that inhibition of vessel development or deterioration of mature vessels was not due to cell death by apoptosis even in the case of iron ions. These are the first data to show the effects of radiation with varying linear energy transfer on a human vessel model.
Radiation Research 01/2011; 175(1):21-8. DOI:10.1667/RR2289.1 · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The array of microbeam cell-irradiation systems, available to users at the Radiological Research Accelerator Facility (RARAF), Center for Radiological Research, Columbia University, is expanding. The HVE 5MV Singletron particle accelerator at the facility provides particles to two focused ion microbeam lines: the sub-micron microbeam II and the permanent magnetic microbeam (PMM). Both the electrostatic quadrupole lenses on the microbeam II system and the magnetic quadrupole lenses on the PMM system are arranged as compound lenses consisting of two quadrupole triplets with "Russian" symmetry. Also, the RARAF accelerator is a source for a proton-induced x-ray microbeam (undergoing testing) and is projected to supply protons to a neutron microbeam based on the (7)Li(p, n)(7)Be nuclear reaction (under development). Leveraging from the multiphoton microscope technology integrated within the microbeam II endstation, a UV microspot irradiator - based on multiphoton excitation - is available for facility users. Highlights from radiation-biology demonstrations on single living mammalian cells are included in this review of microbeam systems for cell irradiation at RARAF.
[Show abstract][Hide abstract] ABSTRACT: Our earlier study demonstrated the induction of PKC isoforms (betaII, PKC-alpha/beta, PKC-theta) by ionizing radiation induced bystander response in human cells. In this study, we extended our investigation to yet another important member of PKC family, PKC epsilon (PKCepsilon). PKCepsilon functions both as an anti-apoptotic and pro-apoptotic protein and it is the only PKC isozyme implicated in oncogenesis. Given the importance of PKCepsilon in oncogenesis, we wished to determine whether or not PKCepsilon is involved in bystander response. Gene expression array analysis demonstrated a 2-3-fold increase in PKCepsilon expression in the bystander human primary fibroblast cells that were co-cultured in double-sided Mylar dishes for 3h with human primary fibroblast cells irradiated with 5Gy of alpha-particles. The elevated PKCepsilon expression in bystander cells was verified by quantitative real time PCR. Suppression of PKCepsilon expression by small molecule inhibitor Bisindolylmaleimide IX (Ro 31-8220) considerably reduced the frequency of micronuclei (MN) induced both by 5Gy of gamma-rays (low LET) and alpha-particles (high LET) in bystander cells. Similar cytoprotective effects were observed in bystander cells after siRNA mediated silencing of PKCepsilon suggestive of its critical role in mediating some of the bystander effects (BE). Our novel study suggests the possibility that PKC signaling pathway may be a critical molecular target for suppression of ionizing radiation induced biological effects in bystander cells.
The international journal of biochemistry & cell biology 08/2009; 41(12):2413-21. DOI:10.1016/j.biocel.2009.06.012 · 4.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Please cite this paper as: Analysis of ionizing radiation-induced DNA damage and repair in three-dimensional human skin model system. Experimental Dermatology 2010; 19: e16–e22.
Abstract: Knowledge of cellular responses in tissue microenvironment is crucial for the accurate prediction of human health risks following chronic or acute exposure to ionizing radiation (IR). With this objective, we investigated the radio responses for the first time in three-dimensional (3D) artificial human skin tissue microenvironment after γ-rays radiation. IR-induced DNA damage/repair response was assessed by immunological analysis of well-known DNA double strand break (DSB) repair proteins, i.e. 53BP1 and phosphorylated ataxia telangiectasia mutatedser1981 (ATMser1981). Efficient 53BP1 and phosphorylated ATM foci formation was observed in human EpiDerm tissue constructs after low and high doses of γ-rays. Interestingly, EpiDerm tissue constructs displayed less 53BP1 and ATM foci number at all radiation doses (0.1, 1, 2.5 and 5 Gy) than that observed for 2D human fibroblasts. DSB repair efficiency judged by the disappearance of 53BP1 foci declined with increasing doses of γ-rays and tissue constructs irradiated with 2.5 and 5 Gy of γ-rays displayed 53BP1 foci persisting up to 72 h of analysis. Pretreatment of EpiDerm tissue constructs with LY294002, [an inhibitor of phosphatidylinositol-3 kinase and PI-3 kinase like kinases (PIKK)] completely abolished IR-induced 53BP1 foci formation and increased the apoptotic death. This observation indicates the importance of PIKK signalling pathway for efficient radiation responses in intact tissue constructs. In summary, we have successfully demonstrated the feasibility of monitoring the DNA damage response in human skin tissue microenvironment. In this system, 53BP1 can be used as a useful marker for monitoring the DSB repair efficiency.
[Show abstract][Hide abstract] ABSTRACT: Recent developments in microbeam technology have made drastic improvements in particle delivery, focusing, image processing and precision to allow for rapid advances in our knowledge in radiation biology. The unequivocal demonstration that targeted cytoplasmic irradiation results in mutations in the nuclei of hit cells and the presence of non-targeted effects, all made possible using a charged particle microbeam, results in a paradigm shift in our basic understanding of the target theory and other radiation-induced low dose effects. The demonstration of a bystander effect in 3D human tissue and whole organisms have shown the potential relevance of the non-targeted response in human health. The demonstration of delayed mutations in the progeny of bystander cells suggest that genomic instability induced following ionizing radiation exposure is not dependent on direct damage to cell nucleus. The identification of specific signaling pathways provides mechanistic insight on the nature of the bystander process.
Journal of Radiation Research 03/2009; 50 Suppl A(Suppl.A):A7-A12. DOI:10.1269/jrr.08135S · 1.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Charged-particle microbeams, developed to provide targeted irradiation of individual cells, and then of sub-cellular components, and then of 3-D tissues and now organisms, have been instrumental in challenging and changing long accepted paradigms of radiation action. However the potential of these valuable tools can be enhanced by integrating additional components with the direct ability to measure biological responses in real time, or to manipulate the cell, tissue or organism of interest under conditions where information gained can be optimized. The RARAF microbeam has recently undergone an accelerator upgrade, and been modified to allow for multiple microbeam irradiation laboratories. Researchers with divergent interests have expressed desires for particular modalities to be made available and ongoing developments reflect these desires. The focus of this review is on the design, incorporation and use of multiphoton and other imaging, micro-manipulation and single cell biosensor capabilities at RARAF. Additionally, an update on the status of the other biology oriented microbeams in the Americas is provided.
Journal of Radiation Research 03/2009; 50 Suppl A(Suppl.A):A21-8. DOI:10.1269/jrr.08134S · 1.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Multiphoton microscopy has been added to the array of imaging techniques at the endstation for the Microbeam II cell irradiator at Columbia University's Radiological Research Accelerator Facility (RARAF). This three-dimensional (3D), laser-scanning microscope functions through multiphoton excitation, providing an enhanced imaging routine during radiation experiments with tissuelike samples, such as small living animals and organisms. Studies at RARAF focus on radiation effects; hence, this multiphoton microscope was designed to observe postirradiation cellular dynamics. This multiphoton microscope was custom designed into an existing Nikon Eclipse E600-FN research fluorescence microscope on the irradiation platform. Design details and biology applications using this enhanced 3D-imaging technique at RARAF are reviewed.
The Review of scientific instruments 01/2009; 79(12):123707. DOI:10.1063/1.3043439 · 1.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The radiation-induced bystander effect represents a paradigm shift in our understanding of the radiobiological effects of ionizing radiation, in that extranuclear and extracellular events may also contribute to the final biological consequences of exposure to low doses of radiation. Although radiation-induced bystander effects have been well documented in a variety of biological systems, the mechanism is not known. It is likely that multiple pathways are involved in the bystander phenomenon, and different cell types respond differently to bystander signalling. Using cDNA microarrays, a number of cellular signalling genes, including cyclooxygenase-2 (COX-2), have been shown to be causally linked to the bystander phenomenon. The observation that inhibition of the phosphorylation of extracellular signal-related kinase (ERK) suppressed the bystander response further confirmed the important role of the mitogen-activated protein kinase (MAPK) signalling cascade in the bystander process. Furthermore, cells deficient in mitochondrial DNA showed a significantly reduced response to bystander signalling, suggesting a functional role of mitochondria in the signalling process. Inhibitors of nitric oxide (NO) synthase (NOS) and mitochondrial calcium uptake provided evidence that NO and calcium signalling are part of the signalling cascade. The bystander observations imply that the relevant target for various radiobiological endpoints is larger than an individual cell. A better understanding of the cellular and molecular mechanisms of the bystander phenomenon, together with evidence of their occurrence in-vivo, will allow us to formulate a more accurate model for assessing the health effects of low doses of ionizing radiation.
Journal of Pharmacy and Pharmacology 08/2008; 60(8):943-50. DOI:10.1211/jpp.60.8.0001 · 2.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Histone H2A variant H2AX is a dose-dependent suppressor of oncogenic chromosome translocations. H2AX participates in DNA double-strand break repair, but its role in other DNA repair pathways is not known. In this study, role of H2AX in cellular response to alkylation DNA damage was investigated. Cellular sensitivity to two monofunctional alkylating agents (methyl methane sulfonate and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)) was dependent on H2AX dosage, and H2AX null cells were more sensitive than heterozygous cells. In contrast to wild-type cells, H2AX-deficient cells displayed extensive apoptotic death due to a lack of cell-cycle arrest at G(2)/M phase. Lack of G(2)/M checkpoint in H2AX null cells correlated well with increased mitotic irregularities involving anaphase bridges and gross chromosomal instability. Observation of elevated poly(ADP) ribose polymerase 1 (PARP-1) cleavage suggests that MNNG-induced apoptosis occurs by PARP-1-dependent manner in H2AX-deficient cells. Consistent with this, increased activities of PARP and poly(ADP) ribose (PAR) polymer synthesis were detected in both H2AX heterozygous and null cells. Further, we demonstrate that the increased PAR synthesis and apoptotic death induced by MNNG in H2AX-deficient cells are due to impaired activation of mitogen-activated protein kinase pathway. Collectively, our novel study demonstrates that H2AX, similar to PARP-1, confers cellular protection against alkylation-induced DNA damage. Therefore, targeting either PARP-1 or histone H2AX may provide an effective way of maximizing the chemotherapeutic value of alkylating agents for cancer treatment.
[Show abstract][Hide abstract] ABSTRACT: Studies over the last several years have revealed the existence of a biological phenomenon known as "bystander effect", wherein cells that are not exposed to radiation elicit a similar response to that of irradiated cells. Understanding the mechanism(s) underlying the bystander effect is important not only for radiation risk assessment but also for evaluation of protocols for cancer radiotherapy. Evaluation of signaling pathways in bystander cells may provide an insight to understand the molecular mechanisms(s) responsible for this complex phenomenon. With this objective, the time course kinetics of intracellular distribution of protein kinase C (PKC isoforms PKC-betaII, PKC-alpha/beta, PKC-theta) was investigated in total and subcellular (cytosolic and nuclear) fractions of human lung fibroblast (MRC-5) cells. MRC-5 cells were either irradiated or treated with the irradiated conditioned medium collected 1h after 1 or 10 Gy of gamma-irradiation. The radiation dose selected was in the range of therapeutic usage of radiation for the human cancer treatment. Unexpectedly, bystander cells showed higher activation of protein kinase C isoforms as compared to irradiated and sham-treated control cells. Protein kinase C isoforms were more enriched in the nuclear fraction than the cytosolic fraction proteins. Induction of PKC isoforms in bystander cells are due to post-translational modifications as shown by the non-phosphorylated protein kinase C level in both irradiated and bystander cells did not differ from the sham-treated control cells. The specific activation of protein kinase C isoforms in bystander cells as demonstrated for the first time in this study may help to identify the effect of therapeutically used radiation exposure for the tumor destructions along with its implications for adjacent non-irradiated cells and organs.
The International Journal of Biochemistry & Cell Biology 02/2008; 40(1):125-34. DOI:10.1016/j.biocel.2007.07.002 · 4.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This investigation is aimed to determine the role of low LET (linear energy transfer, gamma-rays) and high LET (alpha-particles) radiations on bystander effect of using the same type of cells and its implications on colony-forming efficiency from a single cell.
Normal human fetal lung (MRC-5), immortalized repair deficient ataxia telangiectasia mutated (ATM) (GM5,849C) and normal (GM637H) fibroblast cells were used. Colony-forming efficiency in bystander cells (GM637H) was studied using the medium transfer technique from the two donor (MRC-5 and GM5,849C) cells and the procedure followed for bystander treatment is presented schematically in Figure 1. Evidence of change in colony formation in bystander cells, was assessed by scavenging nitric oxide (NO).
Enhancement of 10 - 30% in colony-forming efficiency was observed in bystander GM637H cells treated with irradiated conditioned medium (ICM) from MRC-5 cells collected 1 h after different doses of either gamma-rays (1, 2.5, 5 and 10 Gy) or alpha particles (0.25, 0.5, 1 and 2.5 Gy) irradiation. Similar results were obtained when ICM derived from the ATM (GM5,849C) cells. However, the stimulation was not dose dependent. Furthermore, we also show that the increase in dilutions of ICM (1:1, 1:5 and 1:10) showed an inverse correlation with cloning efficiency. Treatment of MRC-5 cells with PTIO (2-phenyl-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide) a NO scavenger, 1 h prior to irradiation reduced the enhancement of ICM mediated cell survival.
In the present study, though both the low and high LET radiations enhanced the clonogenic potential of the bystander recipient cells, medium from the ATM defective (GM5,849C) cells after gamma-irradiation showed less stimulating effect than the medium from the normal (MRC-5) cells. However, after alpha-irradiation an inverse effect was seen. NO may play an important role in enhancing the growth potential in these bystander cells.
International Journal of Radiation Biology 09/2007; 83(8):551-9. DOI:10.1080/09553000701384499 · 1.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is growing recognition that the effects of ionizing radiation may extend to more than those cells that directly suffer damage to DNA in the cell nucleus. Data from several investigators have indicated that cells neighboring those that are irradiated also demonstrate several responses seen in hit cells--the so-called bystander effect. The microbeam facility at the Center for Radiological Research is particularly well suited for the study of this bystander effect, since it has the ability to place known numbers of charged particles (protons or alpha-particles at LETs from 20 to 180 KeV/microm) at defined positions relative to individual cells. That is, some known fraction of cells in a population can be irradiated through the nucleus, or the cytoplasm or even adjacent to cells through the media. Therefore, using the microbeam it is possible to examine individual cell responses in both hit and nonhit cells in the same population.
Alterations in the cyclin-dependent kinase inhibitor CDKN1a (p21/Cip1/WAF1) were quantified at the mRNA level in single normal human fibroblasts following precise delivery of 0 or 10 alpha-particles per cell at 90 KeV/microm to 50% of cells in a population. Semiquantitative RT-PCR of individual hit cells demonstrated increases in the levels of CDKN1A message that followed the kinetics previously described for irradiated populations. Furthermore, nonhit bystander cells also showed increased (though lesser) levels of CDKN1a message.
Data presented here demonstrate the power of this approach, which combines the ability of the microbeam to irradiate specific cells in a population and the ability to quantify the response to the irradiation in individual targeted and bystander cells.
[Show abstract][Hide abstract] ABSTRACT: Ataxia telangiectasia (AT) is an autosomal recessive multisystem human disorder and patients are characterized by cerebellar
ataxia, oculocutaneous telangiectasia, immunodeficiency, chromosomal instability and radio sensitivity with an increased predisposition
to lymphoid cancer in childhood. The gene responsible for AT, ataxia telangiectasia mutated (ATM), has been cloned and its
protein product has been biochemically characterized as a serine/threonine kinase belonging to the family of phosphatidylinositol
(PI-3) like kinases. Subsequent biochemical studies by several laboratories have identified a number of DNA repair and cell
cycle proteins that are phosphorylated by ATM kinase in response to different DNA damaging agents. One intriguing question that comes to mind is whether the phenotypic
features of AT stem from a DNA repair defect or a cell cycle defect or both. The scope of this review is focused on the potential
functions of ATM in both DNA repair and cell cycle checkpoint regulation and how deficiencies in these overlapping functions can lead to some
of the phenotypic features of AT patients.
DNA Repair and Human Disease, 03/2007: pages 82-97;
[Show abstract][Hide abstract] ABSTRACT: In earlier reports we have shown that a vitamin A analog inhibits radiation-induced transformation in vitro and that retinoids and TPA exert antagonistic effects on vitamin A-binding protein production. Since the tumor promoter TPA has been reported to enhance radiation-induced transformation, it seemed a logical step to follow up these various studies and to investigate whether the antagonism between vitamin A and TPA on cell differentiation in vitro prevails in their effects on radiation-induced transformation in vitro.
Annals of the New York Academy of Sciences 12/2006; 359(1):237 - 250. DOI:10.1111/j.1749-6632.1981.tb12750.x · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cellular response to ionizing radiation is not limited to cells irradiated directly but can be demonstrated in neighboring "bystander" populations. The ability of mouse embryonic stem (ES) cells to express a bystander effect and the role of the radioresistance gene Rad9 were tested. Mouse ES cells differing in Rad9 status were exposed to broad-beam 125 keV/ microm 3He alpha particles. All populations, when confluent, demonstrated a dose-independent bystander effect with respect to cell killing, and the Rad9-/- genotype did not selectively alter that response or cell killing after direct exposure to this high-LET radiation. In contrast, relative to Rad9+/+ cells, the homozygous mutant was sensitive to direct exposure to alpha particles when in log phase, providing evidence of a role for Rad9 in repair of potentially lethal damage. Direct exposure to alpha particles induced an increase in the frequency of apoptosis and micronucleus formation, regardless of Rad9 status, although the null mutant showed high spontaneous levels of both end points. All populations demonstrated alpha-particle-induced bystander apoptosis, but that effect was most prominent in Rad9-/- cells. Minimal alpha-particle induction of micronuclei in bystander cells was observed, except for the Rad9-/- mutant, where a significant increase above background was detected. Therefore, the Rad9 null mutation selectively sensitizes mouse ES cells to spontaneous and high-LET radiation-induced bystander apoptosis and micronucleus formation, but it has much less impact on cell killing by direct or bystander alpha-particle exposure. Results are presented in the context of defining the function of Rad9 in the cellular response to radiation and its differential effects on individual bystander end points.
Radiation Research 12/2005; 164(5):655-61. DOI:10.1667/RR3458.1 · 2.91 Impact Factor