Kevin M. Hopkins

Human homologue of S. pombe Rad9 interacts with BCL-2/BCL-xL and promotes apoptosis.

DNA damage induces apoptosis through a signalling pathway that can be suppressed by the BCL-2 protein, but the mechanism by which DNA damage does this is unknown. Here, using yeast two-hybrid and co-immunoprecipitation studies, we show that RAD9, a human protein involved in the control of a cell-cycle checkpoint, interacts with the anti-apoptotic Bcl-2-family proteins BCL-2 and BCL-xL, but not with the pro-apoptotic BAX and BAD. When overexpressed in mammalian cells, RAD9 induces apoptosis that can be blocked by BCL-2 or BCL-xL. Conversely, antisense RAD9 RNA suppresses cell death induced by methyl methanesulphonate. These findings indicate that RAD9 may have a new role in regulating apoptosis after DNA damage, in addition to its previously described checkpoint-control and other radioresistance-promoting functions.

Mammalian Rad9 Plays a Role in Telomere Stability, S- and G2-Phase-Specific Cell Survival, and Homologous Recombinational Repair

ABSTRACT The protein products of several rad checkpoint genes of Schizosaccharomyces pombe (rad1+, rad3 +, rad9 +, rad17 +, rad26 +, and hus1 +) play crucial roles in sensing changes in DNA structure, and several function in the maintenance of telomeres. When the mammalian homologue of S. pombe Rad9 was inactivated, increases in chromosome end-to-end associations and frequency of telomere loss were observed. This telomere instability correlated with enhanced S- and G2-phase-specific cell killing, delayed kinetics of γ-H2AX focus appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase-specific DNA damage repair. Furthermore, mammalian Rad9 interacted with Rad51, and inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S and G2 phases of the cell cycle. Together, these findings provide evidence of roles for mammalian Rad9 in telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.

Deletion of Mouse Rad9 Causes Abnormal Cellular Responses to DNA Damage, Genomic Instability, and Embryonic Lethality

ABSTRACT The fission yeast Schizosaccharomyces pombe rad9 gene promotes cell survival through activation of cell cycle checkpoints induced by DNA damage. Mouse embryonic stem cells with a targeted deletion of Mrad9, the mouse ortholog of this gene, were created to evaluate its function in mammals. Mrad9−/− cells demonstrated a marked increase in spontaneous chromosome aberrations and HPRT mutations, indicating a role in the maintenance of genomic integrity. These cells were also extremely sensitive to UV light, gamma rays, and hydroxyurea, and heterozygotes were somewhat sensitive to the last two agents relative to Mrad9 +/+ controls. Mrad9 −/− cells could initiate but not maintain gamma-ray-induced G2 delay and retained the ability to delay DNA synthesis rapidly after UV irradiation, suggesting that checkpoint abnormalities contribute little to the radiosensitivity observed. Ectopic expression of Mrad9 or human HRAD9 complemented Mrad9 −/− cell defects, indicating that the gene has radioresponse and genomic maintenance functions that are evolutionarily conserved. Mrad9 +/− mice were generated, but heterozygous intercrosses failed to yield Mrad9 −/− pups, since embryos died at midgestation. Furthermore, Mrad9 −/− mouse embryo fibroblasts were not viable. These investigations establish Mrad9 as a key mammalian genetic element of pathways that regulate the cellular response to DNA damage, maintenance of genomic integrity, and proper embryonic development.

Fission Yeast rad12+ Regulates Cell Cycle Checkpoint Control and Is Homologous to the Bloom’s Syndrome Disease Gene

ABSTRACT The human BLM gene is a member of the Escherichia coli recQ helicase family, which includes the Saccharomyces cerevisiae SGS1 and human WRN genes. Defects inBLM are responsible for the human disease Bloom’s syndrome, which is characterized in part by genomic instability and a high incidence of cancer. Here we describe the cloning ofrad12+, which is the fission yeast homolog ofBLM and is identical to the recently reportedrhq1 + gene. We showed that rad12null cells are sensitive to DNA damage induced by UV light and γ radiation, as well as to the DNA synthesis inhibitor hydroxyurea. Overexpression of the wild-type rad12 + gene also leads to sensitivity to these agents and to defects associated with the loss of the S-phase and G2-phase checkpoint control. We showed genetically and biochemically thatrad12 + acts upstream fromrad9 +, one of the fission yeast G2checkpoint control genes, in regulating exit from the S-phase checkpoint. The physical chromosome segregation defects seen inrad12 null cells combined with the checkpoint regulation defect seen in the rad12 + overproducer implicate rad12 + as a key coupler of chromosomal integrity with cell cycle progression.

The Radiation-Induced Bystander Effect for Clonogenic Survival

Abstract Sawant, S. G., Zheng, W., Hopkins, K. M., Randers-Pehrson, G., Lieberman, H. B. and Hall, E. J. The Radiation-Induced Bystander Effect for Clonogenic Survival. Radiat. Res. 157, 361–364 (2002). It has long been accepted that the radiation-induced heritable effects in mammalian cells are the result of direct DNA damage. Recent evidence, however, suggests that when a cell population is exposed to a low dose of α particles, biological effects occur in a larger proportion of cells than are estimated to have been traversed by α particles. Experiments involving the Columbia University microbeam, which allows a known fraction of cells to be traversed by a defined number of α particles, have demonstrated a bystander effect for clonogenic survival and oncogenic transformation in C3H 10T½ cells. When 1 to 16 α particles were passed through the nuclei of 10% of a C3H 10T½ cell population, more cells were unable to form colonies than were actually traversed by α particles. Both hit and non-hit cells contributed to the outcome of the experiments. The present work was undertaken to assess the bystander effect of radiation in only non-hit cells. For this purpose, Chinese hamster V79 cells transfected with hygromycin- or neomycin-resistance genes were used. V79 cells stably transfected with a hygromycin resistance gene and stained with a nuclear dye were irradiated with the charged-particle microbeam in the presence of neomycin-resistant cells. The biological effect was studied in the neomycin-resistant V79 cells after selective removal of the hit cells with geneticin treatment.

Molecular cloning and analysis of Schizosaccharomyces pombe rad9, a gene involved in DNA repair and mutagenesis

SummaryThe mutant allele rad9-192 renders Schizosaccharomyces pombe cells sensitive to ionizing radiation and UV light. We have isolated from a S. pombe genomic DNA library a unique recombinant plasmid that is capable of restoring wild-type levels of radioresistance to a rad9 192-containing cell population. Plasmid integration studies using the cloned DNA, coupled with mating and tetrad analyses, indicate that this isolated DNA contains the wild-type rad9 gene. We inactivated the repair function of the cloned fragment by a single insertion of the S. pombe ura4 gene. This nonfunctional fragment was used to create a viable disruption mutant, thus demonstrating that the rad9 gene does not encode an essential cellular function. In addition, the rad9-192 mutant population is as radiosensitive as the disruption mutant, indicating that rad9 gene function is severely if not totally inhibited by the molecular defect responsible for the rad9-192 phenotype. DNA sequence analysis of rad9 reveals an open reading frame of 1,278 bp, interrupted by three introns 53 bp, 57 bp, and 56 by long, respectively, and ending in the termination codon TAG. This gene is capable of encoding a protein of 426 amino acids, with a corresponding calculated molecular weight of 47,464 daltons. No significant homology was detected between the rad9 gene or its deduced protein sequence and sequences previously entered into DNA and protein sequence data banks.

Schizosaccharomyces pombe Rad9 contains a BH3‐like region and interacts with the anti‐apoptotic protein Bcl‐2

Here we report that the Schizosaccharomyces pombe Rad9 (SpRad9) protein contains a group of amino acids with similarity to the Bcl‐2 homology 3 death domain, which is required for SpRad9 interaction with human Bcl‐2 and apoptosis induction in human cells. Overexpression of Bcl‐2 in S. pombe inhibits cell growth independently of rad9, but enhances resistance of rad9‐null cells to methyl methanesulfonate, ultraviolet and ionizing radiation. These observations suggest that SpRad9 may represent the first member of the Bcl‐2 protein family identified in yeast, though the cell death pathways in S. pombe may differ from those found in mammals.

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.

Targeted Deletion of Rad9 in Mouse Skin Keratinocytes Enhances Genotoxin-Induced Tumor Development

The Rad9 gene is evolutionarily conserved from yeast to humans and plays crucial roles in genomic maintenance, DNA repair, and cell cycle checkpoint controls. However, the function of this gene with respect to tumorigenesis is not well-understood. A Rad9-null mutation in mice causes embryonic lethality. In this study, we created mice in which mouse Rad9, Mrad9, was deleted only in keratinocytes to permit examination of the potential function of the gene in tumor development. Mice with Mrad9(+/-) or Mrad9(-/-) keratinocytes showed no overt, spontaneous morphologic defects and seemed similar to wild-type controls. Painting the carcinogen 7,12-dimethylbenzanthracene (DMBA) onto the skin of the animals caused earlier onset and more frequent formation of tumors and senile skin plaques in Mrad9(-/-) mice, compared with Mrad9(+/-) and Mrad9(+/+) littermates. DNA damage response genes p21, p53, and Mrad9B were expressed at higher levels in Mrad9(-/-) relative to Mrad9(+/+) skin. Keratinocytes isolated from Mrad9(-/-) skin had more spontaneous and DMBA-induced DNA double strand breaks than Mrad9(+/+) keratinocytes, and the levels were reduced by incubation with the antioxidant epigallocatechin gallate. These data suggest that Mrad9 plays an important role in maintaining genomic stability and preventing tumor development in keratinocytes.

Mouse Rad1 deletion enhances susceptibility for skin tumor development

BackgroundCells are constantly exposed to stresses from cellular metabolites as well as environmental genotoxins. DNA damage caused by these genotoxins can be efficiently fixed by DNA repair in cooperation with cell cycle checkpoints. Unrepaired DNA lesions can lead to cell death, gene mutation and cancer. The Rad1 protein, evolutionarily conserved from yeast to humans, exists in cells as monomer as well as a component in the 9-1-1 protein complex. Rad1 plays crucial roles in DNA repair and cell cycle checkpoint control, but its contribution to carcinogenesis is unknown.ResultsTo address this question, we constructed mice with a deletion of Mrad1. Matings between heterozygous Mrad1 mutant mice produced Mrad1+/+ and Mrad1+/- but no Mrad1-/- progeny, suggesting the Mrad1 null is embryonic lethal. Mrad1+/- mice demonstrated no overt abnormalities up to one and half years of age. DMBA-TPA combinational treatment was used to induce tumors on mouse skin. Tumors were larger, more numerous, and appeared earlier on the skin of Mrad1+/- mice compared to Mrad1+/+ animals. Keratinocytes isolated from Mrad1+/- mice had significantly more spontaneous DNA double strand breaks, proliferated slower and had slightly enhanced spontaneous apoptosis than Mrad1+/+ control cells.ConclusionThese data suggest that Mrad1 is important for preventing tumor development, probably through maintaining genomic integrity. The effects of heterozygous deletion of Mrad1 on proliferation and apoptosis of keratinocytes is different from those resulted from Mrad9 heterozygous deletion (from our previous study), suggesting that Mrad1 also functions independent of Mrad9 besides its role in the Mrad9-Mrad1-Mhus1 complex in mouse cells.