DNMT1 and DNMT3B regulate tumorigenicity of human prostate cancer cells by controlling RAD9 expression through targeted methylation.

Prostate cancer is the second most common type of cancer and the second leading cause of cancer death in American men. RAD9 stabilizes the genome, but prostate cancer cells and tumors often have high quantities of the protein. Reduction of RAD9 level within prostate cancer cells decreases tumorigenicity of nude mouse xenographs and metastasis phenotypes in culture, indicating that RAD9 overproduction is essential for the disease. In prostate cancer DU145 cells, CpG hypermethylation in a transcription suppressor site of RAD9 intron 2 causes high-level gene expression. Herein, we demonstrate that DNA methyltransferases DNMT1 and DNMT3B are highly abundant in prostate cancer cells DU145, CWR22, LNCaP and PC-3; yet, these DNMTs bind primarily to the transcription suppressor in DU145, the only cells where methylation is critical for RAD9 regulation. For DU145 cells, DNMT1 or DNMT3B shRNA reduced RAD9 level and tumorigenicity, and RAD9 ectopic expression restored this latter activity in the DNMT knockdown cells. High levels of RAD9, DNMT1, DNMT3B and RAD9 transcription suppressor hypermethylation were significantly correlated in prostate tumors, and not in normal prostate tissues. Based on these results, we propose a novel model where RAD9 is regulated epigenetically by DNMT1 and DNMT3B, via targeted hypermethylation, and that consequent RAD9 overproduction promotes prostate tumorigenesis.

RAD9A promotes metastatic phenotypes through transcriptional regulation of anterior gradient 2 (AGR2).

RAD9A plays an important role in prostate tumorigenesis and metastasis-related phenotypes. The protein classically functions as part of the RAD9A-HUS1-RAD1 complex but can also act independently. RAD9A can selectively transactivate multiple genes, including CDKN1A and NEIL1 by binding p53-consensus sequences in or near promoters. RAD9A is overexpressed in human prostate cancer specimens and cell lines; its expression correlates with tumor progression. Silencing RAD9A in prostate cancer cells impairs their ability to form tumors in vivo and migrate as well as grow anchorage independently in vitro. We demonstrate herein that RAD9A transcriptionally controls AGR2, a gene aberrantly overexpressed in patients with metastatic prostate cancer. Transient or stable knockdown of RAD9A in PC-3 cells caused downregulation of AGR2 protein abundance. Reduced AGR2 protein levels were due to lower abundance of AGR2 mRNA. The AGR2 genomic region upstream of the coding initiation site contains several p53 consensus sequences. RAD9A bound specifically to the 5'-untranslated region of AGR2 in PC-3 cells at a partial p53 consensus sequence at position +3136 downstream from the transcription start site, determined by chromatin immunoprecipitation, followed by PCR amplification. Binding of RAD9A to the p53 consensus sequence was sufficient to drive AGR2 gene transcription, shown by a luciferase reporter assay. In contrast, when the RAD9A-binding sequence on the AGR2 was mutated, no luciferase activity was detected. Knockdown of RAD9A in PC-3 cells impaired cell migration and anchorage-independent growth. However, ectopically expressed AGR2 in RAD9A-depleted PC-3 cells restored these phenotypes. Our results suggest RAD9A drives metastasis by controlling AGR2 abundance.

Regulation of NEIL1 protein abundance by RAD9 is important for efficient base excision repair.

RAD9 participates in DNA damage-induced cell cycle checkpoints and DNA repair. As a member of the RAD9-HUS1-RAD1 (9-1-1) complex, it can sense DNA damage and recruit ATR to damage sites. RAD9 binding can enhance activities of members of different DNA repair pathways, including NEIL1 DNA glycosylase, which initiates base excision repair (BER) by removing damaged DNA bases. Moreover, RAD9 can act independently of 9-1-1 as a gene-specific transcription factor. Herein, we show that mouse Rad9(-/-) relative to Rad9(+/+) embryonic stem (ES) cells have reduced levels of Neil1 protein. Also, human prostate cancer cells, DU145 and PC-3, knocked down for RAD9 demonstrate reduced NEIL1 abundance relative to controls. We found that Rad9 is required for Neil1 protein stability in mouse ES cells, whereas it regulates NEIL1 transcription in the human cells. RAD9 depletion enhances sensitivity to UV, gamma rays and menadione, but ectopic expression of RAD9 or NEIL1 restores resistance. Glycosylase/apurinic lyase activity was reduced in Rad9(-/-) mouse ES and RAD9 knocked-down human prostate cancer whole cell extracts, relative to controls. Neil1 or Rad9 addition restored this incision activity. Thus, we demonstrate that RAD9 regulates BER by controlling NEIL1 protein levels, albeit by different mechanisms in human prostate cancer versus mouse ES cells.

The DNA damage checkpoint protein RAD9A is essential for male meiosis in the mouse.

In mitotic cells, RAD9A functions in repairing DNA double-strand breaks (DSBs) by homologous recombination and facilitates the process by cell cycle checkpoint control in response to DNA damage. DSBs occur naturally in the germline during meiosis but whether RAD9A participates in repairing such breaks is not known. In this study, we determined that RAD9A is indeed expressed in the male germ line with a peak of expression in late pachytene and diplotene stages, and the protein was found associated with the XY body. As complete loss of RAD9A is embryonic lethal, we constructed and characterized a mouse strain with Stra8-Cre driven germ cell-specific ablation of Rad9a beginning in undifferentiated spermatogonia in order to assess its role in spermatogenesis. Adult mutant male mice were infertile or sub-fertile due to massive loss of spermatogenic cells. The onset of this loss occurs during meiotic prophase, and there was an increase in the numbers of apoptotic spermatocytes as determined by TUNEL. Spermatocytes lacking RAD9A usually arrested in meiotic prophase, specifically in pachytene. The incidence of unrepaired DNA breaks increased, as detected by accumulation of γH2AX and DMC1 foci on the axes of autosomal chromosomes in pachytene spermatocytes. The DNA topoisomerase IIß-binding protein 1 (TOPBP1) was still localized to the sex body, albeit with lower intensity, suggesting that RAD9A may be dispensable for sex body formation. We therefore show for the first time that RAD9A is essential for male fertility and for repair of DNA DSBs during meiotic prophase I.

RAD9 enhances radioresistance of human prostate cancer cells through regulation of ITGB1 protein levels.

BACKGROUND: Mouse embryonic stem cells null for Rad9 are sensitive to deleterious effects of ionizing radiation exposure. Likewise, integrin ß1 is a known radioprotective factor. Previously, we showed that RAD9 downregulation in human prostate cancer cells reduces integrin ß1 protein levels and ectopic expression of Mrad9 restores inherent high levels. METHODS: We used RNA interference to knockdown Rad9 expression in PC3 and DU145 prostate cancer cells. These cells were then exposed to ionizing radiation, and integrin ß1 protein levels were measured by immunoblotting. Survival of irradiated cells was measured by clonogenicity, cell cycle analysis, PARP-1 cleavage, and trypan blue exclusion. RESULTS: The function of RAD9 in controlling integrin ß1 expression is unique and not shared by the other members of the 9-1-1 complex, HUS1 and RAD1. RAD9 or integrin ß1 silencing sensitizes DU145 and PC3 cells to ionizing radiation. Irradiation of DU145 cells with low levels of RAD9 induces cleavage of PARP-1 protein. High levels of ionizing radiation have no effect on integrin ß1 protein levels. However, when RAD9 downregulation is combined with 10 Gy of ionizing radiation in DU145 or PC3 cells, there is an additional 50% downregulation of integrin ß1 compared with levels in unirradiated RAD9 knockdown cells. Finally, PC3 cells growing on fibronectin display increased radioresistance. However, PC3 cells with RAD9 knockdown are no longer protected by fibronectin after treatment with ionizing radiation. CONCLUSIONS: Downregulation of RAD9 when combined with ionizing radiation results in reduction of ITGB1 protein levels in prostate cancer cells, and increased lethality.

PARP inhibition selectively increases sensitivity to cisplatin in ERCC1-low non-small cell lung cancer cells.

Platinum compounds are the foundation of chemotherapy regimens for non-small cell lung cancer (NSCLC) despite poor response rates and limited response duration. It has been reported that tumor expression of excision repair cross-complementation group 1 (ERCC1), a key component in nucleotide excision repair, may correlate with clinical response to platinum agents. We found that most primary lung tumor specimens demonstrated a stronger protein expression of poly (adenosine diphosphate ribose) polymerases 1 (PARP1) than their normal counterparts. Therefore, we hypothesized that combining PARP inhibition with platinum compounds may be an approach to improve platinum-based therapy for NSCLC. Drug combination experiments revealed that two distinct PARP inhibitors, olaparib and veliparib, not only potentiated the cell killing by cisplatin but also conferred cytotoxicity as a single agent specifically in ERCC1-low HCC827 and PC9 but not in ERCC1-high A549 and H157 lung cancer cells. Moreover, small interfering RNA knockdown of ERCC1 in A549 and H157 cells increased their sensitivities to both cisplatin and olaparib in a synergistic manner in our model. Furthermore, mechanistic studies indicated that combined PARP inhibitor and cisplatin could lead to sustained DNA double-strand breaks, prolonged G2/M cell cycle arrest with distinct activation of checkpoint kinase 1 signaling and more pronounced apoptosis preferentially in lung cancer cells with low ERCC1 expression. Collectively, these data suggest that there is a synergistic relationship between PARP inhibition and low ERCC1 expression in NSCLC that could be exploited for novel therapeutic approaches in lung cancer therapy based on tumor ERCC1 expression.

Rad9 protein contributes to prostate tumor progression by promoting cell migration and anoikis resistance.

Rad9 as part of the Rad9-Hus1-Rad1 complex is known to participate in cell cycle checkpoint activation and DNA repair. However, Rad9 can act as a sequence-specific transcription factor, modulating expression of a number of genes. Importantly, Rad9 is up-regulated in prostate cancer cell lines and clinical specimens. Its expression correlates positively with advanced stage tumors and its down-regulation reduces tumor burden in mice. We show here that transient down-regulation of Rad9 by RNA interference reduces DU145 and PC3 prostate cancer cell proliferation and survival in vitro. In addition, transient or stable down-regulation of Rad9 impairs migration and invasion of the cells. Moreover, stable reduction of Rad9 renders DU145 cell growth anchorage-dependent. It also decreases expression of integrin ß1 protein and sensitizes DU145 and LNCaP cells to anoikis and impairs Akt activation. On the other hand, stable expression of Mrad9, the mouse homolog, in DU145/shRNA Rad9 cells restores migration, invasion, anchorage-independent growth, integrin ß1 expression, and anoikis resistance with a concomitant elevation of Akt activation. We thus demonstrate for the first time that Rad9 contributes to prostate tumorigenesis by increasing not only tumor proliferation and survival but also tumor migration and invasion, anoikis resistance, and anchorage-independent growth.

Contributions of Rad9 to tumorigenesis.

Rad9 plays a crucial role in maintaining genomic stability by regulating cell cycle checkpoints, DNA repair, telomere stability, and apoptosis. Rad9 controls these processes mainly as part of the heterotrimeric 9-1-1 (Rad9-Hus1-Rad1) complex. However, in recent years it has been demonstrated that Rad9 can also act independently of the 9-1-1 complex as a transcriptional factor, participate in immunoglobulin class switch recombination, and show 3'-5' exonuclease activity. Aberrant Rad9 expression has been associated with prostate, breast, lung, skin, thyroid, and gastric cancers. High expression of Rad9 is causally related to, at least, human prostate cancer growth. On the other hand, deletion of Mrad9, the mouse homolog, is responsible for increased skin cancer incidence. These results reveal that Rad9 can act as an oncogene or tumor suppressor. Which of the many functions of Rad9 are causally related to initiation and progression of tumorigenesis and the mechanistic details by which Rad9 induces or suppresses tumorigenesis are presently not known, but are crucial for the development of targeted therapeutic interventions.

Mouse Rad9b is essential for embryonic development and promotes resistance to DNA damage.

RAD9 participates in promoting resistance to DNA damage, cell cycle checkpoint control, DNA repair, apoptosis, embryogenesis, and regulation of transcription. A paralogue of RAD9 (named RAD9B) has been identified. To define the function of mouse Rad9b (Mrad9b), embryonic stem (ES) cells with a targeted gene deletion were constructed and used to generate Mrad9b mutant mice. Mrad9b(-/-) embryos are resorbed after E7.5 while some of the heterozygotes die between E12.5 and a few days after birth. Mrad9b is expressed in embryonic brain and Mrad9b(+/-) embryos exhibit abnormal neural tube closure. Mrad9b(-/-) mouse embryonic fibroblasts are not viable. Mrad9b(-/-) ES cells are more sensitive to gamma rays and mitomycin C than Mrad9b(+/+) controls, but show normal gamma-ray-induced G2/M checkpoint control. There is no evidence of spontaneous genomic instability in Mrad9b(-/-) cells. Our findings thus indicate that Mrad9b is essential for embryonic development and mediates resistance to certain DNA damaging agents.

FOXP1 and NDRG1 act differentially as downstream effectors of RAD9-mediated prostate cancer cell functions.

Metastatic progression is the key feature of prostate cancer primarily responsible for mortality caused by this disease. RAD9 is an oncogene for prostate cancer, and the encoded protein enhances metastasis-related phenotypes. RAD9 is a transcription factor with a limited set of regulated target genes, but the complete list of downstream genes critical for prostate carcinogenesis is unknown. We used microarray gene expression profiling and chromatin immunoprecipitation in parallel to identify genes transcriptionally controlled by RAD9 that contribute to this cancer. We found expression of 44 genes altered in human prostate cancer DU145 cells when RAD9 is knocked down by siRNA, and all of them bind RAD9 at their genomic location. FOXP1 and NDRG1 were down regulated when RAD9 expression was reduced, and we evaluated them further. We demonstrate that reduced RAD9, FOXP1 or NDGR1 expression decreases cell proliferation, rapid migration, anchorage-independent growth, anoikis resistance, and aerobic glycolysis. Ectopic expression of FOXP1 or NDRG1 partially restored aerobic glycolysis to prostate cancer cells with reduced RAD9 abundance, but only FOXP1 significantly complemented the other deficiencies. We thus show, for the first time, that RAD9 regulates FOXP1 and NDRG1 expression, and they function differently as downstream effectors for RAD9-mediated prostate cancer cell activities.

Mouse Rad1 deletion enhances susceptibility for skin tumor development.

BACKGROUND: Cells 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. RESULTS: To 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. CONCLUSION: These 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.

Targeting MEK5 impairs nonhomologous end-joining repair and sensitizes prostate cancer to DNA damaging agents.

Radiotherapy is commonly used to treat a variety of solid human tumors, including localized prostate cancer. However, treatment failure often ensues due to tumor intrinsic or acquired radioresistance. Here we find that the MEK5/ERK5 signaling pathway is associated with resistance to genotoxic stress in aggressive prostate cancer cells. MEK5 knockdown by RNA interference sensitizes prostate cancer cells to ionizing radiation (IR) and etoposide treatment, as assessed by clonogenic survival and short-term proliferation assays. Mechanistically, MEK5 downregulation impairs phosphorylation of the catalytic subunit of DNA-PK at serine 2056 in response to IR or etoposide treatment. Although MEK5 knockdown does not influence the initial appearance of radiation- and etoposide-induced γH2AX and 53BP1 foci, it markedly delays their resolution, indicating a DNA repair defect. A cell-based assay shows that nonhomologous end joining (NHEJ) is compromised in cells with ablated MEK5 protein expression. Finally, MEK5 silencing combined with focal irradiation causes strong inhibition of tumor growth in mouse xenografts, compared with MEK5 depletion or radiation alone. These findings reveal a convergence between MEK5 signaling and DNA repair by NHEJ in conferring resistance to genotoxic stress in advanced prostate cancer and suggest targeting MEK5 as an effective therapeutic intervention in the management of this disease.

DUSP1 is controlled by p53 during the cellular response to oxidative stress.

p53 controls the cellular response to genotoxic stress through multiple mechanisms. We report here that p53 regulates DUSP1, a dual-specific threonine and tyrosine phosphatase with stringent substrate specificity for mitogen-activated protein kinase (MAPK). DUSP1 is a potent inhibitor of MAPK activity through dephosphorylation of MAPK. In a colon cancer cell line containing inducible ectopic p53, DUSP1 protein level is significantly increased upon activation of p53, leading to cell death in response to nutritional stress. In mouse embryo fibroblast cells, DUSP1 protein abundance is greatly increased after oxidative stress in a p53-dependent manner and also when apoptosis is triggered. We show that p53 induces the activity of a human DUSP1 regulatory region. Furthermore, p53 can physically interact with the DUSP1 regulatory region in vivo, and p53 binds to a 10-bp perfect palindromic site in this DUSP1 regulatory region. We show that overexpression of DUSP1 or inhibition of MAPK activity significantly increases cellular susceptibility to oxidative damage. These findings indicate that p53 is a transcriptional regulator of DUSP1 in stress responses. Our results reveal a mechanism whereby p53 selectively regulates target genes and suggest a way in which subgroups of those target genes might be controlled independently.

Mammalian Rad9 plays a role in telomere stability, S- and G2-phase-specific cell survival, and homologous recombinational repair.

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 gamma-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.

DNA damage repair and response proteins as targets for cancer therapy.

The cellular response to DNA damage is critical for determining whether carcinogenesis, cell death or other deleterious biological effects will ensue. Numerous cellular enzymatic mechanisms can directly repair damaged DNA, or allow tolerance of DNA lesions, and thus reduce potential harmful effects. These processes include base excision repair, nucleotide excision repair, nonhomologous end joining, homologous recombinational repair and mismatch repair, as well as translesion synthesis. Furthermore, DNA damage-inducible cell cycle checkpoint systems transiently delay cell cycle progression. Presumably, this allows extra time for repair before entry of cells into critical phases of the cell cycle, an event that could be lethal if pursued with damaged DNA. When damage is excessive apoptotic cellular suicide mechanisms can be induced. Many of the survival-promoting pathways maintain genomic integrity even in the absence of exogenous agents, thus likely processing spontaneous damage caused by the byproducts of normal cellular metabolism. DNA damage can initiate cancer, and radiological as well as chemical agents used to treat cancer patients often cause DNA damage. Many genes are involved in each of the DNA damage processing mechanisms, and the encoded proteins could ultimately serve as targets for therapy, with the goal of neutralizing their ability to repair damage in cancer cells. Therefore, modulation of DNA damage responses coupled with more conventional radiotherapy and chemotherapy approaches could sensitize cancer cells to treatment. Alteration of DNA damage response genes and proteins should thus be considered an important though as of yet not fully exploited avenue to enhance cancer therapy.

Mechanism of radiation-induced bystander effects: a unifying model.

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.

Prostate cancer: unmet clinical needs and RAD9 as a candidate biomarker for patient management.

Prostate cancer is a complex disease, with multiple subtypes and clinical presentations. Much progress has been made in recent years to understand the underlying genetic basis that drives prostate cancer. Such mechanistic information is useful for development of novel therapeutic targets, to identify biomarkers for early detection or to distinguish between aggressive and indolent disease, and to predict treatment outcome. Multiple tests have become available in recent years to address these clinical needs for prostate cancer. We describe several of these assays, summarizing test details, performance characteristics, and acknowledging their limitations. There is a pressing unmet need for novel biomarkers that can demonstrate improvement in these areas. We introduce one such candidate biomarker, RAD9, describe its functions in the DNA damage response, and detail why it can potentially fill this void. RAD9 has multiple roles in prostate carcinogenesis, making it potentially useful as a clinical tool for men with prostate cancer. RAD9 was originally identified as a radioresistance gene, and subsequent investigations revealed several key functions in the response of cells to DNA damage, including involvement in cell cycle checkpoint control, at least five DNA repair pathways, and apoptosis. Further studies indicated aberrant overexpression in approximately 45% of prostate tumors, with a strong correlation between RAD9 abundance and cancer stage. A causal relationship between RAD9 and prostate cancer was first demonstrated using a mouse model, where tumorigenicity of human prostate cancer cells after subcutaneous injection into nude mice was diminished when RNA interference was used to reduce the normally high levels of the protein. In addition to activity needed for the initial development of tumors, cell culture studies indicated roles for RAD9 in promoting prostate cancer progression by controlling cell migration and invasion through regulation of ITGB1 protein levels, and anoikis resistance by modulating AKT activation. Furthermore, RAD9 enhances the resistance of human prostate cancer cells to radiation in part by regulating ITGB1 protein abundance. RAD9 binds androgen receptor and inhibits androgen-induced androgen receptor's activity as a transcription factor. Moreover, RAD9 also acts as a gene-specific transcription factor, through binding p53 consensus sequences at target gene promoters, and this likely contributes to its oncogenic activity. Given these diverse and extensive activities, RAD9 plays important roles in the initiation and progression of prostate cancer and can potentially serve as a valuable biomarker useful in the management of patients with this disease.

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

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.

Deletion of mouse rad9 causes abnormal cellular responses to DNA damage, genomic instability, and embryonic lethality.

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 G(2) 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.

Combined haploinsufficiency for ATM and RAD9 as a factor in cell transformation, apoptosis, and DNA lesion repair dynamics.

Loss of function of oncogenes, tumor suppressor genes and DNA damage processing genes has been implicated in the development of many types of cancer, but for the vast majority of cases, there is no link to specific germ line mutations. In the last several years, heterozygosity leading to haploinsufficiency for proteins involved in DNA repair pathways was shown to play a role in genomic instability and carcinogenesis after DNA damage is induced. Because the effect of haploinsufficiency for one protein is relatively small, we hypothesize that predisposition to cancer could be a result of the additive effect of heterozygosity for two or more genes, critical for pathways that control DNA damage signaling, repair or apoptosis. To address this issue, primary mouse cells, haploinsufficient for one or two proteins, ATM and RAD9, related to the cellular response to DNA damage were examined. The results show that cells having low levels of both ATM and RAD9 proteins are more sensitive to transformation by radiation, have different DNA double-strand break repair dynamics and are less apoptotic when compared with wild-type controls or those cells haploinsufficient for only one of these proteins. Our conclusions are that under stress conditions, the efficiency and capacity for DNA repair mediated by the ATM/RAD9 cell signaling network depend on the abundance of both proteins and that, in general, DNA repair network efficiencies are genotype-dependent and can vary within a specific range.