Activated checkpoint kinase 2 provides a survival signal for tumor cells

Tumor cells often become resistant to DNA damage-based therapy; however, the underlying mechanisms are not yet understood. Here, we show that tumor cells exposed to DNA damage counteract cell death by releasing the antiapoptotic protein, survivin, from mitochondria. This is independent of p53, and requires activated checkpoint kinase 2 (Chk2), a putative tumor suppressor. Molecular or genetic targeting of Chk2 prevents the release of survivin from mitochondria, enhances DNA damage-induced tumor cell apoptosis, and inhibits the growth of resistant in vivo tumors. Therefore, activated Chk2 circumvents its own tumor-suppressive functions by promoting tumor cell survival. Inhibiting Chk2 in combination with DNA-damaging agents may provide a rational approach for treating resistant tumors

Essential Role of the Small GTPase Ran in Postnatal Pancreatic Islet Development

The small GTPase Ran orchestrates pleiotropic cellular responses of nucleo-cytoplasmic shuttling, mitosis and subcellular trafficking, but whether deregulation of these pathways contributes to disease pathogenesis has remained elusive. Here, we generated transgenic mice expressing wild type (WT) Ran, loss-of-function Ran T24N mutant or constitutively active Ran G19V mutant in pancreatic islet β cells under the control of the rat insulin promoter. Embryonic pancreas and islet development, including emergence of insulin+ β cells, was indistinguishable in control or transgenic mice. However, by one month after birth, transgenic mice expressing any of the three Ran variants exhibited overt diabetes, with hyperglycemia, reduced insulin production, and nearly complete loss of islet number and islet mass, in vivo. Deregulated Ran signaling in transgenic mice, adenoviral over-expression of WT or mutant Ran in isolated islets, or short hairpin RNA (shRNA) silencing of endogenous Ran in model insulinoma INS-1 cells, all resulted in decreased expression of the pancreatic and duodenal homeobox transcription factor, PDX-1, and reduced β cell proliferation, in vivo. These data demonstrate that a finely-tuned balance of Ran GTPase signaling is essential for postnatal pancreatic islet development and glucose homeostasis, in vivo

Molecular dependence of estrogen receptor-negative breast cancer on a notch-survivin signaling axis

Despite progress in the management of breast cancer, the molecular underpinnings of clinically aggressive subtypes of the disease are not well-understood. Here, we show that activation of Notch developmental signaling in estrogen receptor (ER)-negative breast cancer cells results in direct transcriptional up-regulation of the apoptosis inhibitor and cell cycle regulator survivin. This response is associated with increased expression of survivin at mitosis, enhanced cell proliferation, and heightened viability at cell division. Conversely, targeting Notch signaling with a peptidyl gamma-secretase inhibitor suppressed survivin levels, induced apoptosis, abolished colony formation in soft agar, and inhibited localized and metastatic tumor growth in mice, without organ or systemic toxicity. In contrast, ER+ breast cancer cells, or various normal cell types, were insensitive to Notch stimulation. Therefore, ER- breast cancer cells become dependent on Notch-survivin signaling for their maintenance, in vivo. Therapeutic targeting of this pathway may be explored for individualized treatment of patients with clinically aggressive, ER- breast cancer

IAP regulation of metastasis

Inhibitor-of-Apoptosis (IAP) proteins contribute to tumor progression, but the requirements of this pathway are not understood. Here, we show that intermolecular cooperation between XIAP and survivin stimulates tumor cell invasion and promotes metastasis. This pathway is independent of IAP inhibition of cell death. Instead, a survivin-XIAP complex activates NF-kappaB, which in turn leads to increased fibronectin gene expression, signaling by beta1 integrins, and activation of cell motility kinases FAK and Src. Therefore, IAPs are direct metastasis genes, and their antagonists could provide antimetastatic therapies in patients with cancer

Acute Thrombus Burden on Coated Flow Diverters Assessed by High Frequency Optical Coherence Tomography

PURPOSE: The implantation of flow diverters requires administration of dual anti-platelet therapy, posing the potential for complications. The p48MW HPC (phenox, Bochum, Germany) hydrophilic-coated flow diverting stent is designed to be anti-thrombotic, thus opening the potential for single anti-platelet therapy. We deploy a novel intravascular high-resolution imaging technique, high-frequency optical coherence tomography (HF-OCT), to study in an animal model the acute thrombus formation on coated p48MW devices versus uncoated control devices. METHODS: Three pigs were implanted with 4 flow diverters each, two test hydrophilic-coated devices, and two control uncoated devices (p48MW). Each pig was treated with a different anti-platelet regime: no anti-platelet therapy, aspirin only, aspirin and clopidogrel. Twenty minutes after the flow diverter was implanted, an HF-OCT data set was acquired. Acute clot formed on the flow diverter at each covered side branch was measured from the HF-OCT slices. Factors considered to be important were the device type (pHPC versus bare metal), aspirin, clopidogrel, and vessel location. A linear model was constructed from the significant factors. RESULTS: Both coating (p \u3c 0.001) and aspirin (p = 0.003) were significantly related to reduction in clot burden, leading to an approximate 100-fold and 50-fold reduction in clot, respectively. CONCLUSIONS: This study shows the power of HF-OCT not only in the detection of clot but also the quantification of clot burden. In an animal model, the pHPC-coated p48MW significantly reduced acute thrombus formation over jailed side branches as compared to the bare metal p48MW that was nearly eliminated when combined with aspirin administration

Deregulated PDX-1 expression in Ran-targeted cells.

<p><i>A</i>, Pancreas sections from non-TG, Ran-WT, Ran-G19V or Ran-T24N transgenic mice were stained with an antibody to insulin or PDX-1, by immunohistochemistry. <i>B</i>, The number of insulin- or PDX-1-stained cells was quantified by morphometry in the indicated surface area. Insulin⁺ cells, non-TG (n = 2), 28.2±0.4; Ran-WT (asymptomatic, n = 3), 29.8±1.2; Ran-WT (diabetic, n = 3), 6.2±0.6, ***, p<0.0001; PDX-1⁺ cells, Non-TG (n = 2), 34.2±4.1; Ran-WT (asymptomatic, n = 3), 37.5±2.3; Ran-WT (diabetic, n = 3), 22.8±1.8, **, p = 0.007. <i>C</i>, Islets from 2 mo-old non-TG or a Ran-WT transgenic mouse were analyzed by Western blotting. <i>D</i>, Pancreatic islets isolated from non-TG mice were transduced <u>ex vivo</u> with control pAd-GFP or pAd-GFP-Ran-WT, pAd-GFP-Ran-G19V or pAd-GFP-T24N and analyzed after 48 h by fluorescence microscopy for GFP expression (<i>left</i>), or Western blotting (<i>right</i>). *, non-specific. <i>E</i>, INS-1 cells were left untreated (INS-1) or transduced with control lentivirus (pLKO) or lentivirus encoding Ran-directed shRNA (Ran, 74V1), and analyzed by Western blotting. <i>F</i>, Parental INS-1 cells or four independent clones of INS-1 cells stably transduced with Ran-directed shRNA (77V-2, 74V-1, 75V-1, 75V-2) were analyzed for changes in insulin release in the supernatant. Representative experiment out of at least two independent determinations.</p

Transgenic expression of Ran impairs glucose metabolism.

<p><i>A and B</i>, The indicated PCR-confirmed transgenic mice were analyzed for blood glucose content at 2 mo of age under <u>ad libitum</u> feeding (<i>A</i>) or fasting (<i>B</i>) conditions. Non-TG, non-transgenic mice. Glucose concentrations (mg/dl) in each group in <i>A</i> (number of mice in parentheses), and statistical analyses (unpaired <i>t</i> test) are as follows: Non-TG (n = 24), 125±2; Ran-WT (n = 26), 189.4±17.8, p = 0.013; Ran-G19V (n = 24), 153.7±6.5, p = 0.003; Ran-T24N (n = 21), 172.4±21.1, p = 0.02. Statistical data re-analysis of the groups in <i>A</i> using ANOVA and post-hoc multiple tests with Bonferroni procedure was as follows: Ran-WT, p<0.0001; Ran-G19V, 0.017; Ran-T24N, p = 0.029. <i>C</i>, The indicated non-TG or Ran transgenic mice were analyzed for blood insulin concentrations. Insulin levels (ng/ml) in each group (number of mice in parentheses), and statistical analyses (unpaired <i>t</i> test) are as follows: Non-TG (n = 8), 1.26±0.1; Ran-WT (n = 11), 0.7±0.16, p = 0.019; Ran-G19V (n = 5), 0.61±0.03, p = 0.0008; Ran-T24N (n = 7), 0.49±0.14, p = 0.0009. Statistical data re-analysis using ANOVA and post-hoc multiple tests with Bonferroni procedure was as follows: Ran-WT, p = 0.023; Ran-G19V, p = 0.031; Ran-T24N, p = 0.004. One outlier mouse in the Ran-G19V group with aberrantly high insulin level (2.35 ng/ml) was excluded from the analysis. For panels <i>A–C</i>, each point corresponds to an individual mouse. <i>D</i>, Islets (20/well) isolated from non-TG or Ran-WT transgenic mice were incubated with 5 mM D-glucose, and analyzed for insulin release in the supernatant at the indicated time intervals. Mean±SD of replicates. <i>E</i>, Islets (20/well) isolated from non-TG (<i>black</i>) or Ran-WT (<i>purple</i>), Ran-G19V (<i>grey</i>) or Ran-T24N (<i>blue</i>) transgenic mice were incubated with 16.7 mM glucose, and analyzed for insulin release in the supernatant at the indicated time intervals. Mean±SD of replicates.</p

Longitudinal Monitoring of Flow-Diverting Stent Tissue Coverage After Implant in a Bifurcation Model Using Neurovascular High-Frequency Optical Coherence Tomography

BACKGROUND: Tissue growth over covered branches is a leading cause of delayed thrombotic complications after flow-diverter stenting (FDS). Due to insufficient resolution, no imaging modality is clinically available to monitor this phenomenon. OBJECTIVE: To evaluate high-frequency optical coherence tomography (HF-OCT), a novel intravascular imaging modality designed for the cerebrovascular anatomy with a resolution approaching 10 microns, to monitor tissue growth over FDS in an arterial bifurcation model. METHODS: FDS were deployed in a rabbit model (n = 6), covering the aortic bifurcation. The animals were divided in different groups, receiving dual antiplatelet therapy (DAPT) (n = 4), aspirin only (n = 1), and no treatment (n = 1). HF-OCT data were obtained in vivo at 3 different time points in each animal. For each cross-sectional image, metal and tissue coverage of the jailed ostium was quantified. Scanning electron microscopy images of harvested arteries were subsequently obtained. RESULTS: Good quality HF-OCT data sets were successfully acquired at implant and follow-up. A median value of 41 (range 21-55) cross-sectional images were analyzed per ostium for each time point. Between 0 and 30 d after implant, HF-OCT analysis showed a significantly higher ostium coverage when DAPT was not given. After 30 d, similar growth rates were found in the DAPT and in the aspirin group. At 60 d, a coverage of 90% was reached in all groups. CONCLUSION: HF-OCT enables an accurate visualization of tissue growth over time on FDS struts. The use of FDS in bifurcation locations may induce a drastic reduction of the jailed-branch ostium area

Complete clot ingestion with cyclical ADAPT increases first-pass recanalization and reduces distal embolization

BACKGROUND: Evidence is mounting that first-pass complete recanalization during mechanical thrombectomy is associated with better clinical outcomes in patients presenting with an emergent large vessel occlusion. We hypothesize that aspiration achieving complete clot ingestion results in higher first-pass successful recanalization with quantitative reduction in distal emboli. METHODS: A patient-specific cerebrovascular replica was connected to a flow loop. Occlusion of the middle cerebral artery was achieved with clot analogs. Independent variables were the diameter of the aspiration catheter (0.054-0.088in) and aspiration pattern (static versus cyclical). Outcome measures were the first-pass rates of complete clot ingestion, the extent of recanalization, and the particle-size distribution of distal emboli. RESULTS: All aspiration catheters were successfully navigated to the occlusion. Complete clot ingestion during aspiration thrombectomy resulted in first-pass complete recanalization in every experiment, only achieved in 21% of experiments with partial ingestion (P \u3c 0.0001). Aspiration through the large bore 0.088in device resulted in the highest rates of complete clot ingestion (90%). Cyclical aspiration (18-29 inHg, 0.5 Hz) significantly increased the rate of complete clot ingestion (OR21 [1.6, 266]; P=0.04). In all experiments, complete clot ingestion resulted in fewer and smaller distal emboli. CONCLUSIONS: Complete clot ingestion results in fewer distal emboli and the highest rates of first-pass complete recanalization. The rate of complete ingestion during aspiration thrombectomy is a function of both the inner diameter of the aspiration catheter and use of cyclical aspiration

Defective islet development in Ran transgenic mice.

<p><i>A</i>, Pancreas tissues from non-TG, Ran-WT, Ran-G19V or Ran-T24N transgenic mice were analyzed by H&E or immunohistochemical staining with an antibody to insulin. <i>B</i>, Pancreas sections from non-TG, asymptomatic Ran-WT or diabetic Ran-WT transgenic mice were analyzed for islet number or islet surface area by morphometry of insulin-stained areas. Islet number, non-TG (n = 2), 29.5±1.5; Ran-WT (n = 3), 23±1.73, n.s., not significant; Ran-WT diabetic (n = 3), 9.33±0.88, **, p = 0.001; islet surface area, non-TG (n = 2), 0.51±0.02; Ran-WT (n = 3), 0.35±0.021, *, p = 0.017; Ran-WT diabetic (n = 3), 0.057±0.004, **, p<0.0001. <i>C</i>, Pancreas tissues from Ran-WT, Ran-G19V or Ran-T24N transgenic mice were analyzed by immunohistochemical staining with antibodies to somatostatin or glucagon.</p