Cytosolic SYT/SS18 Isoforms Are Actin-Associated Proteins that Function in Matrix-Specific Adhesion

SYT (SYnovial sarcoma Translocated gene or SS18) is widely produced as two isoforms, SYT/L and SYT/S, that are thought to function in the nucleus as transcriptional coactivators. Using isoform-specific antibodies, we detected a sizable pool of SYT isoforms in the cytosol where the proteins were organized into filamentous arrays. Actin and actin-associated proteins co-immunoprecipitated with SYT isoforms, which also co-sedimented and co-localized with the actin cytoskeleton in cultured cells and tissues. The association of SYT with actin bundles was extensive yet stopped short of the distal ends at focal adhesions. Disruption of the actin cytoskeleton also led to a breakdown of the filamentous organization of SYT isoforms in the cytosol. RNAi ablation of SYT/L alone or both isoforms markedly impaired formation of stress fibers and focal adhesions but did not affect formation of cortical actin bundles. Furthermore, ablation of SYT led to markedly impaired adhesion and spreading on fibronectin and laminin-111 but not on collagen types I or IV. These findings indicate that cytoplasmic SYT isoforms interact with actin filaments and function in the ability cells to bind and react to specific extracellular matrices

How Reliable Is Ki-67 Immunohistochemistry in Grade 2 Breast Carcinomas? A QA Study of the Swiss Working Group of Breast- and Gynecopathologists

Adjuvant chemotherapy decisions in breast cancer are increasingly based on the pathologist's assessment of tumor proliferation. The Swiss Working Group of Gyneco- and Breast Pathologists has surveyed inter- and intraobserver consistency of Ki-67-based proliferative fraction in breast carcinomas. Methods Five pathologists evaluated MIB-1-labeling index (LI) in ten breast carcinomas (G1, G2, G3) by counting and eyeballing. In the same way, 15 pathologists all over Switzerland then assessed MIB-1-LI on three G2 carcinomas, in self-selected or pre-defined areas of the tumors, comparing centrally immunostained slides with slides immunostained in the different laboratoires. To study intra-observer variability, the same tumors were re-examined 4 months later. Results The Kappa values for the first series of ten carcinomas of various degrees of differentiation showed good to very good agreement for MIB-1-LI (Kappa 0.56–0.72). However, we found very high inter-observer variabilities (Kappa 0.04–0.14) in the read-outs of the G2 carcinomas. It was not possible to explain the inconsistencies exclusively by any of the following factors: (i) pathologists' divergent definitions of what counts as a positive nucleus (ii) the mode of assessment (counting vs. eyeballing), (iii) immunostaining technique, and (iv) the selection of the tumor area in which to count. Despite intensive confrontation of all participating pathologists with the problem, inter-observer agreement did not improve when the same slides were re-examined 4 months later (Kappa 0.01–0.04) and intra-observer agreement was likewise poor (Kappa 0.00–0.35). Conclusion Assessment of mid-range Ki-67-LI suffers from high inter- and intra-observer variability. Oncologists should be aware of this caveat when using Ki-67-LI as a basis for treatment decisions in moderately differentiated breast carcinomas

Recombinant Lysyl Oxidase Propeptide Protein Inhibits Growth and Promotes Apoptosis of Pre-Existing Murine Breast Cancer Xenografts

Lysyl oxidase propeptide (LOX-PP) ectopic overexpression inhibits the growth of cancer xenografts. Here the ability and mode of action of purified recombinant LOX-PP (rLOX-PP) protein to inhibit the growth of pre-existing xenografts was determined. Experimental approaches employed were direct intratumoral injection (i.t.) of rLOX-PP protein into murine breast cancer NF639 xenografts, and application of a slow release formulation of rLOX-PP implanted adjacent to tumors in NCR nu/nu mice (n = 10). Tumors were monitored for growth, and after sacrifice were subjected to immunohistochemical and Western blot analyses for several markers of proliferation, apoptosis, and for rLOX-PP itself. Direct i.t. injection of rLOX-PP significantly reduced tumor volume on days 20, 22 and 25 and tumor weight at harvest on day 25 by 30% compared to control. Implantation of beads preloaded with 35 micrograms rLOX-PP (n = 10) in vivo reduced tumor volume and weight at sacrifice when compared to empty beads (p<0.05). A 30% reduction of tumor volume on days 22 and 25 (p<0.05) and final tumor weight on day 25 (p<0.05) were observed with a reduced tumor growth rate of 60% after implantation. rLOX-PP significantly reduced the expression of proliferation markers and Erk1/2 MAP kinase activation, while prominent increases in apoptosis markers were observed. rLOX-PP was detected by immunohistochemistry in harvested rLOX-PP tumors, but not in controls. Data provide pre-clinical findings that support proof of principle for the therapeutic anti-cancer potential of rLOX-PP protein formulations

Vicrostatin – An Anti-Invasive Multi-Integrin Targeting Chimeric Disintegrin with Tumor Anti-Angiogenic and Pro-Apoptotic Activities

Similar to other integrin-targeting strategies, disintegrins have previously shown good efficacy in animal cancer models with favorable pharmacological attributes and translational potential. Nonetheless, these polypeptides are notoriously difficult to produce recombinantly due to their particular structure requiring the correct pairing of multiple disulfide bonds for biological activity. Here, we show that a sequence-engineered disintegrin (called vicrostatin or VCN) can be reliably produced in large scale amounts directly in the oxidative cytoplasm of Origami B E. coli. Through multiple integrin ligation (i.e., αvβ3, αvβ5, and α5β1), VCN targets both endothelial and cancer cells significantly inhibiting their motility through a reconstituted basement membrane. Interestingly, in a manner distinct from other integrin ligands but reminiscent of some ECM-derived endogenous anti-angiogenic fragments previously described in the literature, VCN profoundly disrupts the actin cytoskeleton of endothelial cells (EC) inducing a rapid disassembly of stress fibers and actin reorganization, ultimately interfering with EC's ability to invade and form tubes (tubulogenesis). Moreover, here we show for the first time that the addition of a disintegrin to tubulogenic EC sandwiched in vitro between two Matrigel layers negatively impacts their survival despite the presence of abundant haptotactic cues. A liposomal formulation of VCN (LVCN) was further evaluated in vivo in two animal cancer models with different growth characteristics. Our data demonstrate that LVCN is well tolerated while exerting a significant delay in tumor growth and an increase in the survival of treated animals. These results can be partially explained by potent tumor anti-angiogenic and pro-apoptotic effects induced by LVCN

Distinct mechanisms for diastolic dysfunction in diabetes mellitus and chronic pressure-overload

Chronic pressure-overload and diabetes mellitus are two frequent disorders affecting the heart. We aimed to characterize myocardial structural and functional changes induced by both conditions. Pressure-overload was established in Wistar-han male rats by supra-renal aortic banding. Six-weeks later, diabetes was induced by streptozotocin (65 mg/kg,ip), resulting in four groups: SHAM, banding (BA), diabetic (DM) and diabetic-banding (DB). Six-weeks later, pressure-volume loops were obtained and left ventricular samples were collected to evaluate alterations in insulin signalling pathways, extracellular matrix as well as myofilament function and phosphorylation. Pressure-overload increased cardiomyocyte diameter (BA 22.0 ± 0.4 μm, SHAM 18.2 ± 0.3 μm) and myofilament maximal force (BA 25.7 ± 3.6 kN/m(2), SHAM 18.6 ± 1.4 kN/m(2)), Ca(2+) sensitivity (BA 5.56 ± 0.02, SHAM 5.50 ± 0.02) as well as MyBP-C, Akt and Erk phosphorylation, while decreasing rate of force redevelopment (K (tr); BA 14.9 ± 1.1 s(-1), SHAM 25.2 ± 1.5 s(-1)). At the extracellular matrix level, fibrosis (BA 10.8 ± 0.9%, SHAM 5.3 ± 0.6%), pro-MMP-2 and MMP-9 activities increased and, in vivo, relaxation was impaired (τ; BA 14.0 ± 0.9 ms, SHAM 12.9 ± 0.4 ms). Diabetes increased cardiomyocyte diameter, fibrosis (DM 21.4 ± 0.4 μm, 13.9 ± 1.8%, DB 20.6 ± 0.4 μm, 13.8 ± 0.8%, respectively), myofilament Ca(2+)sensitivity (DM 5.57 ± 0.02, DB 5.57 ± 0.01), advanced glycation end-product deposition (DM 4.9 ± 0.6 score/mm(2), DB 5.1 ± 0.4 score/mm(2), SHAM 2.1 ± 0.3 score/mm(2)), and apoptosis, while decreasing K (tr) (DM 13.5 ± 1.9 s(-1), DB 15.2 ± 1.4 s(-1)), Akt phosphorylation and MMP-9/TIMP-1 and MMP-1/TIMP-1 ratios. Diabetic hearts were stiffer (higher end-diastolic-pressure: DM 7.0 ± 1.2 mmHg, DB 6.7 ± 0.7 mmHg, SHAM 5.3 ± 0.4 mmHg, steeper end-diastolic-pressure-volume relation: DM 0.59 ± 0.18, DB 0.83 ± 0.17, SHAM 0.41 ± 0.10), and hypo-contractile (decreased end-systolic-pressure-volume-relation). DB animals presented further pulmonary congestion (Lungs/body-weight: DB 5.23 ± 0.21 g/kg, SHAM 3.80 ± 0.14 g/kg) as this group combined overload-induced relaxation abnormalities and diabetes-induced stiffness. Diabetes mellitus and pressure overload led to distinct diastolic dysfunction phenotypes: while diabetes promoted myocardial stiffening, pressure overload impaired relaxation. The association of these damages accelerates the progression of diastolic heart failure progression in diabetic-banded animals

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

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