Pathology analysis of the mice injected with Ba/F3 cells, control p185 cells and p185 cells transduced with Abi1 shRNA

() Spleen weight of mice injected with Ba/F3 cells (control) and the p185 cells expressing with (p185 + Abi1 shRNA) or without (p185) Abi1 shRNA. () Histology of spleens and livers from the mice receiving Ba/F3 cells (control) and the p185 cells expressing with (p185 + Abi1 shRNA) or without (p185 ctrl) Abi1 shRNA. Arrowheads indicate the massively expanded p185 cells.<p><b>Copyright information:</b></p><p>Taken from "Abi1 gene silencing by short hairpin RNA impairs Bcr-Abl-induced cell adhesion and migration and leukemogenesis "</p><p></p><p>Carcinogenesis 2008;29(9):1717-1724.</p><p>Published online 2 May 2008</p><p>PMCID:PMC2527646.</p><p></p

Abi1 knockdown inhibited Bcr-Abl-stimulated actin cytoskeleton remodeling, MT1-MMP clustering, as well as cell adhesion and migration on fibronectin-coated surfaces

() Inhibition of Bcr-Abl-induced abnormal actin remodeling by Abi1 knockdown. Ba/F3 cells and the Ba/F3 cells expressing p185, p185 and p185 plus Abi1 shRNA were fixed and stained with TRITC-conjugated phalloidin. The cells with F-actin-rich structures (invadopodia-like structures) were visualized by fluorescence microscopy as shown by arrowheads (upper panel) and were counted (lower panel, represented as mean ± SD percentage of three randomly picked areas). () The p185 cells expressing GFP–MT1-MMP were transduced with either control retrovirus (control) or the retrovirus expressing Abi1 shRNA. The knock down of Abi1 expression was confirmed by western blotting (data not shown). The distribution of GFP–MT1-MMP was visualized by fluorescence microscopy. A similar result has been described previously (). () Effects of Abi1 knockdown on Bcr-Abl-stimulated cell adhesion (lower panel) and migration (upper panel) on fibronectin-coated surfaces. Ba/F3 cells and the p185 cells transduced with either non-silencing shRNA or Abi1 shRNAs were grown in fibronectin-coated six-well plate (2.5 × 10 per well) for 16 h. The total cells and the cells that were adherent to fibronectin-coated surfaces were counted and the percentage of adherent cells calculated. The vertical axis shows the percentage of the adherent cells and is expressed as the mean ± SD of triplicate wells. For cell migration on fibronectin-coated membrane, 1 × 10 cells were tested in Transwell migration assay. The vertical axis shows the percentage of the migrated cells and is expressed as the mean ± SD of triplicate wells; * < 0.01.<p><b>Copyright information:</b></p><p>Taken from "Abi1 gene silencing by short hairpin RNA impairs Bcr-Abl-induced cell adhesion and migration and leukemogenesis "</p><p></p><p>Carcinogenesis 2008;29(9):1717-1724.</p><p>Published online 2 May 2008</p><p>PMCID:PMC2527646.</p><p></p

The knock down of Abi1 expression attenuated Bcr-Abl-induced activation of Lyn kinases

Ba/F3 cells (lanes 1 and 4), control p185 cells (lanes 2 and 5) and p185 Abi1 shRNA cells (lanes 3 and 6) were starved in RPMI 1640 plus 0.1% bovine serum albumin for 6 h. The cells were then treated without (lanes 1–3) or with (lanes 4–6) recombinant rat IL3 (20 ng/ml) for 10 min. The lysates (2 × 10 cells) from indicated cell lines were immunoprecipitated (IP) with anti-Lyn antibodies, separated on 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subjected to western blot (WB) analysis using anti-Src p416 antibody, which recognizes activated Lyn (upper panel). The membrane then was stripped and reprobed with anti-Lyn antibodies (lower panel). Two Lyn isoforms, p53Lyn and p56Lyn, were indicated.<p><b>Copyright information:</b></p><p>Taken from "Abi1 gene silencing by short hairpin RNA impairs Bcr-Abl-induced cell adhesion and migration and leukemogenesis "</p><p></p><p>Carcinogenesis 2008;29(9):1717-1724.</p><p>Published online 2 May 2008</p><p>PMCID:PMC2527646.</p><p></p

Abi1 knockdown did not affect Bcr-Abl-induced protein tyrosine phosphorylation and IL3-independent growth

() Profiles of protein tyrosine phosphorylation in Ba/F3 cells and p185 cells transduced with either non-silencing shRNA (p185) or Abi1 shRNAs (Abi1R). Indicated cell lines were starved in RPMI 1640 plus 0.1% bovine serum albumin for 6 h prior to harvest. Total lysates from 1 × 10 cells were separated on 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subjected to western blot analysis with anti-phosphotyrosine antibody. () IL3-independent growth of Ba/F3 cells as well as the p185 cells transduced with either non-silencing shRNA (p185) or Abi1 shRNA (Abi1R).<p><b>Copyright information:</b></p><p>Taken from "Abi1 gene silencing by short hairpin RNA impairs Bcr-Abl-induced cell adhesion and migration and leukemogenesis "</p><p></p><p>Carcinogenesis 2008;29(9):1717-1724.</p><p>Published online 2 May 2008</p><p>PMCID:PMC2527646.</p><p></p

Increasing extracellular matrix collagen level and MMP activity induces cyst development in polycystic kidney disease

<p>Abstract</p> <p>Background</p> <p>Polycystic Kidney Disease (PKD) kidneys exhibit increased extracellular matrix (ECM) collagen expression and metalloproteinases (MMPs) activity. We investigated the role of these increases on cystic disease progression in PKD kidneys.</p> <p>Methods</p> <p>We examined the role of type I collagen (collagen I) and membrane bound type 1 MMP (MT1-MMP) on cyst development using both <it>in vitro</it> 3 dimensional (3D) collagen gel culture and <it>in vivo</it> PCK rat model of PKD.</p> <p>Results</p> <p>We found that collagen concentration is critical in controlling the morphogenesis of MDCK cells cultured in 3D gels. MDCK cells did not form 3D structures at collagen I concentrations lower than 1 mg/ml but began forming tubules when the concentration reaches 1 mg/ml. Significantly, these cells began to form cyst when collagen I concentration reached to 1.2 mg/ml, and the ratios of cyst to tubule structures increased as the collagen I concentration increased. These cells exclusively formed cyst structures at a collagen I concentration of 1.8 mg/ml or higher. Overexpression of MT1-MMP in MDCK cells significantly induced cyst growth in 3D collagen gel culture. Conversely, inhibition of MMPs activity with doxycycline, a FDA approved pan-MMPs inhibitor, dramatically slowed cyst growth. More importantly, the treatment of PCK rats with doxycycline significantly decreased renal tubule cell proliferation and markedly inhibited the cystic disease progression.</p> <p>Conclusions</p> <p>Our data suggest that increased collagen expression and MMP activity in PKD kidneys may induce cyst formation and expansion. Our findings also suggest that MMPs may serve as a therapeutic target for the treatment of human PKD.</p