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The ubiquitin interaction motif (UIM)-containing protein RAP80 was recently found to play a key role in DNA damage response (DDR) signaling by facilitating the translocation of several DDR mediators, including BRCA1, to ionizing irradiation (IR)-induced foci (IRIF). In this study, we examine the effect of the loss of RAP80 on genomic stability and the susceptibility to cancer development in RAP80 null (RAP80−/−) mice. RAP80−/− mice are viable and did not exhibit any apparent developmental defects. Mouse embryonic fibroblasts (MEFs) derived from RAP80−/− mice underwent premature senescence compared to wild type (WT) MEFs, were more sensitive to IR, and exhibited a higher level of spontaneous and IR-induced genomic instability. RAP80−/− thymocytes were more sensitive to IR-induced cell death than WT thymocytes. RAP80−/− mice were more susceptible to spontaneous lymphoma development and the development of DMBA-induced mammary gland tumors. Moreover, the loss of RAP80 accelerated tumor formation in both p53−/− and p53+/− mice. Our data indicate that RAP80-deficiency promotes genomic instability and causes an increase in cancer risk consistent with the concept that RAP80 exhibits a tumor suppressor function.
A proper response to DNA damage is essential for maintaining genomic stability and to prevent the accumulation and transmission of damaged DNA during cell division (1). Among the different types of DNA damage, DNA double-strand breaks (DSBs) are the most detrimental to mammalian cells. DSBs activate a DNA damage response (DDR) that coordinates the activation of cell cycle checkpoints, apoptosis, and DNA repair networks, to ensure accurate repair and genomic integrity. Defects in DNA repair mechanisms are associated with various human developmental and immune disorders and an increased cancer risk (2–5).
The DDR is initiated by sensors, such as the Mre11-Rad50-Nbs1 (MRN) complex, that detect DSBs and recruit effectors that carry out the repair, and mediators that promote the interactions between effectors and sensors (1, 6–9). At an early stage, the MRN recruits the ATM kinase, which upon activation catalyzes the phosphorylation of many proteins, including the histone variant H2AX (γH2AX), and promotes the recruitment and phosphorylation of MDC1 (1, 6–8, 10). Interaction of MDC1 with RING-finger ubiquitin ligase RNF8 and the subsequent association with the E2 ubiquitin-conjugating enzyme Ubc13 leads to the K63- or K6-linked polyubiquitylation of histones H2AX and H2A at the chromatin flanking DSBs, thereby amplifying the DSBs signals. The accumulation of DDR protein complexes at the break sites results in the formation of distinctive nuclear foci (ionizing radiation (IR)-induced foci or IRIF) (11, 12).
Receptor-Associated Protein 80 (RAP80 or UIMC1) is a Ubiquitin Interaction Motif-containing (UIMC) nuclear protein that is associated with several proteins, including BRCA1 (13–19), MDC1 (20), p53 (18), and the estrogen receptor alpha (ERα) (13). RAP80 facilitates the recruitment of the BRCA1-BARD1-Abraxas/CCDC98-MERIT-BRCC36 complex to IRIF. This translocation is mediated at least in part by the interaction of the UIMs of RAP80 to K63-linked polyubiquitin chains in histones at or near DSBs (11, 12). Cells in which RAP80 is knocked-down by siRNA, are impaired in forming and retaining IRIF containing BRCA1 and other downstream DDR proteins. These cells also exhibit increased radiosensitivity and impaired cell cycle checkpoints. In addition to its link with BRCA1, RAP80 was shown to be directly regulated by p53 and found to be associated with p53 in complex with E3 ubiquitin ligase HDM2 protein (18).
Its role in DDR and the potential link between RAP80 mutations and human mammary and ovarian cancer, suggested that defective RAP80 function/expression might result in genomic instability and increased cancer risk (5, 21). In this study, we investigated the effect of the loss of RAP80 expression on genomic stability and cancer development in RAP80−/− mice using several tumorigenesis models, including IR-induced lymphoma and 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary cancer. Our results demonstrated that RAP80 protects against genomic instability and reduces cancer risk supporting the hypothesis that RAP80 functions as a tumor suppressor.
The formation of spontaneous tumors was monitored in male and female WT and RAP80−/− mice for a period of 20 months. For chemically induced tumor formation, 2 months old female WT and RAP80−/− mice were treated by gavage with DMBA (5 mg/ml in sesame oil; 1 mg/25 g total body weight; Sigma) once per week for 4 weeks and the development of mammary gland tumor formation and other tumors was monitored by weekly palpation for a period of 10 months (22). At 10 months, all mice were euthanized and necropsy performed to examine the presence of additional tumors. To determine the effect of the loss of RAP80 in p53-deficient mice, RAP80−/− mice were crossed with B6.129S2-Trp53tm1Tyj/J (p53+/−) mice (The Jackson Laboratory) to generate RAP80−/− p53+/− and RAP80−/− p53−/− mice. Spontaneous and IR-induced tumor development was monitored and tumor-free survival analyzed by the Kaplan-Meier method using Graphpad Prism software.
RAP80−/− and WT MEFs were generated according to standard procedures (23). For the in vitro proliferation assay, 2 × 105 passage 1 MEFs were seeded onto each 60 mm dishes containing DMEM and 10% FBS and then incubated at 37°C under 5% CO2. After 3–5 days upon confluence, the cells were trypsinized, counted, and reseeded at the same density of 3 × 105 cells/6-cm dish. Given two measurements of a growing quantity, q1 at time t1 and q2 at time t2, doubling time (Td) can be calculated with the following formula, Td = (t2−t1) * log (2 − q2/q1). To measure actively proliferating cells, MEFs at various passages were pulse-labeled with 10 mM BrdU. After fixation cells were incubated with a biotinylated anti-BrdU antibody, and subsequently with a streptavidin-horseradish peroxidase-coupled secondary antibody and diaminobenzidine. Cells were counterstained with Harris hematoxylin (blue staining). Immortalized MEFs (iMEFs) were generated by SV40 T-antigen viral expression as described (24).
Passage 3 MEFs or iMEFs derived from WT and RAP80−/− littermates were seeded onto coverslips and either left untreated or subjected to 4 or 10 Gy of γ radiation. At various time points after IR, the MEFs were fixed with 4% paraformaldehyde, permeabilized in 0.2% Triton X-100, and blocked in Superblock blocking buffer (Piece), followed by staining with rabbit anti-BRCA1 (kind gift from Dr. R Hakem, University of Toronto) and mouse monoclonal anti-γH2AX (Ser139; Upstate). Endogenous RAP80Ser205P was detected with the RAP80Ser205P antibody, prescribed previously (25). Donkey anti-mouse Alexa Fluor 595 and donkey anti-rabbit Alexa Fluor 488 (Invitrogen) were used for the secondary staining, followed by DAPI (Invitrogen) counterstaining. Images were taken on a laser confocal microscope (LSM510; Carl Zeiss, Inc.) under 40X or 60X magnification. For nuclear fragmentation assay, iMEFs were subjected to 4 Gy of γ radiation. Forty-eight hours later, 200 DAPI stained nuclei were counted for each genotype.
WT or RAP80−/− iMEFs were either left untreated or subjected to 10 Gy of γ irradiation. At 10 min and 2 h after IR, cell lysates were collected and analyzed by SDS PAGE. The resolving bands were then transferred to PVDF membranes. Western blot analysis was performed using rabbit RAP80Ser205P (25) and anti-GAPDH (Cell Signaling Technology) antibodies.
iMEFs were seeded on 60-mm culture dishes (3 × 103 cells/dish). Cultures were either left untreated or subjected to various doses of γ radiation or UV and incubated for 14 days at 37°C. Cells were then stained with 1% Coomassie brilliant blue, 5% acetic acid, and 30% methanol and the number of colonies was counted. Senescence-associated (SA-β-gal) staining of MEFs was performed according to the manufacturer’s protocol (BioVision).
MEFs or iMEFs were either left untreated or treated with various doses of γ radiation. At 24 h or 48h after radiation, cells were lifted by trypsin treatment and fixed in ice cold 70% ethanol before store in −20°C for overnight. Cells were then washed once with ice cold PBS and stained with PI for 30 min before subject to Facsort to determine the DNA content. Cell cycle percentages were analyzed using Modfit software (Verity).
MEFs, iMEFs or thymocytes (0.5–1 × 106/ml in RPMI medium supplemented with 10% FCS and 5 × 10−5 M β-mercaptoethanol) were either left untreated or subjected to γ or UV radiation, or treated with doxorubicin, 5-fluorouracil, or nutlin-3a (Sigma) at the doses indicated (18). After 18 h, the number of apoptotic cells was determined using an Annexin-V-FITC/PI Apoptosis Detection Kit (BD Biosciences) following the manufacturer’s protocol. QRT-PCR analysis of Bax, Puma, p21, Siva1, and Gapdh mRNA was performed as described in Supplemental Information. To assess radiosensitivity in vivo, 20 pairs of age-matched WT and RAP80−/− littermates were subjected to IR (8 Gy). Mice were monitored daily for survival and Kaplan-Meier survival curves were established. A log-rank test was used for statistical analysis.
To obtain greater insights into the physiological functions of RAP80, we generated RAP80-null mice by crossing RAP80fx/fx mice with Tg(CMV-Cre) mice as described in Supplemental Information and Fig. 1A and B. QRT-PCR showed that RAP80 mRNA expression was undetectable in testis, spleen and thymus, which normally express high levels of RAP80 (Fig. 1C). Western blot and immunofluorescence analysis showed that after IR treatment phosphorylated RAP80 was undetectable and did not accumulate at IRIFs in RAP80−/− MEFs consistent with the absence of RAP80 protein in these cells (Fig. 1D and E).
RAP80−/− mice exhibited a normal appearance and no anatomical or developmental defects were apparent. Deficiency in several DDR genes have been reported to affect spermatogenesis and to cause a reduction in the number of lymphocytes (26, 27); however, spermatogenesis appeared normal (data not shown) and both male and female RAP80−/− mice were fertile. FACS analysis showed little change in different CD4/CD8 thymocyte subpopulations between RAP80−/− and WT mice suggesting that loss of RAP80 did not affect thymopoiesis in a major way (data not shown).
The doubling time of RAP80−/− MEFs increased more rapidly upon passing than that of WT MEFs (Fig. 2A) and contained a lower number of bromodeoxyuridine (BrdU)-positive cells (Fig. 2B). By passage 3, RAP80−/− MEFs contained more non-dividing giant, β-gal positive senescent cells than WT cultures (Fig. S1A), suggesting that RAP80−/− MEFs underwent premature entry into senescence as was reported for mice deficient in other DDR genes, including BRCA1, Ku80, Ku70, and ATM (28, 29). Immortalized RAP80−/− and WT iMEFs no longer showed significant differences in proliferation and did not contain senescent cells (not shown). However, after UV irradiation, RAP80−/− iMEF cultures contained a larger number of senescent-like cells than those of WT iMEFs (Fig. S1B).
Because of its role in DDR, we examined whether the loss of RAP80 had any effect on radiosensitivity by examining the effect of IR on colony forming efficiency (CFE). Although at 2 and 4 Gy, IR reduced the CFE of RAP80−/− iMEFs to a greater extent than WT iMEFs, the differences were not statistically significant; however, at 6 Gy the reduction in CFE was significantly greater in RAP80−/− than WT iMEFs (Fig. 2C). Consistent with these observations, examination of the effect of IR on isolated thymocytes showed that RAP80−/− thymocytes were significantly more sensitive to IR-induced cell death than WT thymocytes (Fig. 2D). RAP80−/− mice exhibited a slightly greater susceptibility to whole body IR compared to WT littermates (Fig. S2); however, the difference was not statistically significant.
Previous studies indicated that RAP80 facilitated the translocation of BRCA1 to IRIF (14–17). To further evaluate the effect of the loss of RAP80 on the localization of BRCA1 to IRIF, we compared the dynamics of BRCA1 foci formation in response to IR in WT or RAP80−/− MEFs. Although the loss of RAP80 did not prevent the formation of BRCA1 IRIF, a significant decrease in foci intensity and the percentage of cells containing nuclei with > 5 BRCA1+ foci was observed in RAP80−/− MEFs compared to WT MEFs 8 h after IR (Fig. 3A, B). This observation is consistent with a recent study showing that knockdown of RAP80 expression by siRNAs in U2OS cells did not prevent the formation of BRCA1 IRIF, but diminished the intensity of the foci and particularly reduced the retention of the foci at later time points (30).
Previously, we demonstrated that siRNA knockdown of RAP80 expression stabilizes p53 and results in increased transactivation of p53 target genes and increased apoptosis following DNA damage (18). We therefore determined whether RAP80−/− MEFs responded in a similar manner. Except for nutlin, which activates p53 by inhibiting its interaction with mdm2 without causing DNA damage, all treatments resulted in significantly more cell death in RAP80−/− MEFs than in WT MEFs (Fig. 4A). However, this difference in cell death was greatly reduced in WT and RAP80−/− iMEFs, which might be related to the inactivation of p53 by SV40 T-antigen (Fig. 4B). Moreover, IR, UV, doxorubicin and fluorouracil all increased the induction of the p53 target genes, Bax, Siva1 and Puma, to a greater extent in RAP80−/− MEFs than in WT MEFs, while the induction of these genes by nutlin was not significantly different between RAP80−/− and WT MEFs (Fig. 4C, Fig. S3). Little difference was observed in p21 expression.
Regulation of cell-cycle checkpoints is an integral part of the DDR (1, 9, 31, 32). Irradiation-induced checkpoints are activated during the G1 to S transition, the S phase, and the G2/M cell cycle boundary. To determine whether the loss of RAP80 affected cell cycle checkpoints, we compared the cell cycle distribution of RAP80−/− and WT MEFs in response to IR. At 24 h after exposure to 4 Gy IR, both WT and RAP80−/− MEFs exhibited a marked increase in the percentage of cells in G2/M, consistent with a G2/M arrest (Fig. 4D). At 48 h after IR, the percentage of WT MEFs in G2/M decreased, whereas that of RAP80−/− MEFs remained elevated. The prolonged G2/M arrest could be explained by the hypothesis that repair of DNA damage is less efficient in RAP80−/− MEFs.
Premature senescence observed in MEFs deficient in H2AX, ATM or BRCA1 expression has been reported to be associated with chromosomal instability (1, 3, 28, 33). Phosphorylation of H2AX (γH2AX) is thought to be a reliable indicator for DNA damage and genomic instability (2, 34). To examine the effect of the loss of RAP80 on genomic stability, we compared the number of γ-H2AX positive cells in untreated RAP80−/− and WT MEFs Under basal and low stress conditions RAP80−/− MEFs contained a significant higher percentage of cells with γ-H2AX positive nuclei than WT MEFs (Fig. 5A). All RAP80−/− and WT MEFs were γ-H2AX positive upon 4 Gy IR exposure (Fig. 5A, B); however, the γ-H2AX foci persisted longer in RAP80−/− MEFs than in WT MEFs (Fig. 5C). In addition, at 24 hours after IR RAP80−/− MEFs exhibited a greater extent of nuclear fragmentation as well as more micronuclei compared to WT MEFs (Fig. 5D). These observations are consistent with our conclusion that genetic lesions are repaired less efficiently in RAP80−/− MEFs than in WT.
To further examine whether the loss of RAP80 expression resulted in genomic instability, we analyzed metaphase spreads prepared from RAP80−/− and WT iMEFs treated with 4 Gy IR. An increase in the number of sister chromatid exchanges was observed in RAP80−/− MEFs (Fig. S4A, B) consistent with studies examining the effect of RAP80 knockdown by siRNA on genome stability (21, 30).
Mice deficient in several DDR proteins have been reported to exhibit an increased susceptibility to cancer development due to increased genomic instability (2, 35, 36). Because the loss of RAP80 expression results in less efficient DNA damage repair and enhanced genome instability, it has been suggested that it might influence cancer risk as well. We therefore monitored WT and RAP80−/− mice for over 20 months for spontaneous tumor formation. During the first 10 months, none of the WT or RAP80−/− mice developed tumors. However, by 20 months, 52% of the RAP80−/− mice had developed either thymic or splenic lymphomas, whereas only 1 out of 28 WT mice (3.6%) developed lymphoma (Fig. 6A). Occasionally, these lymphomas were found to infiltrate various non-lymphoid organs, including liver, lung, and kidney (Fig. S5A-F). In addition, RAP80−/− mice were more susceptible to IR-induced lymphoma formation. After exposure to 6 Gy IR, about 18% (4/22) of the RAP80−/− mice developed thymic lymphomas over a 6-month period, whereas only 4.7% (1/21) of the WT littermate controls did (Fig. 6B).
Previous studies have shown that the loss of the tumor suppressor p53 greatly enhances tumor development in mice (35, 37). To determine whether the additional loss of RAP80 might further enhance tumor development in p53−/− mice, we examined tumor development in RAP80−/− p53−/− double-KO mice. RAP80−/− p53−/− mice did not show any developmental defects, but exhibited a significant decrease in tumor-free survival compared to p53−/− controls (Fig. 6A).
p53+/− mice have been reported to develop lymphomas and sarcomas at the age of 6–9 months and are widely used in short-term carcinogenicity testing (35, 37). To further examine the role of RAP80 in tumor development, we compared the susceptibility of RAP80−/−, p53+/− RAP80−/−, and p53+/− mice to IR-induced lymphoma formation. Although 42.8% (6/14) of p53+/− mice developed thymic lymphomas over a 4-month period, 78.5% (11/14) of p53+/− RAP80−/− developed thymic lymphomas (Fig. 6C). In addition, the average tumor weight of thymic lymphomas in p53+/− RAP80−/− mice was significantly greater than that of p53+/− mice (Fig. 6D). These results further support our conclusion that RAP80 deficiency enhances lymphoma development.
We next compared the susceptibility of RAP80−/− and WT mice to DMBA-induced tumor formation. DMBA has been reported to induce the development of mammary gland and ovarian carcinomas, as well as lymphomas (22, 38). In our study, female WT and RAP80−/− mice were treated with DMBA once per week for 4 weeks and the development of mammary gland tumor formation was monitored by weekly palpation for a period of 10 months. Over a period of 10 months only 5% of the WT mice developed DMBA-induced mammary gland tumors, which is consistent with reports that the C57BL6 strain is generally resistant to the development of DMBA-induced mammary tumors. In contrast, 40% of RAP80−/− mice developed mammary gland tumors (Fig. 7A). These observations support our hypothesis that the loss of RAP80 function causes an increased risk of mammary cancer development. DMBA treatment also induced lymphomas and ovarian carcinomas in both WT and RAP80−/− mice; however, no statistically significant difference in frequency was observed between WT and RAP80−/− mice. The overall tumor-free survival remained significantly different between RAP80−/− and WT mice (Fig. 7B). Collectively, these observations indicate that a deficiency in RAP80 increases the susceptibility to spontaneous and environmental stress-induced tumor formation in agreement with the conclusion that RAP80 functions as a tumor suppressor.
Previous reports demonstrated that RAP80 plays an important role in DDR signaling. RAP80 is part of a BRCA1 protein complex and facilitates the translocation of the complex to DSBs (13–19, 39). This translocation is at least in part mediated through the interaction of the UIMs of RAP80 with K63- and K6-linked ubiquitinated γH2AX and other histones. Recently, Hu, et al. (30) reported that knockdown of RAP80 by siRNAs affected the retention of BRCA1 at IRIF rather then the initial recruitment of this complex. Our results showing diminished retention of BRCA1 complexes at IRIF at later times after DNA damage in MEFs derived from RAP80−/− embryos is in agreement with these observations. It was suggested that RAP80 complexes contribute to a process that down-modulates BRCA1 homologous recombination function and thereby suppressing exaggerated BRCA1-driven HR (30). This was corroborated by observations showing that the down-regulation of RAP80 by siRNA or expression of a functionally deficient RAP80 mutant in U2OS cells result in a hyper-recombination phenotype accompanied by increased chromosomal abnormalities, the majority of which consist of sister chromatid breaks and sister chromatid exchange (21, 30). Our analysis of chromosome spreads of iMEFs from WT and RAP80−/− mice indicated that the number of sister chromatid exchanges was significantly increased in IR exposed RAP80−/− iMEFs (Fig. S3) and further supports a role for RAP80 in the down-modulation of homologous recombination functions of BRCA1.
We further demonstrated that MEFs derived from RAP80−/− embryos undergo premature senescence. Moreover, RAP80−/− MEFs as well as RAP80−/− thymocytes, exhibited an increased sensitivity to several DNA damaging agents indicative of a less efficient DDR. Interesting the sensitivity to DNA damaging agents was p53 dependent. In addition, cultures of non-irradiated RAP80−/− MEFs contained a higher number of cells with γH2AX foci compared to WT cells and an increase in CtIP and BACH1 foci has been reported in U2OS cells in which RAP80 is down-regulated (21, 30, 40). These observations would be consistent with more extensive end processing of spontaneous single and double strand breaks in RAP80-deficient cells and with the concept that loss of RAP80 function leads to deficient DSB repair and increased chromosome instability.
A major role of DDR proteins in mammalian cells is to maintain genomic integrity. Impairment of the DDR process results in genome instability and increased cancer risk. In this study, we examined whether the loss of RAP80 expression affected cancer risk in several different murine cancer models. Our data showed that by 20 months the percentage of spontaneously developing lymphomas in RAP80−/− mice was significantly greater than in WT mice (52% versus 3.6%, respectively). IR exposure accelerated lymphoma formation in both WT and RAP80−/− mice; however, RAP80−/− mice remained more susceptible. Similar findings were recently reported by Wu et al. (41). These observations indicate that loss of RAP80 enhances the susceptibility to lymphoma development consistent with the hypothesis that RAP80 functions as tumor suppressor.
It is well established that p53 acts as a tumor suppressor and plays an important role in determining heritable cancer risk (31, 35, 42). Inactivation of p53 acts cooperatively with genetic alterations in other tumor suppressor or oncogenes to increase cancer risk (43). In this study, we showed that RAP80−/− p53−/− double knockout mice exhibited a greater decrease in tumor-free survival compared to p53−/− single knockout mice indicating that the loss of RAP80 acts synergistically with the loss of p53 in promoting spontaneous lymphoma development. No statistically significant difference was observed in tumor-free survival between irradiated p53−/− and RAP80−/− p53−/− mice, which is likely due to the very small latency in tumor development. Heterozygous p53 knockout mice also exhibit a predisposition for developing lymphoma, sarcoma, and carcinoma and exposure to IR considerably reduces the latency to tumor formation and survival (37). We showed that p53+/− RAP80−/− mice were significantly more susceptible to the development of IR-induced thymic lymphomas than p53+/− mice (78.5% versus 42.8%, respectively). These observations suggest that inactivating RAP80 and p53 mutations cooperate in promoting tumorigenesis. The latter is consistent with the concept that RAP80 functions as a tumor suppressor. In agreement with previous observations (18), we found that the ability of several p53 activators to induce cell death and the expression of p53 target genes was significantly enhanced in RAP80−/− MEFs. As proposed for its modulation of BRCA1 (30), RAP80 might suppress the ability of p53 to transactivate apoptotic target genes in the presence of DNA stress and damage signals thereby increasing the time to repair.
In humans, germline mutations in BRCA1 and BRCA2 account for 5–7% of familial breast cancers (10, 44). Because RAP80 is part of a BRCA1 complex, we were particularly interested in determining whether RAP80−/− mice were more susceptible to DMBA-induced mammary cancer development. We found that RAP80−/− mice were significantly more susceptible to DMBA-induced mammary cancer development than WT mice. In addition to mammary cancer, DMBA treatment also enhanced the formation of lymphomas and ovarian cancer. Our results show that total tumor-free survival was significantly reduced in RAP80−/− mice. These observations are consistent with our conclusion that loss of RAP80 enhances cancer risk. Several recent studies investigating an association between RAP80 mutations and breast cancer risk in humans reported that truncating mutations of the RAP80 gene are not associated with familial breast cancer, while RAP80 haplotype or rare missense mutations may be weakly or moderately associated with elevated risk of breast cancer (45–47). A separate study found an association between a RAP80 haplotype and elevated ovarian cancer risk (5). Screening of Finnish BRCA1/BRCA2 mutation-negative breast cancer families revealed the presence of several RAP80 variants (21); however, whether these are associated with increased cancer risk remains to be established. Overall these studies appear to indicate that the mutations identified within RAP80 to-date do not play a major role in increasing the risk of breast cancer. In summary, our study indicates that the loss of RAP80 enhances genome instability and increases the susceptibility to cancer development in several mouse models of human cancer. The impact of RAP80 on DNA repair and maintenance of genomic stability are consistent with the concept that RAP80 can function as a tumor suppressor.
The authors would like to thank Drs. Michael Fessler and Alex Merrick for providing the p53 KO mouse strain, Dr. Hong Soon Kang with qRT-PCR analysis, Laura Miller for her help with colony management and dosing the mice, and Grace Liao for her assistance with the genotyping. This research was supported by the Intramural Research Program of the National Institute of Environmental Health Sciences, the National Institutes of Health (Z01-ES-101586 to AMJ and Z01-ES065079 to MAR).
None of the authors state a conflict of interest.