Molecular characterisation of murine acute myeloid leukaemia induced by 56Fe ion and 137Cs gamma ray irradiation.

Exposure to sparsely ionising gamma- or X-ray irradiation is known to increase the risk of leukaemia in humans. However, heavy ion radiotherapy and extended space exploration will expose humans to densely ionising high linear energy transfer (LET) radiation for which there is currently no understanding of leukaemia risk. Murine models have implicated chromosomal deletion that includes the hematopoietic transcription factor gene, PU.1 (Sfpi1), and point mutation of the second PU.1 allele as the primary cause of low-LET radiation-induced murine acute myeloid leukaemia (rAML). Using array comparative genomic hybridisation, fluorescence in situ hybridisation and high resolution melt analysis, we have confirmed that biallelic PU.1 mutations are common in low-LET rAML, occurring in 88% of samples. Biallelic PU.1 mutations were also detected in the majority of high-LET rAML samples. Microsatellite instability was identified in 42% of all rAML samples, and 89% of samples carried increased microsatellite mutant frequencies at the single-cell level, indicative of ongoing instability. Instability was also observed cytogenetically as a 2-fold increase in chromatid-type aberrations. These data highlight the similarities in molecular characteristics of high-LET and low-LET rAML and confirm the presence of ongoing chromosomal and microsatellite instability in murine rAML.

Genetic susceptibility: radiation effects relevant to space travel.

Genetic variation in the capacity to repair radiation damage is an important factor influencing both cellular and tissue radiosensitivity variation among individuals as well as dose rate effects associated with such damage. This paper consists of two parts. The first part reviews some of the available data relating to genetic components governing such variability among individuals in susceptibility to radiation damage relevant for radiation protection and discusses the possibility and extent to which these may also apply for space radiations. The second part focuses on the importance of dose rate effects and genetic-based variations that influence them. Very few dose rate effect studies have been carried out for the kinds of radiations encountered in space. The authors present here new data on the production of chromosomal aberrations in noncycling low passage human ATM+/+ or ATM+/- cells following irradiations with protons (50 MeV or 1 GeV), 1 GeV(-1) n iron ions and gamma rays, where doses were delivered at a high dose rate of 700 mGy(-1) min, or a lower dose rate of 5 mGy min(-1). Dose responses were essentially linear over the dose ranges tested and not significantly different for the two cell strains. Values of the dose rate effectiveness factor (DREF) were expressed as the ratio of the slopes of the dose-response curves for the high versus the lower (5 mGy min(-1)) dose rate exposures. The authors refer to this as the DREF5. For the gamma ray standard, DREF5 values of approximately two were observed. Similar dose rate effects were seen for both energies of protons (DREF5 ≈ 2.2 in both cases). For 1 GeV(-1) n iron ions [linear energy transfer (LET) ≈ 150 keV µ(-1)], the DREF5 was not 1 as might have been expected on the basis of LET alone but was approximately 1.3. From these results and conditions, the authors estimate that the relative biological effectiveness for 1 GeV(-1) n iron ions for high and low dose rates, respectively, were about 10 and 15 rather than around 20 for low dose rates, as has been assumed by most recommendations from radiation protection organizations for charged particles of this LET. The authors suggest that similar studies using appropriate animal models of carcinogenesis would be valuable.

Caspase 3-mediated stimulation of tumor cell repopulation during cancer radiotherapy.

In cancer treatment, apoptosis is a well-recognized cell death mechanism through which cytotoxic agents kill tumor cells. Here we report that dying tumor cells use the apoptotic process to generate potent growth-stimulating signals to stimulate the repopulation of tumors undergoing radiotherapy. Furthermore, activated caspase 3, a key executioner in apoptosis, is involved in the growth stimulation. One downstream effector that caspase 3 regulates is prostaglandin E(2) (PGE(2)), which can potently stimulate growth of surviving tumor cells. Deficiency of caspase 3 either in tumor cells or in tumor stroma caused substantial tumor sensitivity to radiotherapy in xenograft or mouse tumors. In human subjects with cancer, higher amounts of activated caspase 3 in tumor tissues are correlated with markedly increased rate of recurrence and death. We propose the existence of a cell death-induced tumor repopulation pathway in which caspase 3 has a major role.

Differential role of DNA-PKcs phosphorylations and kinase activity in radiosensitivity and chromosomal instability.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is the key functional element in the DNA-PK complex that drives nonhomologous end joining (NHEJ), the predominant DNA double-strand break (DSB) repair mechanism operating to rejoin such breaks in mammalian cells after exposure to ionizing radiation. It has been reported that DNA-PKcs phosphorylation and kinase activity are critical determinants of radiosensitivity, based on responses reported after irradiation of asynchronously dividing populations of various mutant cell lines. In the present study, the relative radiosensitivity to cell killing as well as chromosomal instability of 13 DNA-PKcs site-directed mutant cell lines (defective at phosphorylation sites or kinase activity) were examined after exposure of synchronized G(1) cells to (137)Cs γ rays. DNA-PKcs mutant cells defective in phosphorylation at multiple sites within the T2609 cluster or within the PI3K domain displayed extreme radiosensitivity. Cells defective at the S2056 cluster or T2609 single site alone were only mildly radiosensitive, but cells defective at even one site in both the S2056 and T2609 clusters were maximally radiosensitive. Thus a synergism between the capacity for phosphorylation at the S2056 and T2609 clusters was found to be critical for induction of radiosensitivity.

Some unsolved problems and unresolved issues in radiation cytogenetics: a review and new data on roles of homologous recombination and non-homologous end joining.

New data and historical evidence from our own and other laboratories are summarized and discussed bearing on several issues relating to mechanisms and processes involved in the formation of chromosomal aberrations following exposure to ionizing radiations. Specifically addressed are: (1) the lesions and processes affecting the appearance of chromatid-type and/or chromosome-type aberrations after radiation, (2) DNA double strand break rejoining processes and the restitution of breaks vs. the formation of exchanges, (3) the role of homologous recombinational repair in protecting cells from induction of chromatid-type aberrations after irradiation of late S/G2 cells, (4) the role of interphase chromatin structure and nuclear organization in aberration induction, (5) cellular responses for aberration induction in relation to their tissue context, and (6) approaches to the detection of aberrations previously known as "cryptic".

Apoptotic cells activate the "phoenix rising" pathway to promote wound healing and tissue regeneration.

The ability to regenerate damaged tissues is a common characteristic of multicellular organisms. We report a role for apoptotic cell death in promoting wound healing and tissue regeneration in mice. Apoptotic cells released growth signals that stimulated the proliferation of progenitor or stem cells. Key players in this process were caspases 3 and 7, proteases activated during the execution phase of apoptosis that contribute to cell death. Mice lacking either of these caspases were deficient in skin wound healing and in liver regeneration. Prostaglandin E(2), a promoter of stem or progenitor cell proliferation and tissue regeneration, acted downstream of the caspases. We propose to call the pathway by which executioner caspases in apoptotic cells promote wound healing and tissue regeneration in multicellular organisms the "phoenix rising" pathway.

Variations in radiosensitivity among individuals: a potential impact on risk assessment?

To have an impact on risk assessment for purposes of radiation protection recommendations, significantly broad variations in carcinogenic radiosensitivity would have to exist in significant proportions in the human population. Even if we knew all the genes where mutations would have major effects, individual genome sequencing does not seem useful, since we do not know all these genes, nor can we be certain of the phenotypic effect of polymorphisms discovered. Further, sequencing would not reveal epigenetic changes in gene expression. Another approach to develop phenotypic biomarkers for cells or tissues for which variations in radiation response may reflect the variations in carcinogenic sensitivity. To be useful, experimental evidence for such a correlation would be crucial, and it is also evident that correlations may be tissue or tumor specific. Some cellular markers are discussed that have shown promise in this regard. They include chromosome aberration induction and DNA repair assays that are sufficiently sensitive to measure after modest or low doses or dose rates. To this end we summarize here some of these assays and review the results of a number of experiments from our laboratory that show clear differences in DNA repair capacity reflected by gamma-H2AX foci formation in cells from a high proportion (perhaps 1/3) of apparently normal individuals. A low dose-rate assay was used to amplify such differences. Another promising assay combines G(2) chromosomal radiosensitivity with the above gamma-H2AX foci on mitotic chromosomes. There are other potentially useful assays as well.

Comparison of several radiation effects in human MCF10A mammary epithelial cells cultured as 2D monolayers or 3D acinar stuctures in matrigel.

It has been argued that the cell-cell and cell-matrix interaction networks in normal tissues are disrupted by radiation and that this largely controls many of the most important cellular radiation responses. This has led to the broader assertion that individual cells in normal tissue or a 3D normal-tissue-like culture will respond to radiation very differently than the same cells in a 2D monolayer culture. While many studies have shown that, in some cases, cell-cell contact in spheroids of transformed or tumor cell lines can alter radiation responses relative to those for the same cells in monolayer cultures, a question remains regarding the possible effect of the above-mentioned disruption of signaling networks that operate more specifically for cells in normal tissues or in a 3D tissue-like context. To test the generality of this notion, we used human MCF-10A cells, an immortalized mammary epithelial cell line that produces acinar structures in culture with many properties of human mammary ducts. We compared the dose responses for these cells in the 2D monolayer and in 3D ductal or acinar structures. The responses examined were reproductive cell death, induction of chromosomal aberrations, and the levels of gamma-H2AX foci in cells after single acute gamma-ray doses and immediately after 20 h of irradiation at a dose rate of 0.0017 Gy/min. We found no significant differences in the dose responses of these cells in 2D or 3D growth conditions. While this does not mean that such differences cannot occur in other situations, it does mean that they do not generally or necessarily occur.

Molecular analysis of genetic instability caused by chronic inflammation.

Genetic instability is a hallmark of human cancers. It is the driving force for tumor development as it facilitates the accumulation of mutations in genes that regulate cell death and proliferation and therefore promotes malignant transformation. Chronic inflammation is a common underlying condition for human tumor development, accounting for approximately 20% of human cancers. TNFalpha is an important inflammation cytokine and is crucial to the development of inflammation-associated cancers. We have shown that TNFalpha can cause DNA damages through reactive oxygen species (ROS). TNFalpha treatment in cultured cells resulted in increased gene mutations, gene amplification, micronuclei formation and chromosomal instability. Antioxidants significantly reduced TNFalpha-induced genetic damage. In addition, TNFalpha treatment alone led to increased malignant transformation of mouse embryo fibroblasts, which could be partially suppressed by antioxidants. Therefore, genetic instability plays an important role in inflammation-associated cancers.

Radiation leukemogenesis in mice: loss of PU.1 on chromosome 2 in CBA and C57BL/6 mice after irradiation with 1 GeV/nucleon 56Fe ions, X rays or gamma rays. Part I. Experimental observations.

Since deletion of the PU.1 gene on chromosome 2 is a crucial acute myeloid leukemia (AML) initiating step in the mouse model, we quantified PU.1 deleted cells in the bone marrow of gamma-, X- and 56Fe-ion-irradiated mice at various times postirradiation. Although 56Fe ions were initially some two to three times more effective than X or gamma rays in inducing PU.1 deletions, by 1 month postirradiation, the proportions of cells with PU.1 deletions were similar for the HZE particles and the sparsely ionizing radiations. These results indicate that while 56Fe ions are more effective in inducing PU.1 deletions, they are also more effective in causing collateral damage that removes hit cells from the bone marrow. After X, gamma or 56Fe-ion irradiation, AML-resistant C57BL/6 mice have fewer cells with PU.1 deletions than CBA mice, and those cells do not persist in the bone marrow of the C57B6/6 mice. Our findings suggest that quantification of PU.1 deleted bone marrow cells 1 month postirradiation can be used as surrogate for the incidence of radiation-induced AML measured in large-scale mouse studies. If so, PU.1 loss could be used to systematically assess the potential leukemogenic effects of other ions and energies in the space radiation environment.

Radiation leukemogenesis in mice: loss of PU.1 on chromosome 2 in CBA and C57BL/6 mice after irradiation with 1 GeV/nucleon 56Fe ions, X rays or gamma Rays. Part II. Theoretical considerations based on microdosimetry and the initial induction of chromosome aberrations.

Chromosome aberrations in mitotic bone marrow cells of CBA/Ca and C57BL/6 mice were measured 1 day after exposure to 1 Gy of 1 GeV/nucleon 56Fe ions or 3 Gy of gamma rays. The proportion that have lost a region of chromosome 2 containing the PU.1 gene could be explained by a model based on these measurements. The distribution of aberrations among cells was close to the expected Poisson for the gamma-irradiated cells, but for the HZE 56Fe ions the distribution was highly dispersed. The observations were consistent with the results of an analysis similar to that of Edwards and co-workers in 1980 after ex vivo irradiation of human blood with alpha particles. The analysis used to fit the current data was based on a compound Poisson process, also used previously by others, but in addition included the random nature of parameters involved such as cell nuclear diameter, particle traversal lengths through cell nuclei, production of aberrations, and cell cycle arrest per traversal. From the measured numbers of acentric fragments produced, the relative size of chromosome 2 and the region associated with PU.1 deletions, an independent prediction of PU.1 loss agreed well with measurements described in the accompanying paper.

Abnormal gene expression profiles in unaffected parents of patients with hereditary-type retinoblastoma.

The hereditary form of retinoblastoma (Rb) is associated with a germ line mutation in one RB allele and is characterized by the occurrence of multiple, bilateral Rb tumors and a predisposition to the development of second cancers. In an earlier study, we observed an unexpected hypersensitivity to ionizing radiation in skin fibroblasts derived from unaffected parents of children with hereditary Rb. In at least four of these five families, there was no family history of Rb, indicating a new germ line mutation. We hypothesize that the increased parental cell sensitivity to radiation may reflect the presence of an as yet unrecognized genetic abnormality occurring in one or both parents of children with Rb. In the present study, we use DNA microarray technology to determine whether differences in gene expression profiles occurred in the unaffected parents of patients with hereditary Rb relative to normal individuals. Microarray analyses were validated by quantitative reverse transcription-PCR measurements. A distinct difference was observed in the patterns of gene expression between unaffected Rb parents and normal controls. By use of the prediction analysis for microarrays and principal component analysis methodologies, significant differences between the two groups were identified when as few as nine genes were analyzed. Further study of this phenomenon may offer a new insight into the genetic mechanisms of Rb and perhaps more broadly in cancer biology.

A unique role of the DNA fragmentation factor in maintaining genomic stability.

DNA fragmentation is a hallmark of apoptosis (programmed cell death). However, the biological function of apoptotic DNA fragmentation remains unclear. Here, we show that DNA fragmentation factor plays an important role for maintaining genomic stability. Inhibition or loss of the DNA fragmentation factor (DFF)/caspase-activated DNase (CAD), whose nuclease activity is responsible for digesting genomic DNA during apoptosis, led to significant increases in spontaneous or induced gene mutations, gene amplifications, and chromosomal instability in primary mouse cells and transformed human cell lines. The mechanism underlying genetic instability in DFF/CAD-deficient cells, at least in part, involves a small but significant elevation in the survival of cells exposed to ionizing radiation, suggesting that apoptotic DNA fragmentation factor contributes to genomic stability by ensuring the removal of cells that have suffered DNA damage. In support of this hypothesis are the observations of increased cellular transformation of mouse embryonic cells from the DFF/CAD-null mice and significantly enhanced susceptibility to radiation-induced carcinogenesis in these mice. These data, in combination with published reports on the existence of tumor-specific gene mutations/deletions in the DFF/CAD genes in human cancer samples, suggest that apoptotic DNA fragmentation factor is required for the maintenance of genetic stability and may play a role in tumor suppression.

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.

Deficiency in the catalytic subunit of DNA-dependent protein kinase causes down-regulation of ATM.

Previous reports have suggested a connection between reduced levels of the catalytic subunit of DNA-dependent protein kinases (DNA-PKcs), a component of the nonhomologous DNA double-strand breaks end-joining system, and a reduction in ATM. We studied this possible connection in other DNA-PKcs-deficient cell types, and following knockdown of DNA-PKcs with small interfering RNA, Chinese hamster ovary V3 cells, lacking DNA-PKcs, had reduced levels of ATM and hSMG-1, but both were restored after transfection with PRKDC. Atm levels were also reduced in murine scid cells. Reduction of ATM in a human glioma cell line lacking DNA-PKcs was accompanied by defective signaling through downstream substrates, post-irradiation. A large reduction of DNA-PKcs was achieved in normal human fibroblasts after transfection with two DNA-PKcs small interfering RNA sequences. This was accompanied by a reduction in ATM. These data were confirmed using immunocytochemical detection of the proteins. Within hours after transfection, a decline in PRKDC mRNA was seen, followed by a more gradual decline in DNA-PKcs protein beginning 1 day after transfection. No change in ATM mRNA was observed for 2 days post-transfection. Only after the DNA-PKcs reduction occurred was a reduction in ATM mRNA observed, beginning 2 days post-transfection. The amount of ATM began to decline, starting about 3 days post-treatment, then it declined to levels comparable to DNA-PKcs. Both proteins returned to normal levels at later times. These data illustrate a potentially important cross-regulation between the nonhomologous end-joining system for rejoining of DNA double-strand breaks and the ATM-dependent damage response network of pathways, both of which operate to maintain the integrity of the genome.

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.