Lubomir B. Smilenov

Cell cycle activation linked to neuronal cell death initiated by DNA damage.

Increasing evidence indicates that neurodegeneration involves the activation of the cell cycle machinery in postmitotic neurons. However, the purpose of these cell cycle-associated events in neuronal apoptosis remains unknown. Here we tested the hypothesis that cell cycle activation is a critical component of the DNA damage response in postmitotic neurons. Different genotoxic compounds (etoposide, methotrexate, and homocysteine) induced apoptosis accompanied by cell cycle reentry of terminally differentiated cortical neurons. In contrast, apoptosis initiated by stimuli that do not target DNA (staurosporine and colchicine) did not initiate cell cycle activation. Suppression of the function of ataxia telangiectasia mutated (ATM), a proximal component of DNA damage-induced cell cycle checkpoint pathways, attenuated both apoptosis and cell cycle reentry triggered by DNA damage but did not change the fate of neurons exposed to staurosporine and colchicine. Our data suggest that cell cycle activation is a critical element of the DNA damage response of postmitotic neurons leading to apoptosis.

Ionizing radiation induces DNA double-strand breaks in bystander primary human fibroblasts.

That irradiated cells affect their unirradiated ‘bystander’ neighbors is evidenced by reports of increased clonogenic mortality, genomic instability, and expression of DNA-repair genes in the bystander cell populations. The mechanisms underlying the bystander effect are obscure, but genomic instability suggests DNA double-strand breaks (DSBs) may be involved. Formation of DSBs induces the phosphorylation of the tumor suppressor protein, histone H2AX and this phosphorylated form, named γ-H2AX, forms foci at DSB sites. Here we report that irradiation of target cells induces γ-H2AX focus formation in bystander cell populations. The effect is manifested by increases in the fraction of cells in a population that contains multiple γ-H2AX foci. After 18 h coculture with cells irradiated with 20 α-particles, the fraction of bystander cells with multiple foci increased 3.7-fold. Similar changes occurred in bystander populations mixed and grown with cells irradiated with γ-rays, and in cultures containing media conditioned on γ-irradiated cells. DNA DSB repair proteins accumulated at γ-H2AX foci, indicating that they are sites of DNA DSB repair. Lindane, which blocks gap-junctions, prevented the bystander effect in mixing but not in media transfer protocols, while c-PTIO and aminoguanidine, which lower nitric oxide levels, prevented the bystander effect in both protocols. Thus, multiple mechanisms may be involved in transmitting bystander effects. These studies show that H2AX phosphorylation is an early step in the bystander effect and that the DNA DSBs underlying γ-H2AX focus formation may be responsible for its downstream manifestations.

Atm heterozygous mice are more sensitive to radiation-induced cataracts than are their wild-type counterparts

It is important to know whether the human population includes genetically predisposed radiosensitive subsets. In vitro studies have shown that cells from individuals homozygous for ataxia telangiectasia (A-T) are much more radiosensitive than cells from unaffected individuals. Although cells heterozygous for the ATM gene (ATM+/−) may be slightly more radiosensitive in vitro, it remained to be determined whether the greater susceptibility of ATM+/− cells translates into an increased sensitivity for late effects in vivo, though there is a suggestion that radiotherapy patients that are heterozygous for the ATM gene may be more at risk of developing late normal tissue damage. We chose cataractogenesis in the lens as a means to assay for the effects of ATM deficiency in a late-responding tissue. One eye of wild-type, Atm heterozygous and homozygous knockout mice was exposed to 0.5-, 1.0-, 2.0-, or 4.0-Gy x rays. The animals were followed weekly for cataract development by conventional slit-lamp biomicroscopy. Cataract development in the animals of all three groups was strongly dependent on dose. The lenses of homozygous mice were the first to opacify at any given dose. Most important in the present context is that cataracts appeared earlier in the heterozygous versus wild-type animals. The data suggest that ATM heterozygotes in the human population may also be radiosensitive. This may influence the choice of individuals destined to be exposed to higher than normal doses of radiation, such as astronauts, and may also suggest that radiotherapy patients who are ATM heterozygotes could be predisposed to increased late normal tissue damage.

Influence of ATM function on telomere metabolism

The ATM gene product, which is defective in the cancer-prone disorder ataxia telangiectasia, has been implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination and cell cycle control. The ATM gene has homology with the TEL1 gene of yeast, mutations of which lead to shortened telomeres. To test the hypothesis that the ATM gene product is involved in telomere metabolism, we examined telomeric associations (TA), telomere length, and telomerase activity in human cells expressing either dominant-negative or complementing fragments of the ATM gene. The phenotype of RKO colorectal tumor cells expressing ATM fragments containing a leucine zipper (LZ) motif mimics that of ataxia telangiectasia (A-T) cells. These transfected RKO cells relative to transfected controls had a higher frequency of cells with TA and shortened telomeres, but no detectable change in telomerase activity. In addition, the percentage of cells with TA after gamma irradiation was higher in the transfected RKO cells with dominant negative activity of the ATM gene, compared to control cells. SV40 transformed fibroblasts derived from an A-T patient and transfected with a complementing carboxyl terminal kinase region of the ATM gene had a reduced frequency of cells with TA, with no effect on the telomere length or telomerase activity. The present studies using isogenic cells with manipulated ATM function demonstrate a role for the ATM gene product in telomere metabolism.

Radiation-Induced Micro-RNA Expression Changes in Peripheral Blood Cells of Radiotherapy Patients

PURPOSE MicroRNAs (miRNAs), a class of noncoding small RNAs that regulate gene expression, are involved in numerous physiologic processes in normal and malignant cells. Our in vivo study measured miRNA and gene expression changes in human blood cells in response to ionizing radiation, to develop miRNA signatures that can be used as biomarkers for radiation exposure. METHODS AND MATERIALS Blood from 8 radiotherapy patients in complete remission 1 or 2 was collected immediately before and 4 hours after total body irradiation with 1.25 Gy x-rays. Both miRNA and gene expression changes were measured by means of quantitative polymerase chain reaction and microarray hybridization, respectively. Hierarchic clustering, multidimensional scaling, class prediction, and gene ontology analysis were performed to investigate the potential of miRNAs to serve as radiation biomarkers and to elucidate their likely physiologic roles in the radiation response. RESULTS The expression levels of 45 miRNAs were statistically significantly upregulated 4 hours after irradiation with 1.25 Gy x-rays, 27 of them in every patient. Nonirradiated and irradiated samples form separate clusters in hierarchic clustering and multidimensional scaling. Out of 223 differentially expressed genes, 37 were both downregulated and predicted targets of the upregulated miRNAs. Paired and unpaired miRNA-based classifiers that we developed can predict the class membership of a sample with unknown irradiation status, with accuracies of 100% when all 45 upregulated miRNAs are included. Both miRNA control of and gene involvement in biologic processes such as hemopoiesis and the immune response are increased after irradiation, whereas metabolic processes are underrepresented among all differentially expressed genes and the genes controlled by miRNAs. CONCLUSIONS Exposure to ionizing radiation leads to the upregulation of the expression of a considerable proportion of the human miRNAome of peripheral blood cells. These miRNA expression signatures can be used as biomarkers of radiation exposure.

ALTERED TELOMERE NUCLEAR MATRIX INTERACTIONS AND NUCLEOSOMAL PERIODICITY IN ATAXIA TELANGIECTASIA CELLS BEFORE AND AFTER IONIZING RADIATION TREATMENT

ABSTRACT Cells derived from ataxia telangiectasia (A-T) patients show a prominent defect at chromosome ends in the form of chromosome end-to-end associations, also known as telomeric associations, seen at G1, G2, and metaphase. Recently, we have shown that the ATM gene product, which is defective in the cancer-prone disorder A-T, influences chromosome end associations and telomere length. A possible hypothesis explaining these results is that the defective telomere metabolism in A-T cells are due to altered interactions between the telomeres and the nuclear matrix. We examined these interactions in nuclear matrix halos before and after radiation treatment. A difference was observed in the ratio of soluble versus matrix-associated telomeric DNA between cells derived from A-T and normal individuals. Ionizing radiation treatment affected the ratio of soluble versus matrix-associated telomeric DNA only in the A-T cells. To test the hypothesis that the ATM gene product is involved in interactions between telomeres and the nuclear matrix, we examined such interactions in human cells expressing either a dominant-negative effect or complementation of the ATMgene. The phenotype of RKO colorectal tumor cells expressing ATM fragments containing a leucine zipper motif mimics the altered interactions of telomere and nuclear matrix similar to that of A-T cells. A-T fibroblasts transfected with wild-type ATM gene had corrected telomere-nuclear matrix interactions. Further, we found that A-T cells had different micrococcal nuclease digestion patterns compared to normal cells before and after irradiation, indicating differences in nucleosomal periodicity in telomeres. These results suggest that the ATM gene influences the interactions between telomeres and the nuclear matrix, and alterations in telomere chromatin could be at least partly responsible for the pleiotropic phenotypes of the ATM gene.

Cell Cycle Activation in Postmitotic Neurons is Essential for DNA Repair

Increasing evidence indicates that maintenance of neuronal homeostasis involves theactivation of the cell cycle machinery in postmitotic neurons. Our recent findings suggestthat cell cycle activation is essential for DNA damage-induced neuronal apoptosis.However, whether the cell division cycle also participates in DNA repair and survival ofpostmitotic, terminally differentiated neurons, is unknown. Here, we tested thehypothesis that G1 phase components contribute to the repair of DNA and are involved inthe DNA damage response of postmitotic neurons. In cortical terminally differentiatedneurons, treatment with subtoxic concentrations of hydrogen peroxide (H2O2) causedrepairable DNA double-strand breaks (DSBs) and the activation of G1 components of thecell cycle machinery. Importantly, DNA repair was attenuated if cyclin-dependentkinases CDK4 and CDK6, essential elements of G0→G1 transition, were suppressed.Our data suggest that G1 cell cycle components are involved in DNA repair and survivalof postmitotic neurons.

Whole mouse blood microRNA as biomarkers for exposure to γ-rays and (56)Fe ion.

Purpose: Biomarkers of ionising radiation exposure are useful in a variety of scenarios, such as medical diagnostic imaging, occupational exposures, and spaceflight. This study investigates to what extent microRNA (miRNA) expression signatures in mouse peripheral blood can be used as biomarkers for exposures to radiation with low and high linear energy transfers. Materials and methods: Mice were irradiated with doses of 0.5, 1.5, or 5.0 Gy γ-rays (dose rate of 0.0136 Gy/s) or with doses of 0.1 or 0.5 Gy 56Fe ions (dose rate of 0.00208 Gy/s). Total RNA was isolated from whole blood at 6 h or 24 h after irradiation. Three animals per irradiation condition were used. Differentially expressed miRNA were determined by means of quantitative real-time polymerase chain reaction. Results: miRNA expression signatures were radiation type-specific and dose- and time-dependent. The differentially expressed miRNA were expressed in either one condition (71%) or multiple conditions (29%). Classifiers based on the differentially expressed miRNA predicted radiation type or dose with accuracies between 75% and 100%. Gene-ontology analyses show that miRNA induced by irradiation are involved in the control of several biological processes, such as mRNA transcription regulation, nucleic-acid metabolism, and development. Conclusion: miRNA signatures induced by ionising radiation in mouse blood are radiation type- and radiation dose-specific. These findings underline the complexity of the radiation response and the importance of miRNA in it.

Mrad9 and atm haploinsufficiency enhance spontaneous and X-ray-induced cataractogenesis in mice.

Abstract Kleiman, N. J., David, J., Elliston, C. D., Hopkins, K. M., Smilenov, L. B., Brenner, D. J., Worgul, B. V., Hall, E. J. and Lieberman, H. B. Mrad9 and Atm Haploinsufficiency Enhance Spontaneous and X-Ray-Induced Cataractogenesis in Mice. Radiat. Res. 168, 567–573 (2007). Rad9 and Atm regulate multiple cellular responses to DNA damage, including cell cycle checkpoints, DNA repair and apoptosis. However, the impact of dual heterozygosity for Atm and Rad9 is unknown. Using 50 cGy of X rays as an environmental insult and cataractogenesis as an end point, this study examined the effect of heterozygosity for one or both genes in mice. Posterior subcapsular cataracts, characteristic of radiation exposure, developed earlier in X-irradiated double heterozygotes than in single heterozygotes, which were more prone to cataractogenesis than wild-type controls. Cataract onset time and progression in single or double heterozygotes were accelerated even in unirradiated eyes. These findings indicate that the cataractogenic effect of combined heterozygosity is greater than for each gene alone and are the first to demonstrate the impact of multiple haploinsufficiency on radiation effects in an intact mammal. These observations may help explain observed interindividual differential radiosensitivity in human populations and have important implications for those undergoing radiotherapy or exposed to elevated levels of cosmic radiation, such as the astronaut corps. These findings demonstrate that Mrad9 and Atm are important determinants of lens opacification and, given the roles of Atm and Rad9 in maintaining genomic stability, are consistent with a genotoxic basis for radiation cataractogenesis.

Integrin Functions Play a Key Role in the Differentiation of Thymocytes In Vivo

T cells express a variety of surface proteins as they develop to maturity in the thymus. In addition to the TCR-CD3 complex and the two major coreceptors, CD4 and CD8, other surface proteins expressed include receptors for cytokines, growth factors, counterreceptors, and extracellular matrix molecules. To determine the role of integrin adhesion receptors in T cell development, we have expressed a trans-dominant inhibitor of integrin function in the thymus. This inhibitor leads to a block of adhesion to fibronectin due to reduced activation of integrin receptors. This reduced adhesion leads to a partial block in differentiation from CD4−CD8− cells to CD4+CD8+ cells, after the CD25+ stage, suggesting that integrins are important during Lck-mediated differentiation. Furthermore, the overall production of CD4+ cells is reduced compared with that of CD8+ cells without changes in negative selection, suggesting that integrins may be involved in the determination of the fate of the cell as well. These results demonstrate that integrin receptor function is required for proper thymocyte development in vivo.