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Published in Oncotarget V8N52, Oct 27, 2017
BMI1 reduces ATR activation and signalling caused by hydroxyurea
Oncotarget. 2017; 8:89707-89721. https://doi.org/10.18632/oncotarget.21111
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Xiaozeng Lin, Fengxiang Wei, Peter Whyte and Damu Tang _
Xiaozeng Lin1,2,3, Fengxiang Wei1,2,3,4, Peter Whyte5 and Damu Tang1,2,3
1Division of Nephrology, Department of Medicine, McMaster University, Hamilton, Ontario, Canada
2Father Sean O'Sullivan Research Institute, Hamilton, Ontario, Canada
3The Hamilton Center for Kidney Research, St. Joseph's Hospital, Hamilton, Ontario, Canada
4The Genetics Laboratory, Longgang District Maternity and Child Healthcare Hospital, Longgang District, Shenzhen, Guangdong, P.R. China
5Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
Damu Tang, email: damut@mcmaster.ca
Keywords: BMI1, DNA damage response, ATR, CHK1, S checkpoint activation
Received: February 14, 2017 Accepted: September 03, 2017 Published: September 20, 2017
BMI1 facilitates DNA damage response (DDR) induced by double strand DNA breaks; however, it remains unknown whether BMI1 functions in single strand DNA (ssDNA) lesions-initiated DDR. We report here that BMI1 reduces hydroxyurea-elicited ATR activation, thereby reducing the S-phase checkpoints. Hydroxyurea induces ssDNA lesions, which activate ATR through binding TOPBP1 as evidenced by phosphorylation of ATR at threonine 1989 (ATRpT1989). ATR subsequently phosphorylates H2AX at serine 139 (γH2AX) and CHK1 at serine 345 (CHK1pS345), leading to phosphorylation of CDK1 at tyrosine 15 (CDK1pY15) and S-phase arrest. BMI1 overexpression reduced γH2AX, CHK1pS345, CDK1pY15, S-phase arrest, and ATR activation in HU-treated MCF7 and DU145 cells, whereas BMI1 knockdown enhanced these events. BMI1 contains a ring finger, helix-turn, proline/serine domain and two nuclear localization signals (NLS). Individual deletion of these domains did not abolish BMI1-derived reductions of CHK1pS345 in MCF7 cells following HU exposure, suggesting that these structural features are not essential for BMI1 to attenuate ATR-mediated CHK1pS345. BMI1 interacts with both TOPBP1 and ATR. Furthermore, all of our BMI1 mutants associate with endogenous TOPBP1. It has previously been established that association of TOPBP1 and ATR is required for ATR activation. Thus, our results suggest that BMI1 decreases ATR activation through a mechanism that involves binding to TOPBP1 and/or ATR.
BMI1 reduces ATR activation and signalling caused by hydroxyurea | Lin | Oncotarget
BMI1 is a polycomb group (PcG) protein of the polycomb repressive complex 1 (PRC1) [1], and is required for formation of E3 ubiquitin ligase activity of PRC1 via binding to the catalytic subunit RING2 [2–5]. The E3 ubiquitin ligase activity underlies PRC1-mediated suppression of gene expression. For example, BMI1 represses the INK4A/ARF and E4F1 loci [6–9]. The INK4A/ARF locus encodes two tumor suppressors, p16INK4A and p19ARF/p14ARF, via alternative splicing and using differential promoters [10, 11]. E4F1 inhibits cell proliferation, in part, through promoting p53 and CHK1 functions [12–14]. Suppression of these loci contributes to BMI1-derived maintenance of the self-renewal of hematopoietic and neural stem cells [8, 15, 16]. In some cases, inhibition of INK4A and ARF-mediated tumor suppression is critical for tumorigenesis [10, 11] and upregulation of BMI1 occurs in numerous cancer types including non-small cell lung cancer [17], colon cancer [18], breast cancer [19], and nasopharyngeal carcinoma [20]. BMI1 overexpression is able to transform lymphocytes [21, 22] and its upregulation in lymphomas associates with poor prognosis [23–25]. Also, expression of BMI1 can synergize with c-Myc in transgenic mouse models for leukemogenesis [26, 27].
In addition to inhibition of the pRB and p53 tumor suppressors through repression of the INK4A/ARF locus [10, 11], BMI1 is involved in DNA damage response (DDR) [28–31]. DDR is essential in maintenance of genomic integrity and accurate passage of genetic materials to the daughter cells [32]. Compromising DDR leads to genomic instability, a hallmark of cancer [33, 34] and a major cause of tumorigenesis [35–37]. Enhancing repair of DDR lesions contributes to therapy resistance in cancer [38, 39]. DDR is initiated by a variety of DNA lesions [40] through activation of three apical PI3 kinase-related kinases (PIKKs) ATM, ATR, and DNA-PK [41, 42]. PIKKs coordinate DDR via checkpoint activation to prevent cell cycle progression and preparation for DNA lesion repair [32, 43]. Double strand DNA breaks (DSBs) activate ATM, leading to phosphorylation of downstream targets, including CHK2 and γH2AX [41, 42]. CHK2 activation subsequently results in G2/M arrest [32, 43] and the formation of γH2AX nuclear foci around DSBs initiates DSB repair [44, 45]. As part of the repair process, BMI1 rapidly associates with DSBs, ubiquitinates γH2AX, and contributes to homologous recombination (HR)-facilitated DSB repair [28–30]. Additionally, BMI1 also compromises DSB-induced checkpoint activation by reducing ATM activation [31].
Another major arm of DDR is initiated by single-strand DNA (ssDNA) lesions, which are typically produced by stalled replication forks. These lesions are first coated with replication protein A (RPA). RPA-ssDNA independently recruits the ATR-ATRIP complex and TOPBP1, where TOPBP1 activates ATR through a physical association. ATR subsequently phosphorylates and activates CHK1, leading to S-phase arrest [46, 47]. In view of these similarities between ATM and ATR activation, we have examined whether BMI1 also decreases ssDNA-initiated ATR activation.
Hydroxyurea (HU) is a potent DNA synthesis inhibitor [48], and causes stalled replication forks through depletion of the dNTP pool, leading to accumulation of ssDNA and activation of the ATR-dependent S-phase checkpoints [49]. We report here that BMI1 delays S-phase checkpoint activation induced by HU. In MCF7 cells treated with HU, BMI1 overexpression reduced ATR activation, phosphorylation of CHK1, and S-phase arrest, while BMI1 knockdown had the opposite effect. BMI1 interacted with TOPBP1 and ATR in co-immunoprecipitation experiments suggesting a possible mechanism.
BMI1 delays HU-induced activation of the S-phase checkpoints
BMI1 has been reported to enhance HR-mediated DSB repair [28–30], and reduce DSB-initiated G2/M checkpoints caused by etoposide [31]. To investigate whether BMI1 is involved in ssDNA-stimulated DDR, we have constructed MCF7 breast cancer and DU145 prostate cancer cell lines in which BMI1 was either stably overexpressed or knocked-down (Supplementary Figure 1). Using these lines, we examined the impact of BMI1 expression levels on HU-induced S-phase checkpoints. Previous studies have demonstrated that HU causes stalled replication forks as a result of depletion of cellular dNTP pools, leading to activation of the S-phase checkpoints in MCF7 and DU145 cells [50, 51] through CHK1 activation. CHK1 contributes to CDK1 inactivation via sustaining CDK1 phosphorylation at tyrosine 15 (Y15) [52], an event that prevents mitotic entry [53]. Accordingly, treatment of the cells with HU resulted in a dose-dependent stimulation of CHK1 phosphorylation at serine 345 (CHK1pS345), indicative of CHK1 activation, and CDK1pY15 in both MCF7 (Figure 1A, 1C, and 1E) and DU145 cells (Supplementary Figure 2). Both events were substantially reduced in MCF7 BMI1 (Figure 1A, 1C, and 1E). Although there was an unexpectedly high level of CDK1pY15 in DU145 BMI1 cells treated with 0.5mM HU, it is apparent that DU145 BMI1 cells displayed a lower kinetics of CDK1pY15 in response to HU exposure compared to DU145 EV cells (Supplementary Figure 2). Collectively, this evidence suggests that enforced BMI1 expression in both MCF7 and DU145 cells results in reductions in CHK1 activation and CDK1 inactivation following HU treatment. Conversely, knockdown of BMI1 elevated HU-induced CHK1pS345 and CDK1pY15 in MCF7 cells (Figure 1B, 1D, and 1F). Furthermore, we have stably re-expressed murine BMI1 into MCF7 shBMI1 cells [31] and confirmed its expression (Figure 1G); re-expression of murine BMI1 reversed the elevation of CHK1pS345 in MCF7 shBMI1 cells treated with HU (compare the CHK1pS345 profile in Figure 1B to that in Figure 1G), indicating that the increase in CHK1pS345 following BMI1 knockdown in MCF7 cells (Figure 1B) was not caused by potential off-target effects. Examination of HU-induced checkpoint activation has been commonly performed by treating cells for 24 hours at concentrations of 1mM or less [50, 51]. This condition does not result in substantial collapse of the DNA replication forks, as cells renewed proliferation upon releasing from 1mM HU-24 hour treatment with minimal adverse effects in comparison to mock-treated cells (Supplementary Figure 3).
Figure 1: BMI1 decreases HU-induced CHK1 activation in MCF7 cells. MCF7 EV (empty vector), BMI1, shCtrl (Ctrl: control), and shBMI1 stable cell lines were established (see Supplementary Figure 1). These cell lines were treated with HU at the indicated doses for 24 hours, followed by Western blot examination for CHK1 phosphorylation at S345 (CHK1pS345), CHK1, CDK1 phosphorylation at Y15 (CDK1pY15), CDK1, and actin. Experiments were carried out three times; typical results from a single repeat are shown (A, B); means ± S.E (standard error) were graphed (C–F). *p < 0.05 in comparison to the respective control (EV and shCtrl) cells (two-tailed Student's t-test). (G) MCF7 shBMI1 cells were stably transfected with EV or mouse BMI1 (mBMI1) (left panel) [31], followed by the examination of HU-induced CHK1pS345. The experiments were repeated once; typical images from a single repeat are included.
To further evaluate conditions involving 1mM HU or less for 24 hours, we determined the kinetics of CHK1 activation in MCF7 cells following HU treatment. HU elicited early (1–8 h) and late phases (14–24 h) of CHK1 activation (Supplementary Figure 4). As cell cycle arrest, a major consequence of checkpoint activation, is commonly examined at 24 hour following treatment [50, 51], we focused this study on the late phase of CHK1 activation and DDR induced by HU at doses ≤ 1 mM. Nevertheless, because HU also induces an early onset of CHK1pS345 (Supplementary Figure 4), we have determined whether BMI1 affected CHK1pS345 in this early phase. It was clear that overexpression of BMI1 in both MCF7 and DU145 cells robustly reduced the dose-dependent kinetics of CHK1pS345 during a 2-hour HU treatment (Figure 2A, 2B, left panels), whereas knockdown of BMI1 in both lines elevated this event (Figure 2A, 2B, right panels). Furthermore, knockdown of BMI1 in DU145 cells also reduced HU-induced late-phase CHK1pS345 in compared to DU145 shCtrl cells (Figure 2B, bottom right panel). Similar results were also obtained in MCF7 shBMI1 cells (Figure 1B, 1D). BMI1 overexpression in MCF7 and DU145 cells reduced HU-induced CHK1pS345 at 24-hour HU treatment (Figure 1A, 1C; Supplementary Figure 2). Nonetheless, the effects of modulation of BMI1 on CHK1pS345 in cells treated with high doses of HU, particularly 1mM HU appeared less robust compared to cells treated with low doses of HU in response to either late (24 h) or early phase (2h) treatment (Figures 1 and 2). These observations indicate that BMI1 attenuates CHK1 activation and, collectively, the above experiments provide a comprehensive set of data demonstrating a role of BMI1 in reducing CHK1 activation following HU treatment.
Figure 2: BMI1 reduces the early onset of CHK1pS345 caused by HU. The MCF7 (A) and DU145-based (B) EV, BMI1, shCtrl, and shBMI1 cells were treated with the indicated doses of HU for 2 hours, followed by determination of CHK1pS345, CHK1, and actin by Western blot. For DU145 shCtrl and shBMI1 cells, the indicated treatments were also continued for 24 hours (bottom right panel). Experiments were repeated once; typical images from a single repeat are shown.
BMI1 reduces ATR activation caused by HU
The above observations led us to hypothesise that BMI1 reduces ATR activation. ATR activation depends on autophosphorylation at threonine 1989 (ATRpT1989) [54]. In this regard, BMI1 overexpression reduced ATRpT1989 in HU-treated MCF7 cells (Figure 3A, left panel; Figure 3B), whereas BMI1 knockdown enhanced the event (Figure 3A, right panel; Figure 3C). These observations support the concept that BMI1 attenuates HU-induced ATR activation.
Figure 3: BMI1 reduces ATR activation. Examination of phosphorylation of ATR at T1989 (ATRpT1989) in the indicated MCF7 cell lines. Experiments were repeated three times; typical images from a single repeat are shown (A). (B, C) ATR phosphorylation was normalized to the respective ATR and expressed as fold changes to the normalized ATR phosphorylation levels in the respective (EV and shCtrl) control (untreated) cells. Means ± SD (standard derivation) were graphed; *p < 0.05 and **p < 0.01 by 2-tailed Student's t-test in comparison to the respective controls.
This notion is further supported by the effects of BMI1 on γH2AX, a target of ATR, in MCF7 cells [47]. Western blot analysis revealed a reduction of γH2AX in MCF7 BMI1 cells in comparison to MCF7 EV cells in response to HU treatment (Figure 4A, 4C). Similar results were also obtained in DU145 BMI1 cells (Figure 4B). Conversely, knockdown of BMI1 in both MCF7 and DU145 cells elevated HU-induced γH2AX levels (Figure 4A, 4B, 4D). Additionally, formation of γH2AX nuclear foci, an apical and essential event for DSB repair [55, 56], was respectively reduced and enhanced in MCF7 BMI1 and shBMI1 cells in comparison to the respective control cells following HU exposure (Figure 4E, 4F; Supplementary Figure 5).
Figure 4: BMI1 attenuates γH2AX in cells treated with HU. (A, B) The indicated MCF7 and DU145 cell lines were exposed to HU at the indicated doses for 24 hours. The indicated events were examined by Western blot. Experiments were repeated three times. Typical results from a single repeat were included (A, B). γH2AX results for MCF7 EV, BMI1 (C), shCtrl, and shBMI1 (D) cells were quantified. Means ± S.E were graphed. *p < 0.05 in comparison to the respective control cells (two-tailed Student's t-test). (E, F) MCF7 EV, BMI1 (E), shCtrl, and shBMI1 (F) cells were treated with PBS or HU for 24 hours. IF staining for γH2AX was performed. Typical images are presented in Supplementary Figure 5. Experiments were repeated three times; γH2AX-positive nuclei were quantified; mean percentages ± S.E are graphed. *p < 0.05 in comparison to the respective control cells (two-tailed Student's t-test).
BMI1 attenuates HU-induced S phase arrest
ATR is required for S-phase arrest in response to HU-induced DNA damage [50, 51]. To determine whether BMI1 affected this process, HU treated cells were examined for S phase cell cycle arrest. In MCF7 EV cells, HU treatment resulted in the expected dose-dependent accumulation in S phase that is indicative of S phase arrest (Table 1; Supplementary Figure 6). In the MCF7 BMI1 cells, the accumulation of S phase cells was significantly reduced (Table 1; Supplementary Figure 6); whereas, BMI1 knockdown increased the proportion of S phase cells (Table 2; Supplementary Figure 6) suggesting that BMI1 reduces HU-induced S phase arrest.
Table 1: BMI1 reduces HU-induced S arrest in MCF7 cells
64.1 ± 1.8
59.7± 0.1
52.9 ± 1.5*
G2/M
Note: Cell cycle distributions were derived from three independent experiments and were presented as means ± S.E.; Ctrl: control; * p < 0.05 (in comparison to the respective cell cycle distributions of EV cells)
Table 2: Knockdown of BMI1 enhances HU-induced S arrest in MCF7 cells
shCtrl
shBMI1
9.8 ± 1.9*
Note: Cell cycle distributions were derived from three independent experiments and were presented as means ± S.E.; Ctrl: control; * p < 0.05 (in comparison to the respective cell cycle distributions of shCtrl cells)
To further examine BMI1's influence on S-phase arrest in HU-treated cells, MCF7 cells were blocked in mitosis by nocodazole treatment in combination with HU exposure. In these experiments, BMI1-derived reduction of S phase arrest should allow cell cycle progression to mitosis, despite the presence of HU, resulting in mitotic arrest due to the effects of nocodozole. As expected, nocodazole treatment resulted in the accumulation of mitotic cells with condensed chromosomes as detected by immunofluorescence using an antibody to histone H3 phospho-S10 (Figure 5A). HU treatment reduced the number of mitotic nuclei in MCF7 EV cells and this reduction was compromised in MCF7 BMI1 cells, i.e. more mitotic nuclei were observed in HU-treated MCF7 BMI1 cells compared to MCF7 EV cells (Figure 5A, 5B). In contrast, BMI1 knockdown reduced the number of mitotic nuclei in response to HU (Figure 5A, 5B). Collectively, the above observations support the notion that BMI1 reduces S-phase arrest in HU-treated MCF7 cells.
Figure 5: BMI1 decreases HU-elicited S-phase arrest. (A) MCF7 EV, BMI1, shCtrl, and shBMI1 cells were treated with nocodazole (0.1 μg/ml) without and with HU (0.1 mM) for 24 hours. Histone H3 S10 phosphorylation was determined using IF staining; nuclei were counter-stained with DAPI. Experiments were repeated 3 times; typical images from a single repeat are included (B). More than 8,000 nuclei from 12 randomly selected areas per slide were counted to determine the percentage of nuclei positive for histone H3 S10 phosphorylation. Means ± SD (standard deviation) were graphed; *p < 0.05 (two-tailed Student's t-test) in comparison to the individual mock treatments, #p < 0.05 in comparison to HU-treated EV cells; and $p < 0.05 in comparison to HU-treated shCtrl cells.
The involvement of BMI1's structural elements in HU-elicited ATR activation and ATR signalling
Previously, we have described a set of MCF7 cell lines that stably express individual BMI1 mutants with deletions of the RF, PS, HT, or one of NLS sites [31]. Using these lines, we investigated the generation of ATRpT1989 and two ATR targets (CHK1pS345 and γH2AX) upon HU treatment. While some variation occurs in ATRpT1989 in HU treated MCF7 EV cells, the level of phosphorylation plateaued at 0.5mM HU and remained high at 0.8 and 1mM HU (Figure 6). Within this dose range, all mutants had either a minimal or modest effect on HU-induced ATRpT1989 in comparison to MCF7 EV cells (Figure 6), suggesting that these domains contribute to BMI1-derived inhibition of ATRpT1989. This notion is supported further by the minimal impact of these mutants on HU-induced γH2AX (Figure 6). Nonetheless, all mutants reduced CHK1pS345 in cells treated with HU, although the levels of reductions differed among the mutants (Figure 6). As CHK1pS345 is the best characterized target of ATR [57], these domains may not be essential for BMI1 to reduce ATR signalling (see Discussion for details).
Figure 6: Characterization of BMI1-derived inhibition of ATR activation and ATR signalling. MCF7-based EV, BMI1ΔNLS, BMI1ΔHT, BMI1ΔRF, and BMI1ΔPS stable lines have been previously established [31]. These lines were treated as indicated and examined for ATRpT1989, ATR, CHK1pS345, CHK1, actin, γH2AX, and H2AX. Experiments were performed twice. Typical images from a single repeat are shown.
BMI1 binds TOPBP1
ATR activation involves binding to ssDNA and TOPBP1 [41, 58, 59]. The above observations raise the possibility that BMI1 may reduce ATR activation via binding to TOPBP1. In 293T cells co-transfected with BMI1 and TOPBP1, immunoprecipitation (IP) of BMI1 led to co-precipitation of TOPBP1 with and without HU treatment, while HU treatment appeared to reduce the association (Figure 7A, see the ethidium bromide/EB minus lanes). The presence of EB (50 μg/ml) (Figure 7A), a condition that is commonly used to release DNA-associated proteins from DNA [31], did not reduce the efficiency of the co-IP (Figure 7A, compare the respective EB- and EB+ lanes), making it unlikely that the presence of DNA was the cause for the interaction. In fact, the presence of EB apparently enhanced the association, suggesting that interaction between BMI1 and TOPBP1 is possibly mediated by a hydrophobic force, which is enhanced by the presence of positively charged EB.
Figure 7: BMI1 associates with TOPBP1. (A) 293T cells were transiently co-transfected with BMI1 and TOPBP1 for 36 hours, and treated with HU (1 mM for 24 hours). Cell lysates were then immunoprecipitated (IP) with anti-BMI1 (αBMI1) or control IgG (IgG) in the presence or absence of ethidium bromide (EB, 50 μg/ml; note: cell lysates were pre-incubated with EB for 10 minutes on ice prior to IP) and analyzed by Western blot for TOPBP1, BMI1, and ATR. 1/14 of cell lysates used for IP were also analyzed. (B, C) MCF7 cells were treated without or with HU (1 mM for 24 hours). Cell lysates were prepared and treated with benzonase at 1 U/μl on ice for 1 hour, followed by IP for BMI1 (B) or ATR (C), and Western blot examination as indicated. All experiments have been repeated once. Typical images from a single repeat are shown.
We further demonstrated the binding of BMI1 and TOPBP1 using the endogenous proteins. In MCF7 cells, immunoprecipitation of BMI1 resulted in co-precipitation of TOPBP1 in the presence of DNase (benzonase endonuclease), further demonstrating that the association was unlikely to be attributable to the presence of DNA (Figure 7B). Similarly, the association between BMI1 and TOPBP1 could be demonstrated with and without HU exposure (Figure 7B). The observed decreases in co-immunoprecipitation of TOPBP1 with BMI1 in HU-treated cells compared to non-treated cells (Figure 7A, 7B) suggest that reductions in this interaction may free TOPBP1 for ATR activation. Collectively, the above observations suggest that BMI1 exists in a protein complex that includes TOPBP1.
Formation of a potential BMI1/TOPBP1/ATR complex
The binding of BMI1 to TOPBP1 raises the possibility of a BMI1 presence in the TOPBP1/ATR complex. This possibility is supported by the co-IP of endogenous BMI1 through IP of endogenous ATR in the presence of benzonase in MCF7 cells (Figure 7C). The association was observed with and without HU treatment (Figure 7C). Furthermore, the endogenous ATR was co-immunoprecipitated together with the ectopic complex of BM1I-TOPBP1 in 293T cells (Figure 7A) and the endogenous BMI1-TOPBP1 complex in MCF7 cells (Figure 7B). Collectively, this evidence supports the formation of an endogenous BMI1/TOPBP1/ATR complex.
Characterization of the association of BMI1 and TOPBP1
To further study the interaction between BMI1 and TOPBP1, we took advantage of our established MCF7 cell lines stably expressing BMI1ΔRF, BMI1ΔNLS, BMI1ΔHT, or BMI1ΔPS. The expression of the individual BMI1 mutants has been shown in our previous report [31] and has been re-demonstrated here (Figure 8A–8D). As we have reported previously [31], BMI1ΔRF was expressed at a low level in MCF7 BMI1ΔRF cells, which is likely attributable to the mutant protein being unstable. This made detection of the BMI1ΔRF mutant protein difficult in cell lysates [31]. Consistent with our previous report, we observed the mutant in MCF7 BMI1ΔRF cells by real time PCR (data not shown) [31] and IP (Figure 8A) [31]. Importantly, immunoprecipitation of the individual BMI1 mutants through their FLAG tags (using the M2 antibody) resulted in detection of the individual mutant proteins, as expected, along with endogenous TOPBP1 (Figure 8A–8D). With the exception of BMI1ΔRF (Figure 8A), HU treatment either did not affect the association of BMI1ΔHT and TOPBP1 (Figure 8C) or reduced the interaction of TOPBP1 with BMI1ΔNLS (Figure 8B) or BMI1ΔPS (Figure 8D). Collectively, we demonstrated that all BMI1 mutants are capable of interaction with endogenous TOPBP1.
Figure 8: Characterization of BMI1 association with TOPBP1 and ATR. Cell lysate from the indicated MCF7 BMI1ΔRF, BMI1ΔNLS, BMI1ΔHT, or BMI1ΔPS cells (A–E) were pre-treated with benzonase at 1 U/μl on ice for 1 hour, followed by IP with a monoclonal anti-FLAG (M2) (A–D) or anti-ATR antibodies (E). Western blot analyses were performed for ATR, TOPBP1, and the indicated BMI1 mutants (using a polyclonal anti-FLAG antibody). 1/10 of cell lysates used for IP were also analyzed. Experiments were repeated at least three times; typical results from a single repeat are shown. (D) LS: long exposure; SS: short exposure.
Although we were unable to demonstrate an interaction between ATR and BMI1ΔHT or BMI1ΔPS, we observed co-IP of ATR through BMI1ΔRF or BMI1ΔNLS in cells without exposure to HU (Figure 8A, 8B). Consistent with HU treatment enhancing BMI1ΔRF binding to TOPBP1 (Figure 8A) or reducing TOPBP1 interaction with BMI1ΔNLS (Figure 8B), HU exposure respectively elevated the co-IP of ATR via BMI1ΔRF (Figure 8A) and reduced the co-IP of ATR through BMI1ΔNLS (Figure 8B). Due to a low level of the BMI1ΔRF protein in MCF7 BMI1ΔRF cells (see discussion above for details), the system does not support a reverse co-IP, e.g. co-IP of BMI1ΔRF through ATR. Nonetheless, we have demonstrated co-IP of BMI1ΔNLS through endogenous ATR in MCF7 BMI1ΔNLS cells (Figure 8E). Collectively, these observations further support that BMI1 is able to associate with the TOPBP1/ATR complex.
BMI1 is thought to play an important role in maintaining self-renewal of stem cell populations and it may function in tumorigenesis by repressing tumor suppressor genes through its associated ubiquitin E3 ligase activity. More recent studies have provided evidence of a role for BMI1 in DDR regulation by enhancing HR-mediated DSB repair in mouse embryonic fibroblasts (MEFs), HCT116, and U2OS cells [28, 29]. This function may contribute to resistance towards genotoxic drug-based cancer therapies [29, 60–63]. Additionally, BMI1 may attenuate ATM activation following etoposide-caused DSBs, thereby reducing the duration of the etoposide-initiated G2/M checkpoint [31]. By attenuating DDR signalling, normal levels of BMI1 may favour a return to the cell cycle following DNA lesion repair. In cancer cells with BMI1 upregulation, the elevated levels of BMI1 potentially may compromise DDR, thereby contributing to genome instability, a hallmark of tumorigenesis [33, 34]. Our present results extend these observations by demonstrating that BMI1 can downregulate S-phase checkpoints initiated by ssDNA damage induced by HU in MCF7 and DU145 cells. These observations support a broader role for BMI1 in attenuating DNA damage-induced checkpoint activation.
ATM and ATR are related kinases that share a number of structural features such as the FRAP-ATM-TRRAP (FAT) motif, a kinase domain, and a C-terminal FAT (FATC) domain and presumably they have similar mechanisms of activation [47, 64]. While ATM activation requires the presence of DSBs and binding to NBS1, ATR is activated through association with TOPBP1 on RPA-coated ssDNA [41, 58, 59]. BMI1 attenuates ATM activation through binding to NBS1 [31] and ATR activation through its interaction with TOPBP1 (this study) suggesting that it affects the two kinases through similar mechanisms. In addition, several studies support a role for NBS1 in promoting ATR activation [65–67], raising the possibility that BMI1 can influence both ATM and ATR activation through its interaction with NBS1. However, mutational analysis of BMI1 indicates that BMI1 reduces HU-elicited ATR activation in a different manner from its action in inhibiting etopside-induced ATM activation [31]. Individual deletion of RF, HT, PS or NLS does not compromise BMI1's ability to regulate ATM activation nor the subsequent CHK2 phosphorylation under etoposide-initiated DDR [31], whereas some mutants were largely incapable of inhibiting HU-elicited ATRpT1989. Surprisingly, the mutants are competent in inhibiting ATR-mediated phosphorylation of CHK1 at S345, demonstrating specificity of these domains in reducing ATR activation. These results highlight novel properties of BMI1 in inhibiting ATR signalling. It is possible that BMI1 adopts two different structures or uses different structural elements in inhibiting ATR activation and ATR's ability to phosphorylate CHK1. This possibility is not in contrast to the observations that these mutants are incapable of reducing γH2AX in HU-treated MCF7 cells. A possible explanation is that ATM and DNA-PK also produce γH2AX [46, 68] and DNA-PK can be activated by HU-induced replication stress [69]. Additionally, ATR may phosphorylate S139 of H2AX (γH2AX) in a different manner from phosphorylation of CHK1 at S345. Besides DNA-PK, HU can also activate ATM [70]. ATM and DNA-PK may contribute to CHK1 activation in response to HU treatment; this possibility is consistent with the structural similarities among the three PIKKs: ATM, ATR, and DNA-PK [41, 42]. It is possible that full length and mutant BMI1 proteins reduce HU-induced CHK1 phosphorylation through attenuation of ATM and/or DNA-PK activities. This notion is supported by our experiments demonstrating inhibitory activities of BMI1 towards DSB-induced ATM activation [31]. Nonetheless, despite the above possibilities, ATR is the dominant upstream kinase phosphorylating CHK1 in response to stalled replication forks. In this regard, it is tempting to speculate that BMI1 possesses two properties in reducing ATR activation and decreasing ATR's ability to phosphorylate and activate CHK1.
BMI1 is well studied for its associated E3 ubiquitin ligase activity, a property that requires BMI1's RF domain [2–5]. In the DDR process, BMI1 facilitates DSB repair through the E3 ligase activity [28–30], and reduces ATM activation independently of the enzyme activity [31]. For inhibiting HU-induced ATR activation, the RF motif is required. However, for reducing ATR-mediated CHK1pS345, the RF domain is not essential, which is in accordance with the RF domain being dispensable for BMI1 to associate with either TOPBP1 or ATR. These observations share similarity with the RF domain being non-essential in reducing ATM signaling [31]. It is thus possible that BMI1 reduces ATR activation and signalling in E3 ligase-dependent and -independent processes, respectively. Further research will be required to investigate this issue and the structural elements involved in inhibiting ATR activation and ATR signalling.
BMI1 binds TOPBP1 and ATR. While we cannot distinguish whether BMI1 primarily associates with TOPBP1 or ATR, we favour a model that involves the former. This is because the association of BMI1 with TOPBP1 was consistently detected in comparison to its interaction with ATR. However, this issue should be further investigated. Furthermore, this research is essentially based on enforced expression and knockdown of BMI1. Future research should investigate the impact of endogenous BMI1 on ATR activation and functions.
Materials, cell lines, and cell cycle determination
HU and propidium iodide (PI) were purchased from Sigma Aldrich (Oakville, ON). MCF7 breast cancer and DU145 prostate cancer cell lines were obtained from ATCC, and cultured in DMEM (MCF7) and MEM (DU145) supplemented with 10% FBS (Sigma Aldrich, Oakville, ON) and 1% Penicillin-Streptomycin (Life Technologies, Carlsbad, CA). Cell cycle distribution was examined according to our published procedure [71].
Immunofluorescence staining
Immunofluorescence staining was performed by fixing cells with prechilled (–20oC) acetone-methanol for 15 minutes, followed by addition of primary antibodies to anti-γH2AX (Cell Signaling, 1:100) or anti-histone H3 S10 phosphorylation (Upstate, 1:250) at 4ºC overnight. After rinsing, FITC-Donkey anti-rabbit IgG (1:200, Jackson Immuno Research Lab) was applied for 1 hour at room temperature. Slides were subsequently covered with the DAPI mounting medium (VECTOR Lab Inc.). Images were then acquired with a fluorescent microscope (Carl Zeiss, Axiovert 200).
Quantification of γH2AX-positive nuclei
More than 200 nuclei from several randomly selected fields of focus were analyzed. Nuclei with ≥ 10 γH2AX foci and those with < 10 γH2AX foci were respectively defined as positive and negative.
Cell lysates were prepared in a buffer consisting of 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 25mM sodium pyrophosphate, 1 mM NaF, 1mM b-glycerophosphate, 0.1 mM sodium orthovanadate, 1mM PMSF, 2mg/ml leupeptin and 10 mg/ml aprotinin. Cell lysates containing 50mg protein were separated on SDS-PAGE gel, and transferred onto Hybond ECL nitrocellulose membranes (Amersham), followed by treatment with 5% skim milk at room temperature for one hour as well as incubation with individual primary and secondary antibodies. Signals were then developed (ECL Western Blotting Kit, Amersham). Primary antibodies used were: monoclonal anti-BMI1 (1:1000, Invitrogen), anti-H2AX (1:1000, Millpore), anti-γH2AX (1:1000, Cell Signaling), anti-T1989 phosphorylated ATR (1:1000, Abcam), anti-ATR (1:500, Santa Cruz), anti- phospho-CHK1 (S345) (1:500, Cell Signaling), anti-CHK1 (1:1000, Cell signaling), anti-TOPBP1 (1:500, Bethyl), anti-phospho-CDK1 (Y15) (1:1000, Cell Signaling), anti-CDK1 (1:1000, Santa Cruz), anti-tubulin (1:1000, Santa Cruz), and anti-actin (1:1000, Santa Cruz).
IP were carried out by incubating 1mg cell lysate proteins with individual antibodies in the presence of Protein G agarose (Invitrogen) or Dynabeads (Invitrogen) overnight at 4oC, and washing 8 times with a buffer [50mM Tris (PH 7.5), 100 mM NaCl, 7.5mM EGTA, and 0.1% Triton X-100]. Lysates were treated with Benzonase (Sigma, 1 U/μl) for one hour on ice. Antibodies used for IP were polyclonal anti-BMI1 (Santa Cruz, 1 μg), anti-M2 (Sigma, 1 μg), and anti-ATR (Santa Cruz, 1 μg). The immunoprecipitations were analyzed by Western blot using anti-BMI1 (1:1000, Invitrogen), anti-TOPBP1 (1:500, Bethyl), polyclonal anti-FLAG (Sigma, 1:500) and anti-ATR (1:500, Santa Cruz).
Retroviral infection
Retroviral infection was performed according to our published procedure [72]. In brief, a gag-pol expressing vector and an envelope-expressing plasmid (VSV-G) (Stratagene) were co-transfected with a specific retroviral construct into 293T cells for 48 hours. The virus-containing medium was then filtered (0.45 mM filter) and centrifuged (50,000 g for 90 minutes). The viral pellet was resuspended in a specific medium containing 10mg/ml of polybrene (Sigma) prior to infection of cells. Infection was selected using specific antibiotics.
BMI1 overexpression and knockdown in MCF7 and DU145 cells
MCF7 and DU145 cells were transfected with pBabe and pBabe-based BMI1 retrovirus to establish the respective EV and BMI1 stable cell lines. Control and BMI1 retroviral plasmids expressing short hairpin-based RNAs (shRNA) were purchased from Santa Cruz. Retrovirus was packed and used to generate the respective shCtrl and shBMI1 lines.
Student's t-test (2-tails) was used. A p-value < 0.05 was regarded as statistically significant.
X.L. is a recipient of Chinese Government Award for Outstanding Self-Financed Student Abroad.
The authors declare no conflicts of interest
This work was supported in part by a CIHR grant (MOP-84381), the GAP funding from McMaster University and St. Joseph's Hospital at Hamilton, and a grant from Teresa Cascioli Charitable Foundation Research Award in Women's Health to D.T. as well as by grants from Shenzhen Science and Technology Program (Grant No. 20150403094227974), the National Natural Science Foundation of China (Grant No. 81201568), the Natural Science Foundation of Guangdong Province (Grant No.2014A030313749), the National Natural Science Foundation of Heilongjiang Province (grant NO.C2015033), and the Shenzhen Program of Innovation and Entrepreneurship for Overseas Elites (grant No. KQCX20120814150420241) to F.W.
1. Levine SS, King IF, Kingston RE. Division of labor in polycomb group repression. Trends Biochem Sci. 2004; 29:478–85. https://doi.org/10.1016/j.tibs.2004.07.007.
2. de Napoles M, Mermoud JE, Wakao R, Tang YA, Endoh M, Appanah R, Nesterova TB, Silva J, Otte AP, Vidal M, Koseki H, Brockdorff N. Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation. Dev Cell. 2004; 7:663–76. https://doi.org/10.1016/j.devcel.2004.10.005.
3. Wang H, Wang L, Erdjument-Bromage H, Vidal M, Tempst P, Jones RS, Zhang Y. Role of histone H2A ubiquitination in Polycomb silencing. Nature. 2004; 431:873–8. https://doi.org/10.1038/nature02985.
4. Cao R, Tsukada Y, Zhang Y. Role of Bmi-1 and Ring1A in H2A ubiquitylation and Hox gene silencing. Mol Cell. 2005; 20:845–54. https://doi.org/10.1016/j.molcel.2005.12.002.
5. Li Z, Cao R, Wang M, Myers MP, Zhang Y, Xu RM. Structure of a Bmi-1-Ring1B polycomb group ubiquitin ligase complex. J Biol Chem. 2006; 281:20643–9. https://doi.org/10.1074/jbc.M602461200.
6. Bruggeman SW, Valk-Lingbeek ME, van der Stoop PP, Jacobs JJ, Kieboom K, Tanger E, Hulsman D, Leung C, Arsenijevic Y, Marino S, van Lohuizen M. Ink4a and Arf differentially affect cell proliferation and neural stem cell self-renewal in Bmi1-deficient mice. Genes Dev. 2005; 19:1438–43. https://doi.org/10.1101/gad.1299305.
7. Molofsky AV, He S, Bydon M, Morrison SJ, Pardal R. Bmi-1 promotes neural stem cell self-renewal and neural development but not mouse growth and survival by repressing the p16Ink4a and p19Arf senescence pathways. Genes Dev. 2005; 19:1432–7. https://doi.org/10.1101/gad.1299505.
8. Chagraoui J, Niessen SL, Lessard J, Girard S, Coulombe P, Sauvageau M, Meloche S, Sauvageau G. E4F1: a novel candidate factor for mediating BMI1 function in primitive hematopoietic cells. Genes Dev. 2006; 20:2110–20. https://doi.org/10.1101/gad.1453406.
9. Akala OO, Park IK, Qian D, Pihalja M, Becker MW, Clarke MF. Long-term haematopoietic reconstitution by Trp53-/-p16Ink4a-/-p19Arf-/- multipotent progenitors. Nature. 2008; 453:228–32. https://doi.org/10.1038/nature06869.
10. Quelle DE, Zindy F, Ashmun RA, Sherr CJ. Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell. 1995; 83:993–1000.
11. Sherr CJ. Tumor surveillance via the ARF-p53 pathway. Genes Dev. 1998; 12:2984–91.
12. Le Cam L, Linares LK, Paul C, Julien E, Lacroix M, Hatchi E, Triboulet R, Bossis G, Shmueli A, Rodriguez MS, Coux O, Sardet C. E4F1 is an atypical ubiquitin ligase that modulates p53 effector functions independently of degradation. Cell. 2006; 127:775–88. https://doi.org/10.1016/j.cell.2006.09.031.
13. Grote D, Moison C, Duhamel S, Chagraoui J, Girard S, Yang J, Mayotte N, Coulombe Y, Masson JY, Brown GW, Meloche S, Sauvageau G. E4F1 is a master regulator of CHK1-mediated functions. Cell Rep. 2015; 11:210–9. https://doi.org/10.1016/j.celrep.2015.03.019.
14. Rodier G, Kirsh O, Baraibar M, Houles T, Lacroix M, Delpech H, Hatchi E, Arnould S, Severac D, Dubois E, Caramel J, Julien E, Friguet B, et al. The transcription factor E4F1 coordinates CHK1-dependent checkpoint and mitochondrial functions. Cell Rep. 2015; 11:220–33. https://doi.org/10.1016/j.celrep.2015.03.024.
15. Park IK, Qian D, Kiel M, Becker MW, Pihalja M, Weissman IL, Morrison SJ, Clarke MF. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature. 2003; 423:302–5. https://doi.org/10.1038/nature01587.
16. Molofsky AV, Pardal R, Iwashita T, Park IK, Clarke MF, Morrison SJ. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature. 2003; 425:962–7. https://doi.org/10.1038/nature02060.
17. Vonlanthen S, Heighway J, Altermatt HJ, Gugger M, Kappeler A, Borner MM, van Lohuizen M, Betticher DC. The bmi-1 oncoprotein is differentially expressed in non-small cell lung cancer and correlates with INK4A-ARF locus expression. Br J Cancer. 2001; 84:1372–6. https://doi.org/10.1054/bjoc.2001.1791.
18. Kim JH, Yoon SY, Kim CN, Joo JH, Moon SK, Choe IS, Choe YK, Kim JW. The Bmi-1 oncoprotein is overexpressed in human colorectal cancer and correlates with the reduced p16INK4a/p14ARF proteins. Cancer Lett. 2004; 203:217–24.
19. Kim JH, Yoon SY, Jeong SH, Kim SY, Moon SK, Joo JH, Lee Y, Choe IS, Kim JW. Overexpression of Bmi-1 oncoprotein correlates with axillary lymph node metastases in invasive ductal breast cancer. Breast. 2004; 13:383–8. https://doi.org/10.1016/j.breast.2004.02.010.
20. Song LB, Zeng MS, Liao WT, Zhang L, Mo HY, Liu WL, Shao JY, Wu QL, Li MZ, Xia YF, Fu LW, Huang WL, Dimri GP, et al. Bmi-1 is a novel molecular marker of nasopharyngeal carcinoma progression and immortalizes primary human nasopharyngeal epithelial cells. Cancer Res. 2006; 66:6225–32. https://doi.org/10.1158/0008-5472.CAN-06-0094.
21. Haupt Y, Bath ML, Harris AW, Adams JM. bmi-1 transgene induces lymphomas and collaborates with myc in tumorigenesis. Oncogene. 1993; 8:3161–4.
22. Alkema MJ, Jacobs H, van Lohuizen M, Berns A. Pertubation of B and T cell development and predisposition to lymphomagenesis in Emu Bmi1 transgenic mice require the Bmi1 RING finger. Oncogene. 1997; 15:899–910. https://doi.org/10.1038/sj.onc.1201262.
23. Bea S, Tort F, Pinyol M, Puig X, Hernandez L, Hernandez S, Fernandez PL, van Lohuizen M, Colomer D, Campo E. BMI-1 gene amplification and overexpression in hematological malignancies occur mainly in mantle cell lymphomas. Cancer Res. 2001; 61:2409–12.
24. van Galen JC, Muris JJ, Oudejans JJ, Vos W, Giroth CP, Ossenkoppele GJ, Otte AP, Raaphorst FM, Meijer CJ. Expression of the polycomb-group gene BMI1 is related to an unfavourable prognosis in primary nodal DLBCL. J Clin Pathol. 2007; 60:167–72. https://doi.org/10.1136/jcp.2006.038752.
25. Mihara K, Chowdhury M, Nakaju N, Hidani S, Ihara A, Hyodo H, Yasunaga S, Takihara Y, Kimura A. Bmi-1 is useful as a novel molecular marker for predicting progression of myelodysplastic syndrome and patient prognosis. Blood. 2006; 107:305–8. https://doi.org/10.1182/blood-2005-06-2393.
26. Haupt Y, Alexander WS, Barri G, Klinken SP, Adams JM. Novel zinc finger gene implicated as myc collaborator by retrovirally accelerated lymphomagenesis in E mu-myc transgenic mice. Cell. 1991; 65:753–63.
27. van Lohuizen M, Verbeek S, Scheijen B, Wientjens E, van der Gulden H, Berns A. Identification of cooperating oncogenes in E mu-myc transgenic mice by provirus tagging. Cell. 1991; 65:737–52.
28. Ismail IH, Andrin C, McDonald D, Hendzel MJ. BMI1-mediated histone ubiquitylation promotes DNA double-strand break repair. J Cell Biol. 2010; 191:45–60. https://doi.org/10.1083/jcb.201003034.
29. Chagraoui J, Hebert J, Girard S, Sauvageau G. An anticlastogenic function for the Polycomb Group gene Bmi1. Proc Natl Acad Sci U S A. 2011; 108:5284–9. https://doi.org/10.1073/pnas.1014263108.
30. Ginjala V, Nacerddine K, Kulkarni A, Oza J, Hill SJ, Yao M, Citterio E, van Lohuizen M, Ganesan S. BMI1 is recruited to DNA breaks and contributes to DNA damage-induced H2A ubiquitination and repair. Mol Cell Biol. 2011; 31:1972–82. https://doi.org/10.1128/MCB.00981-10.
31. Wei F, Ojo D, Lin X, Wong N, He L, Yan J, Xu S, Major P, Tang D. BMI1 attenuates etoposide-induced G2/M checkpoints via reducing ATM activation. Oncogene. 2015; 34:3063–75. https://doi.org/10.1038/onc.2014.235.
32. Zhou BB, Elledge SJ. The DNA damage response: putting checkpoints in perspective. Nature. 2000; 408:433–9. https://doi.org/10.1038/35044005.
33. Sieber OM, Heinimann K, Tomlinson IP. Genomic instability--the engine of tumorigenesis? Nat Rev Cancer. 2003; 3:701–8. https://doi.org/10.1038/nrc1170.
34. Kops GJ, Weaver BA, Cleveland DW. On the road to cancer: aneuploidy and the mitotic checkpoint. Nat Rev Cancer. 2005; 5:773–85. https://doi.org/10.1038/nrc1714.
35. Hoeijmakers JH. Genome maintenance mechanisms for preventing cancer. Nature. 2001; 411:366–74. https://doi.org/10.1038/35077232.
36. Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers. Nature. 1998; 396:643–9. https://doi.org/10.1038/25292.
37. Rouse J, Jackson SP. Interfaces between the detection, signaling, and repair of DNA damage. Science. 2002; 297:547–51. https://doi.org/10.1126/science.1074740.
38. Burrows N, Williams J, Telfer BA, Resch J, Valentine HR, Fitzmaurice RJ, Eustace A, Irlam J, Rowling EJ, Hoang-Vu C, West CM, Brabant G, Williams KJ. Phosphatidylinositide 3-kinase (PI3K) and PI3K-related kinase (PIKK) activity contributes to radioresistance in thyroid carcinomas. Oncotarget. 2016; 7:63106–23. https://doi.org/10.18632/oncotarget.11056.
39. Gaponova AV, Deneka AY, Beck TN, Liu H, Andrianov G, Nikonova AS, Nicolas E, Einarson MB, Golemis EA, Serebriiskii IG. Identification of evolutionarily conserved DNA damage response genes that alter sensitivity to cisplatin. Oncotarget. 2017; 8:19156–19171. https://doi.org/10.18632/oncotarget.13353.
40. Mordasini V, Ueda S, Aslandogmus R, Berger C, Gysin C, Huhn D, Sartori AA, Bernasconi M, Nadal D. Activation of ATR-Chk1 pathway facilitates EBV-mediated transformation of primary tonsillar B-cells. Oncotarget. 2017; 8:6461–74. https://doi.org/10.18632/oncotarget.14120.
41. Wei F, Yan J, Tang D. Extracellular signal-regulated kinases modulate DNA damage response - a contributing factor to using MEK inhibitors in cancer therapy. Curr Med Chem. 2011; 18:5476–82.
42. Lin X, Yan J, Tang D. ERK kinases modulate the activation of PI3 kinase related kinases (PIKKs) in DNA damage response. Histol Histopathol. 2013; 28:1547–54.
43. Shiloh Y. ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer. 2003; 3:155–68. https://doi.org/10.1038/nrc1011.
44. Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol. 2000; 10:886–95.
45. Celeste A, Petersen S, Romanienko PJ, Fernandez-Capetillo O, Chen HT, Sedelnikova OA, Reina-San-Martin B, Coppola V, Meffre E, Difilippantonio MJ, Redon C, Pilch DR, Olaru A, et al. Genomic instability in mice lacking histone H2AX. Science. 2002; 296:922–7. https://doi.org/10.1126/science.1069398.
46. Awasthi P, Foiani M, Kumar A. ATM and ATR signaling at a glance. J Cell Sci. 2015; 128:4255–62. https://doi.org/10.1242/jcs.169730.
47. Cimprich KA, Cortez D. ATR: an essential regulator of genome integrity. Nat Rev Mol Cell Biol. 2008; 9:616–27. https://doi.org/10.1038/nrm2450.
48. Timson J. Hydroxyurea. Mutat Res. 1975; 32:115–32.
49. Ivessa AS, Lenzmeier BA, Bessler JB, Goudsouzian LK, Schnakenberg SL, Zakian VA. The Saccharomyces cerevisiae helicase Rrm3p facilitates replication past nonhistone protein-DNA complexes. Mol Cell. 2003; 12:1525–36.
50. Wu D, Chen B, Parihar K, He L, Fan C, Zhang J, Liu L, Gillis A, Bruce A, Kapoor A, Tang D. ERK activity facilitates activation of the S-phase DNA damage checkpoint by modulating ATR function. Oncogene. 2006; 25:1153–64. https://doi.org/10.1038/sj.onc.1209148.
51. Wei F, Xie Y, He L, Tao L, Tang D. ERK1 and ERK2 kinases activate hydroxyurea-induced S-phase checkpoint in MCF7 cells by mediating ATR activation. Cell Signal. 2011; 23:259–68. https://doi.org/10.1016/j.cellsig.2010.09.010.
52. O'Connell MJ, Raleigh JM, Verkade HM, Nurse P. Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation. EMBO J. 1997; 16:545–54. https://doi.org/10.1093/emboj/16.3.545.
53. Chen Y, Sanchez Y. Chk1 in the DNA damage response: conserved roles from yeasts to mammals. DNA Repair (Amst). 2004; 3:1025–32. https://doi.org/10.1016/j.dnarep.2004.03.003.
54. Liu S, Shiotani B, Lahiri M, Marechal A, Tse A, Leung CC, Glover JN, Yang XH, Zou L. ATR autophosphorylation as a molecular switch for checkpoint activation. Mol Cell. 2011; 43:192–202. https://doi.org/10.1016/j.molcel.2011.06.019.
55. Lin X, Ojo D, Wei F, Wong N, Gu Y, Tang D. A Novel Aspect of Tumorigenesis-BMI1 Functions in Regulating DNA Damage Response. Biomolecules. 2015; 5:3396–415. https://doi.org/10.3390/biom5043396.
56. van Attikum H, Gasser SM. Crosstalk between histone modifications during the DNA damage response. Trends Cell Biol. 2009; 19:207–17. https://doi.org/10.1016/j.tcb.2009.03.001.
57. Zhao H, Piwnica-Worms H. ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1. Mol Cell Biol. 2001; 21:4129–39. https://doi.org/10.1128/MCB.21.13.4129-4139.2001.
58. Kumagai A, Lee J, Yoo HY, Dunphy WG. TopBP1 activates the ATR-ATRIP complex. Cell. 2006; 124:943–55. https://doi.org/10.1016/j.cell.2005.12.041.
59. Mordes DA, Glick GG, Zhao R, Cortez D. TopBP1 activates ATR through ATRIP and a PIKK regulatory domain. Genes Dev. 2008; 22:1478–89. https://doi.org/10.1101/gad.1666208.
60. Crea F, Duhagon Serrat MA, Hurt EM, Thomas SB, Danesi R, Farrar WL. BMI1 silencing enhances docetaxel activity and impairs antioxidant response in prostate cancer. Int J Cancer. 2011; 128:1946–54. https://doi.org/10.1002/ijc.25522.
61. Facchino S, Abdouh M, Chatoo W, Bernier G. BMI1 confers radioresistance to normal and cancerous neural stem cells through recruitment of the DNA damage response machinery. J Neurosci. 2010; 30:10096–111. https://doi.org/10.1523/JNEUROSCI.1634-10.2010.
62. Gieni RS, Ismail IH, Campbell S, Hendzel MJ. Polycomb group proteins in the DNA damage response: a link between radiation resistance and "stemness". Cell Cycle. 2011; 10:883–94.
63. Liu ZG, Liu L, Xu LH, Yi W, Tao YL, Tu ZW, Li MZ, Zeng MS, Xia YF. Bmi-1 induces radioresistance in MCF-7 mammary carcinoma cells. Oncol Rep. 2012; 27:1116–22. https://doi.org/10.3892/or.2011.1615.
64. Lempiainen H, Halazonetis TD. Emerging common themes in regulation of PIKKs and PI3Ks. EMBO J. 2009; 28:3067–73. https://doi.org/10.1038/emboj.2009.281.
65. Kobayashi M, Hayashi N, Takata M, Yamamoto K. NBS1 directly activates ATR independently of MRE11 and TOPBP1. Genes Cells. 2013; 18:238–46. https://doi.org/10.1111/gtc.12031.
66. Shiotani B, Nguyen HD, Hakansson P, Marechal A, Tse A, Tahara H, Zou L. Two distinct modes of ATR activation orchestrated by Rad17 and Nbs1. Cell Rep. 2013; 3:1651–62. https://doi.org/10.1016/j.celrep.2013.04.018.
67. Manthey KC, Opiyo S, Glanzer JG, Dimitrova D, Elliott J, Oakley GG. NBS1 mediates ATR-dependent RPA hyperphosphorylation following replication-fork stall and collapse. J Cell Sci. 2007; 120:4221–9. https://doi.org/10.1242/jcs.004580.
68. Mukherjee B, Kessinger C, Kobayashi J, Chen BP, Chen DJ, Chatterjee A, Burma S. DNA-PK phosphorylates histone H2AX during apoptotic DNA fragmentation in mammalian cells. DNA Repair (Amst). 2006; 5:575–90. https://doi.org/10.1016/j.dnarep.2006.01.011.
69. Wang CY, Huang EY, Huang SC, Chung BC. DNA-PK/Chk2 induces centrosome amplification during prolonged replication stress. Oncogene. 2015; 34:1263–9. https://doi.org/10.1038/onc.2014.74.
70. Stiff T, Walker SA, Cerosaletti K, Goodarzi AA, Petermann E, Concannon P, O'Driscoll M, Jeggo PA. ATR-dependent phosphorylation and activation of ATM in response to UV treatment or replication fork stalling. EMBO J. 2006; 25:5775–82. https://doi.org/10.1038/sj.emboj.7601446.
71. Tang D, Wu D, Hirao A, Lahti JM, Liu L, Mazza B, Kidd VJ, Mak TW, Ingram AJ. ERK activation mediates cell cycle arrest and apoptosis after DNA damage independently of p53. J Biol Chem. 2002; 277:12710–7. https://doi.org/10.1074/jbc.M111598200.
72. Tang D, Okada H, Ruland J, Liu L, Stambolic V, Mak TW, Ingram AJ. Akt is activated in response to an apoptotic signal. J Biol Chem. 2001; 276:30461–6. https://doi.org/10.1074/jbc.M102045200.
PII: 21111


Well…that was a crappy way to start the morning. Thanks, Jessica at Friendly Atheist!
By the end of 2012, Uganda's internationally contentious "Kill the Gays" bill will become law.
The highly religious, highly Christian country has been trying to get this bill passed since 2009, and it faced a great deal of criticism on an international level along the way: several European countries threatened to reduce aid to the country if the bill was passed.
Speaker Rebecca Kadaga described the bill's passing as a "Christmas gift" for the Ugandan people.
Passing a bill that would kill or imprison for life homosexuals in Uganda – just in time for Christmas! Dead gay Ugandans – the perfect Christmas gift to celebrate Christian love and compassion!
Nope, no way that could go wrong.
Jason Thibeault from Lousy Canuck has an article up with information on how to contact your local MP (if you're in Canada) to ask for an end to Ugandan aid from Canada.
Walking into the family home.
It's warm with light and laughter and decoration.
No awkwardness from the core group.
Old patterns immediately, comfortably assumed.
a flurry of clanging tags whipping tails breathless panting.
I reacquaint with the leather-bound library and tattered children's books.
suspended in the air, magic as ever.
I am overjoyed to reconnect but leery of small talk.
wonder when it will be polite to break into the olive tapenade.
I am drawn to the photos over the fireplace.
All at the height of teenhood, teetering on independence.
He's so young in this photo.
We never see him anymore.
only that he met a woman – a servant of God.
if she thinks Dad might be too warm in that sweater.
his inappropriateness lightening the sorrow for a brief moment.
It's my only story that I have left of him.
when his sister was suffering.
When my mother was grieving.
He and I – we found ourselves at opposite ends of belief.
he would think me damned to hell for eternity.
Now the room fills – constant family mingling with family friends.
Cross-Country Connections is a Biodork weekly blog entry dedicated to telling stories in pictures of three family members – me, my sister and Mom – living in very different locations across the country. Every week we choose a different theme and then take or contribute a personal photo that fits the theme. This week's theme is Lucky.
Another great moment during my first week in DC. Herons were flying around everywhere at the Tidal Basin to north of the mall. As I don't consider myself a good photographer, I consider myself lucky to have even gotten this shot!


Grace Read pictured with her puppy.
Auckland police have made arrests and reunited a woman with her puppy which was allegedly snatched from her arms on Monday.
North Shore woman Grace Read was begging for the safe return of her 10-week-old rottweiler Rosko who was allegedly snatched by two people who came on to her driveway.
She had only had the 10-week old puppy for just over a week when he was taken.
In a Facebook post on Friday evening Waitemata police said they had retrieved the puppy safe and well.
Three people have been arrested in relation to the incident including a 26-year-old man who was charged with aggravated robbery and will appear in North Shore District Court on Saturday.
A 19-year-old woman has been summonsed to court for making a false statement, and a 20-year-old woman has also been summonsed for attempting to procure the commission of an offence and offering to supply cannabis.
Police were still seeking two more people and were continuing to make inquiries.
Describing the "woofing good news" police said Read was "overjoyed".
The investigation was led by the North Shore Crime Investigation Branch whose inquiries alongside the Rodney CIB and Public Safety Team lead them to a Stanmore Bay home where the puppy was located.


JACKSONVILLE, N.C. (July 7, 2015) — Rockstar Energy Drink driver Tanner Foust was sidelined early after a hard hit from a rival driver during the Global Rallycross Championship race at Marine Corps Air Station New River this Independence Day weekend.
So, the two-time series champion and host of Top Gear on History spent much of Sunday's competition as a guest commentator on the live NBC telecast, joining play-by-play announcer Toby Moody and analyst Townsend Bell in the broadcast booth.
Sunday's race saw an estimated 10,000 spectators, who overflowed grandstands to take in the action on the temporary track, built into the runway at MCAS New River. Specialized aircraft, including a V-22 Osprey and a CH-53 Super Stallion, were on display alongside the rallycross cars in the paddock. It was the first time that a championship points-paying motorsport competition took place on an active military installation and the event was the highlight of a big Fourth of July celebration for many military families in the area.
He planned to return to competition for the Last Chance Qualifier and fight his way back into the final. Once the car was back in the paddock, however, the Volkswagen Andretti Rallycross team discovered that the impact had been so hard that it not only tore part of the suspension off the car but also damaged the engine. Foust's race day was over.


text:
Bidding war for murphy
IPSWICH Town are facing a real battle to hold on to their Ireland striker Daryl Murphy as Championship rivals Middlesbrough and Burnley are ready to start a bidding war for the £3million-rated Waterford native.
Murphy, who was the leading scorer in English club football last season with 27 league goals for the Tractor Boys, has increased his stature after being given a starting role by Martin O'Neill in Ireland's last two internationals.
Middlesbrough, desperate to earn promotion next season after losing out to Norwich in the playoff final last month, have had a bid of £2.5m rejected by Ipswich.
But Burnley, who have money to spend following the sale of Danny Ings, are also keen on Murphy and they are expected to make a bid of £3m this week.
Ipswich boss Mick McCarthy is not keen to sell Murphy, and the club rejected a £7m bid for fellow Irishman David McGoldrick last season, McCarthy repeatedly stressing that his club are more interested in winning promotion than banking cash from the sale of players.
But seeing as Ipswich signed Murphy on a free transfer from Celtic in 2013, selling the Ireland international now could be good business.
Murphy, 32, won a place in the starting XI against Scotland last week ahead of rivals strikers like Shane Long and Robbie Keane and he knows he needs the guarantee of first team football to keep him in the frame at international level.
Meanwhile, Huddersfield Town are tracking ex-Bohemians keeper Brian Murphy following his release from QPR. The Waterford man was included in a mass clear-out of players from Loftus Road, Richard Dunne also allowed leave, and despite a lack of first team football, Murphy is in demand and Huddersfield will try to complete a deal if their current No 2 Joe Murphy moves on. Brian Murphy made just one appearance for QPR last season.

redpajama_set_name: RedPajamaCommonCrawl


text:
Bryce Dallas Howard Suggests the 'Jurassic Park' Cast Will Appear in 'Jurassic World 3'
By Josiah Hughes
While Torontonians are obsessed with their own Jurassic Park at Maple Leaf Square as the Raptors push through the NBA Finals, there are still plenty of people thinking about the original Jurassic Park. They might be in for some wish fulfillment of their own, as it looks as if the original film's cast will appear in Jurassic World 3.
While Jeff Goldblum previously appeared in Jurassic World: Fallen Kingdom, both Sam Neill and Laura Dern have not popped up in the new franchise. That will likely change with the third instalment, however.
Speaking with MTV News on the Rocketman red carpet, Bryce Dallas Howard spilled the beans on the cast reunion. "I don't know what's been confirmed or whatever, but I'll blink if it's happening… maybe…" she said before blinking in an exaggerated fashion.
Watch Bryce Dallas Howard seemingly confirm the cast reunion below.
Jurassic World 3 will arrive from director Colin Trevorrow in June 2021.
Will the original #JurassicPark characters come back to help in #JurassicWorld3? If only @BryceDHoward could kind of, maybe, sort of comment on it 😉 pic.twitter.com/ad0ST4ENmt
— MTV NEWS (@MTVNEWS) June 1, 2019
More Colin Trevorrow
More Jeff Goldblum
More Bryce Dallas Howard


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text:
Beit Ya'acov Messianic Jewish Society
A Theologically Messianic Jewish Group In Southern Wisconsin Worshiping Yeshua Where We Live.
Apostolic Writings
Jewish Midrash
Other Texts
Midrash Tehillim 1:1: The Good Fellow, The Bad Fellow: Psalm 1:1
September 26, 2021 September 27, 2021 / Brian P.
Whomever you choose for allies will give you counsel, but it is your heart's true intention that will be revealed by whomever you have chosen. It is the principle of Middah k'neged Middah, measure for measure. Bad fellows love to give bad counsel. A good fellow will love to give good counsel. Fortunate is the man who did not walk in the counsel of the wicked.
The midrash pulls from Proverbs 11:27 to help illustrate. King David is compared to the one who seeks good. King David went out to diligently find good counsel from qualified priests and Levites to serve HaShem in the soon to be constructed Temple. HaShem blessed him to find 24 divisions of priests and 14 divisions of Levites! The text is recalling 1Chr 23-26. Concerning the Levites, David desired to give them new duties in the Temple since they would no longer need to attend to the Tabernacle (See 23:25-32). Likewise, the Aaronites received their duties (See 24:19). David essentially insured that the Levites and Priests would have sustenance and work. What an awesome job to be blessed with, to serve HaShem in His Temple Courts!
And in bringing together these leaders and servants of HaShem, David was able to unite the people to do Divine service and give counsel in cooperation with David's son, Solomon. Solomon was going to build the Temple as HaShem commanded (See 1Chr 22:6-17). But Solomon would need good counsel and his father David blessed him with everything he could. This is what it means to seek out the good. Bring together people who desire to serve HaShem with you.
David also desired to bless Israel, and asked HaShem in prayer to have The Holy Spirit guide him in blessing the people of Israel. This really reveals David's heart to bless and love his nation and his God. Indeed, all blessings come from HaShem and He uses people who are filled with His Spirit to bless others. How fortunate is the person who walks in the Holy Spirit's leading through study and prayer.
The second half of Prov 11:27 says, "He who is bent on evil, upon him it shall com." The midrash personifies evil to be Doeg the Edomite (See 1Sam 21, 22:9-10) and Ahitophel (See 2Sam 15:32-17:23). These men gave evil counsel to people who had ill intent in their hearts. King Saul was exposed for his evil intent. Likewise, Absalom's evil was laid bare before the nation. His counselor and the usurper met eerily similar ends.
The counsel of HaShem is best. When the heart is focused on serving Him, not our own desires, how fortunate are we and those who are around us! Our Master Yeshua, as was His custom, (see Luke 22:39), always sought the Father's Will and Spirit to guide Him through prayer. In this, King David was very much of the same custom. The study of Scripture and prayer with Good Fellows brings blessings.
On the other hand Study and prayer are the defense against the bad fellow. Yeshua always sought the will of the Father. When Peter took Yeshua aside to give bad counsel, Yeshua quickly rebuked him (Matt. 6:22-23). Likewise, when the bad fellow himself attempted to counsel Yeshua, The Master had come fully prepared with prayer and fasting to discern and rebuke the bad counsel (see Matt 4).
Let's follow King David's and King Messiah's example: Study Scripture and prayer, thereby seeking the good counsel of HaShem and bringing goodness to all we see!
Midrash Tehillim 1:1 a midrash on Psalm 1:1
My Translation
Fortunate is the man who did not walk…
It is written, saying: (quoting Proverbs 11:27)
"The seeker of good, he will desire goodwill…"
Who is this seeker of good? This is King David of Israel.
He was seeking out good priests and Levites
from Israel in order to set up the 24 divisions of the
priesthood and the 14 divisions of Levites.
"He will desire goodwill": King David was desiring
Compassion; that May the Divine presence rest
Upon him in order to bless Israel.
Where is their blessing? With the fortunate man!
"But he who seeks an evil fellow, upon him will he come."
This is Doeg and Ahitophel.
← T. Bavli Sanhedrin 90b: Where Is The Resurrection Of The Dead Derived From The Torah?
A Series in Hebrews 8:6-8a: To What Covenant Are The Translators Referring? Part 2 →
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Learn to read the Bible in Hebrew! Serious students who are committed to the hard work of acquiring a new language are encouraged to inquire! Tuition fees will apply for Hebrew lessons to cover cost of materials.
We will be starting a Hebrew class this fall 2022 semester at Fulton Church. Classes will be Monday evenings. Start time will be determined as students enroll. Expect a start time Monday, between 5-7PM, the TBD start time will be determined from the student survey done with enrollment. More details to follow soon!
Join Our Torah Club
Madison Torah Club Meets Every Saturday From 3-5pm. Follow link for more information.
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Beit Ya'acov Messianic Jewish Society serves Rock and Dane County, Wisconsin.
Vision: To train Jewish and Christian disciples of Yeshua, in order to establish Messianic Jewish Congregations in the Dane and Rock county area.
Mission: To form small groups for regular discipleship and to come together for public Messianic Jewish festival celebrations


It seems the filling in of those ditches and widening the pathways is finally going to happen!
It was apparently promised before Christmas – but, alas, that will not happen. We've also been liaising with the Rodney Local Board over this, and they've advised they are waiting on an update on this re final design, budget, and completion date … although it looks like March 2019!
Sadly it will also cover less area than was wanted, with the swales culverted and paved between the Market and JinJin's on the Market side. On the other side the swales will be grasses and edged.
Well done to those stalwarts in Matakana for their perseverance, working alongside the Rodney Local board.


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text:
(MUST WATCH) Jim Rogers Shocking Explanations about Donald Trump – YouTube
February 13, 2017 by 1EarthUnited
Bernie Sanders Calls CNN "Fake News" – And His Feed Is Cut Off – YouTube
Hacker News: "Fileless Malware" Targets Banks and Organization
More than a hundred banks and financial institutions across the world have been infected with a dangerous sophisticated, memory-based malware that's almost undetectable, researchers warned.
Newly published report by the Russian security firm Kaspersky Lab indicates that hackers are targeting banks, telecommunication companies, and government organizations in 40 countries, including the US, South America, Europe and Africa, with Fileless malware that resides solely in the memory of the compromised computers.
Fileless malware was first discovered by the same security firm in 2014, has never been mainstream until now.
Fileless malware is a piece of nasty software that does not copy any files or folder to the hard drive in order to get executed. Instead, payloads are directly injected into the memory of running processes, and the malware executes in the system's RAM.
Since the malware runs in the memory, the memory acquisition becomes useless once the system gets rebooted, making it difficult for digital forensic experts to find the traces of the malware.
The attack was initially discovered by a bank's security team after they found a copy of Meterpreter — an in-memory component of Metasploit — inside the physical memory of a Microsoft domain controller.
After conducting a forensic analysis, Kaspersky researchers found that the attackers leveraged Windows PowerShell to load the Meterpreter code directly into memory rather than writing it to the disk.
The cyber crooks also used Microsoft's NETSH networking tool to set up a proxy tunnel for communicating with the command and control (C&C) server and remotely controlling the infected host.
They also stashed the PowerShell commands into the Windows registry in an effort to reduce nearly all traces of the attacks left in logs or hard drive after a reboot of the device, making detection and forensic analysis difficult.
The ultimate goal of the attackers was apparently aimed at compromising computers that control ATMs so that they could steal money.
Kaspersky Lab researchers plan to reveal more details in April about the attack, which is occurring on an industrial scale worldwide.
The attack has already hit more than 140 enterprise networks in business sectors, with most victims located in the US, France, Ecuador, Kenya, the UK, and Russia. And since the threat is so hard to spot, the actual number is likely much higher.
Swati Khandelwal
Technical Writer, Security Blogger and IT Analyst. She is a Technology Enthusiast with a keen eye on the Cyberspace and other tech related developments.


Are they busy watching the news or the latest viral video? How do they sneak a peek—under their cube or while they pretend to tie their shoes? Find out.
Workplaces may block YouTube, Funny or Die, and other video sites, but that doesn't mean employees don't stream videos on their mobile devices.
To investigate this sneaky streaming, take a look at this infographic from Column Five and Wistia.
A whopping 64 percent of Americans watch online videos at work. Of those, only 53 percent of men and 34 percent of women admit to it.


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text:
Патагонијски опосум или патагонијски пацов (-{Lestodelphys halli}-) је врста сисара из породице опосума (-{Didelphidae}-) и истоименог реда (-{Didelphimorphia}-).

Распрострањење
Подручје Патагоније у Аргентини је једино познато природно станиште врсте.

Популација ове врсте се смањује, судећи по расположивим подацима.

Станиште
Патагонијски опосум има станиште на копну.

Угроженост
Ова врста није угрожена, и наведена је као последња брига јер има широко распрострањење.

Референце

Литература
 http://www.iucnredlist.org/apps/redlist/details/11856/0

Спољашње везе

Опосуми
Фауна Аргентине
Сисари Јужне Америке

redpajama_set_name: RedPajamaWikipedia


Parents, has your child come home talking about the new Book Club, Animal Club, or Cartoonist Club? If so, it's because some entrepreneurial students in the EDS After School program have decided to start their own clubs, held from 4-4:30, during After School. These student-led clubs cover a variety of topics and students are learning valuable leadership skills. While not as structured as an enrichment, I believed it was important to let the students run with their ideas, and am excited to build these clubs into our program as best I can. In the meantime, if your student would like to start a club, or join a club, but you are not sure how that will fit into your afternoon schedule, please reach out and let me know. This is a brand new program, and I am excited to try it out and see what we can make it! I'm really enjoying their spirit, leadership, and ingenuity.


text:
Ventricular Tachycardia: Catheter Ablation, Who When and How?
Jason Bradfield, MD, discusses ventricular tachycardia and catheter ablation.
Thank you all for being here and thank you to the organizers for having me. My topic for today is to trick your tech cardia catheter ablation. Who when and how my name is Jason Bradfield from U. C. L. A cardiac arrhythmia center. Have no disclosures. So we're gonna talk a little bit today about VT ablation. I realize this is a mixed audience of folks interested in cardiology. Not necessarily all electro physiologists. We'll talk about who we oblate both idiopathic and empathic patients and those with cardiomyopathy when we intervene. Generally, this is when meds fail or not desired by the patient and we'll talk a little bit about the techniques we use, which varies depending on the substrate idiopathic PVCS or BT occur in patients with structurally normal hearts. We treat them predominantly for one of two reasons, either symptoms or a high burden of PVCS that leads to a risk of PVC induced cardiomyopathy. Typically in the 15 to 20% or more range on a 24 hour. Holter monitor any empathic PVCS and VT typically are focal and origin as opposed to structural VTS which are more typically reentry in origin in terms of their mechanism. So when we're doing an ablation for a patient with idiopathic PVcs, we're typically looking for electrical signals that are early relative to the beat that we're mapping. So we use electron atomic maps to localize the earliest sign of activation. So the earlier earlier you are with these local signals on the ablation catheter or on a mapping. Catheter. The more likely you are in the site of origin. And then we can use both the three D. Map and flores coptic images as shown here. And this is the ablation catheter in the right ventricular outflow track to map and oblate those vocal arrhythmias. We can also use pace mapping where we pace at the site of early activation and see if that matches the clinical PBC. So this is pacing and this is the clinical PVC and they're very similar to suggest we're in the right place. Idiopathic arrhythmias tend to cluster in certain regions of the heart. We typically think of the right ventricular outflow tract is the most common side of origin. Some of that may be an over representation of early studies that were successfully a bladed in the R. V. O. T. P. V. C. S in reality can come from a variety of different regions but they tend to cluster in what we call the superior subtle process and the inferior septal process that includes areas like the R. V. O. T. The aortic root, the LV summit and that are kind of different regions within what we would call the superior septal process. And then the inferior process process includes things like the cardiac crux often involving the venous system in other areas when you're mapping idiopathic ventricular arrhythmias. The key is really to be comprehensive and your mapping and not assume that you're always going to be coming from one place. These are very highly related and closely related anatomical regions in this image. You see an aortic root shot with a simultaneously with an R. V. O. T. Angiogram. And you can see how anatomically close these regions are with a coronary sinus and A I. V. Catheter as well as his catheter and RV catheter by mapping comprehensively in a number of different regions we can hone in on the area of the earliest activation. A lot of these arrhythmias come from the left side of the heart. It probably more than was originally thought. This is an example of a map of the aortic root as you see here. And these are the coronary cusp in the L. B. Body or risk optically superimposed is an eric route shot onto that map again with the coronary sinus catheter in place. And by mapping retrograde through the aorta, we can put a catheter into the coronary cusp of late successfully. Many arrhythmias that come from this region anatomically, as I mentioned, the arctic route as shown here and the post here right ventricular outflow tract are highly related and only separated by a very small wall thin wall. And so sometimes people think that something's coming from the RV. O. T. But in reality it may be coming from their root coronary cusp region because they're so anatomically close and you can see that from this pathologic specimen coronary customs are here. Our bot is here in this thin walled structure of P. V. C. Or V. T. Could be coming from anywhere on either side of this and what you label it as really depends on where you were successful at blading it and sometimes the PVCS can exit on different sides of this wall. So in this case this PVC was a bladed successfully in the R. V. O. T. And a similar but slightly different PVC a bladed across that wall in the coronary cusp. We're learning a lot about idiopathic PVcs and that there are some circadian or diurnal variations that occur in this patient population. And this helps us understand which patients are likely to respond to certain medications like beta blockers but also helps us understand when we're doing a PVC or Bt ablation and empathic patient. What drugs will likely help us bring out those extra beats. Because in this patient population it is often true that the PVCS are hard to elicit and bring out and we use medications like ice april to bring those PVcs out for sufficient mapping. Knowing their diurnal variation helps us determine which drugs will be successful. And that's something that we published a while back moving towards structural meaty again, typically occurring in patients with underlying karma empathy. The mechanism is re entrant because of scars as shown in this MRI you get areas of slow conduction through the scar which allow for re entrance circuits to occur. All Vts and structural heart disease have some form of substrate. Whether that's systemic heart disease or infiltrated disease or some other substrate. But they also often have triggers whether that's changes in autonomic tone related to their karma apathy. Whether it's some stimulant or drug, whether it's an electrolyte abnormality that combined with the substrate to cause VT and sometimes VF. And in the worst case scenario said cardiac death when it comes to ablation of structural heart disease, we know that it works. The international matricular cardio ablation center collaborative group which we run out of U. C. L. A. Showed in retrospective data. Multi center international data that the success rate at one year. VT freedom was approximately 70% in all comers. That includes a skeptic and non ischemic retinopathy. What we also know from numerous studies is that patients need to be referred earlier in their disease course to have the best chance of success if we wait too long. Their crime apathy may progress to the point that ablation may be less likely to be successful. So referring when they have their first event as opposed to when they've had recurrent admissions for VT storm is in the patient's best interest additionally adding more than one anti arrhythmic drug rarely is beneficial. And ablation has been shown to be superior to adding a second drug as in the banished trial ablation doesn't work as well for non ischemic as it does for ischemic patients. And there's high variability in the success rate for non ischemic patients. But the way we like to look at non ischemic cardiomyopathy is there are certain patient populations within the non ischemic cohort that do very well with ablation, RBC and sharks disease. Or two of those. They have success rates that are very similar to patient population with ischemic heart disease. There are some patients that have less predictable substrates but still highly beneficial results from ablation such as myocarditis. It's chronic sarcoidosis without inflammation, et cetera. There are others that are even more challenging, like hypertrophic cardiomyopathy, amyloid heart or if there's active inflammation of any kind. Those patients have a much lower success rate in each of these patient populations. To be taken into consideration as you're determining the best treatment course and likelihood of success and the non ischemic patient population. Epic cardio mapping where we stick a needle in like a parakeet synthesis but without any fluid around the heart and put catheters into the pericardial space. And you can see the needle down here tempting of the pericardium here with a little bit of contrast and the wire going in and around the heart safely to allow for catheter manipulation in the pericardial space. We determine in a lot of these patients what the best treatment modality is or mapping technique is based on preoperative imaging. So M. R. I. Is done frequently at our institution looking for the scar that is associated with the patients VT if they have septal scar. As shown here. Perhaps epic are real mapping wouldn't be a benefit because the epic cardio surface is not an issue. It's the septal surface. If we're seeing trans mural or epic cardio or mid myocardial scar then having both endo karniol inside of the heart mapping and epic are ideal outside of the heart mapping can be beneficial in terms of treatment outcomes. But again, knowing the substrate matters because the central substrates wouldn't be benefited. Likewise, sometimes patients can even have scars on their popular muscles which is an inter caboto restructure. And again, knowing where you're going in terms of your mapping strategy is highly important. So imaging it plays a key role. We've done a lot of work with pet scans. So in the non ischemic patient population, understanding if patients have an inflammatory cardiomyopathy and if that inflammatory cardiomyopathy is active. So if there's active inflammation on a pet scan, patients do very poorly with ablation and they should be treated with anti inflammatory drugs and then considered for ablation. If they have matricula arrhythmias despite control of the active inflammation inflammation on an electron atomic map during VT mapping and ablation looks very much like scar and therefore differentiating your target becomes very challenging and information cannot be easily a bladed. In our experience we find about a 25% hit rate in the non ischemic population for active inflammation for patients presenting with V. T. V. T. Or B. T. Storm. And we find that by treating that inflammation, those patients do approximately as well as someone without inflammation treated with ablation. So we can avoid ablation in some patients simply by treating the active inflammation. When we do map any of these patients, the techniques involved looking for these areas of slow conduction that I mentioned and we do that by looking at the local electro graham signals. So we make scar maps as shown on the top right here, grading scarf purple, being normal tissue. And we make functional maps which are maps that look at slow areas of slow conduction. So we can assign a color to timeframe of activation and see that when we see bunching of colors that shows areas of slowing of conduction and that suggests areas of functional delay that may lead to re entering arrhythmias. This is an epic arial maps a scar map again grading scar probably being normal. And so these maps can be made on either surface and this is an example of a functional map where we see this bunching of colors and the target is where these colors come together because that's where the areas of slow conduction reside. This is an example of a scar map with activation where you can see the activation into that scar and then the activation into the area of slow conduction. We use newer technology has very high density multi polar mapping which can be very advantageous for quickly obtaining thousands of points on an activation map and decreasing the duration of the procedure for patients that are often very sick. And in this case you see the grid map of the grid catheter here on that Bacardi um and we can quickly map this VT. And we're seeing what we call diastolic potentials, which is slow conduction between the QRS complexes that suggest the area of the isthmus of the VT or the critical region. That needs to be a bladed for control of the ventricular. With me by using this newer technology, we can, in addition to making our scar map here of an inferior basal inferior scar. BTS induced. We see the late potentials that I mentioned that that suggests areas of slow conduction in sinus rhythm that then translate to potentially areas of critical slow conduction during VT. The mid diastolic potentials. Most BTS are not well tolerated for patients with cardiomyopathy because there have low ejection fractions that are quite sick. And so by using all that data that is showing the scar maps the functional maps. Even though we can't map up the entire circuit of the BT because the patient becomes hypertensive, we can get a quick activation map in the critical area and we can see the conduction coming through this little isthmus here and that allows us to target the appropriate area without keeping the patient in VT for very long so that they don't decompensate ablation in that region of the scar and the critical isthmus makes the patient non irreducible and it's a good end point for VT ablation. Now there is a lot of art to the that goes with the science of VT ablation. Many things can't be completely studied in large numbers due to the you know how sick these patients are. So we will often map patients in VT if they can tolerate it. But that often may depend on the injection fraction. Well others we will just look at the substrate in the functional maps. Some patients need human every support meaning balloon pumps and paella even ECMO to allow for mapping. There's no strict cut off for that. A lot of it depends on understanding how sick the individual patient is. Um But every patient is different. Um And so experience plays a big role in understanding which patients need that extra support form a safety standpoint because in the long run their goal is to get these patients through these procedures safely. Here's one more example of an interesting case where a number of decisions had to be made. There were somewhat atypical an older patient for our patient population thought to be needing hospice by referring doctors. His family insisted to be transferred to our institution had been incessant, slow tolerated VT for months, suggestion fraction had dropped substantially. He came in in two B. T. N. D. T. To the cath lab had been in Bt so long that he developed left appendage clots and so we went retrograde for activation instead transept and you see the catholic coming through his bio prosthetic aortic developed here. We found mid diastolic potentials on the anterior wall and terminated the VT with the initial abrasions in this area. Here on the anterior wall had an extensive scar on the mid typical part of his ventricle. We consolidated the ablation in the region of the termination. We did not try to re induce the V. T. To the clots and the risk of needing to shock the patient in the risk of stroke related to those clots and the patient did fantastic. His he was without Bt for many years in just a fraction improved and he did it very well. In some patients with VF we can do ablation typically that's for patients that have a triggered PVC, a consistent PVC that can be reproduced. That then leads to VF because BF itself can't typically be mapped. But if you can find the triggering PVC, you can often control of VF by targeting that PVC in rare instances were also considering more of a de bulking of the Perkin Gee system. This is very early data looking at the fact that the Perkins, the system in some patients is hyperactive and reentry within this system can lead to VF and so oftentimes in small numbers. Again this early data, potentially we can control the arrhythmias by de bulking the distal Birkin gee system. It often involves patients that have some scarring in the area at the base of the popular muscles where the break in the system lives. Hybrid surgical ablation is something that we do a lot of work in for patients that need that have had previous cardiac surgery. They often will need ablation using surgical techniques where the surgeon will open up access with the economy or subsidize avoid window. In this case the patient had a double mechanical valve. The access is shown here, the beating heart. That's the l the impacts that you're seeing there. We can make activation maps and see diastolic potentials in the operating room. In this case, the VT was mapped and you can start to see the circuit here. Here's the VT and we use cryo ablation and you'll see VT terminate as you go along here on the first cry a lesion. This is the cryo pro and that we often use freezes to the surface of the my accordion. And it's very effective informing leach notes. We can do these procedures on pump Western economy, but we prefer not to. It's only rare instances that we do that when no other options are available. We are looking at understanding lesion technology and how we can best deliver deep lesions for these patients while protecting the coronary arteries. One last note sP R. T. Or radiation for VT is an area of interest for a number of centers looking at external beam treatment finding ways to make VT treatment noninvasive still in the early stages but a number of promising reports in this area. We still have a lot to learn, however, about how to optimize these lesions sets and how to deliver effective non invasive lesions over the next few years will get a lot more data in that area. In conclusion, VT ablation works. Comprehensive planning, however, is essential for the best outcomes. Ablation should be considered earlier in the treatment of patients before the african storm or long term side effects from anti arrhythmic drugs. Again, thank you to the organizers for having me and thank you to my team at UCLA.
Jason Bradfield, MD, FACC FHRS
Director, Specialized Program for Ventricular Tachycardia
UCLA Cardiac Arrhythmia Center


text:
Narratives of Mandarin-speaking patients with schizophrenia
Ning HSU, Hin Tat CHEUNG, Enan WANG, Tai-Li CHOU
Department of Linguistics and Modern Language Studies (LML)
Research output: Chapter in Book/Report/Conference proceeding › Chapters
Schizophrenia is characterized by abnormalities in perception and expression. The traditional view of their disordered speech as a reflection of their disorganized thinking (Bleuler, 1950; Kraepelin, 1919/1992) has been questioned for the reason that language and thought are not in total isomorphic relationship. The disordered speech observed in individuals with schizophrenia could be more than a mere thought disorder or a mere language disorder (Harvey & Neale, 1983; McKenna & Oh, 2005). The present study investigated the narrative ability of Mandarin-speaking patients with schizophrenia. A group of 22 patients with schizophrenia as well as 20 normal controls participated in a story-telling task. Participants were asked to narrate three picture-books and their performance was evaluated by a Mandarin version of Narrative Assessment Profile (Tsou, Chang, & Cheung, 2009), which examines eight dimensions of a narrative: topic maintenance, event sequencing, reasonableness, referential skill, background information, evaluation, conjunctive cohesion, and the overall narrative pattern. Lexical choices of connectives (causal connectives and contrastive connectives) as well as verbal predicates (perceptual verbs, psychological verbs, and mental verbs) were also analyzed. Results showed that participants in the schizophrenic group preserved an intact ability to produce basic elements in narratives, performing comparatively well in dimensions of event sequencing and referential skill. However, their performance in the other six dimensions was significantly weaker than the controls. It is proposed that individuals with schizophrenia display an overall lack of structure in narratives, as reported in previous literature (Chaika & Alexander, 1986; Lysaker et al., 2005). They seemed to have difficulties in coping with the communication needs of their listeners, as revealed by lower scores in reasonableness, background information, and evaluation. Further evidence came from lexical analyses which also showed that the group with schizophrenia used fewer contrastive connectives and mental verbs when telling their stories. Discussions of the implications of these findings are considered. Copyright © 2013 John Benjamins Publishing Company.
Chinese language narration: Culture, cognition, and emotion
Allyssa MCCABE, Chien-ju CHANG
Amsterdam; Philadelphia
John Benjamins Publishing Company
https://doi.org/10.1075/sin.19.09hsu
Hsu, N., Cheung, H., Wang, E., & Chou, T.-L. (2013). Narratives of Mandarin-speaking patients with schizophrenia. In A. McCabe, & C.-j. Chang (Eds.), Chinese language narration: Culture, cognition, and emotion (pp. 181-206). Amsterdam; Philadelphia: John Benjamins Publishing Company.
Narrative assessment
10.1075/sin.19.09hsu
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Schizophrenia Medicine & Life Sciences 100%
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