Tenascin-C Enhances Pancreatic Cancer Cell Growth and Motility and Affects Cell Adhesion through Activation of the Integrin Pathway

Background: Pancreatic cancer (PDAC) is characterized by an abundant fibrous tissue rich in Tenascin-C (TNC), a large ECM glycoprotein mainly synthesized by pancreatic stellate cells (PSCs). In human pancreatic tissues, TNC expression increases in the progression from low-grade precursor lesions to invasive cancer. Aim of this study was the functional characterization of the effects of TNC on biologic relevant properties of pancreatic cancer cells. Methods: Proliferation, migration and adhesion assays were performed on pancreatic cancer cell lines treated with TNC or grown on a TNC-rich matrix. Stable transfectants expressing the large TNC splice variant were generated to test the effects of endogenous TNC. TNC-dependent integrin signaling was investigated by immunoblotting, immunofluorescence and pharmacological inhibition. Results: Endogenous TNC promoted pancreatic cancer cell growth and migration. A TNC-rich matrix also enhanced migration as well as the adhesion to the uncoated growth surface of poorly differentiated cell lines. In contrast, adhesion to fibronectin was significantly decreased in the presence of TNC. The effects of TNC on cell adhesion were paralleled by changes in the activation state of paxillin and Akt. Conclusion: TNC affects proliferation, migration and adhesion of poorly differentiated pancreatic cancer cell lines and migh

HIV-1 IN/Pol recruits LEDGF/p75 into viral particles

Background: The dynamic interaction between HIV and its host governs the replication of the virus and the study of the virus-host interplay is key to understand the viral lifecycle. The host factor lens epithelium-derived growth factor (LEDGF/p75) tethers the HIV preintegration complex to the chromatin through a direct interaction with integrase (IN). Small molecules that bind the LEDGF/p75 binding pocket of the HIV IN dimer (LEDGINs) block HIV replication through a multimodal mechanism impacting early and late stage replication including HIV maturation. Furthermore, LEDGF/p75 has been identified as a Pol interaction partner. This raised the question whether LEDGF/p75 besides acting as a molecular tether in the target cell, also affects late steps of HIV replication. Results: LEDGF/p75 is recruited into HIV-1 particles through direct interaction with the viral IN (or Pol polyprotein) and is a substrate for HIV-1 protease. Incubation in the presence of HIV-1 protease inhibitors resulted in detection of full-length LEDGF/p75 in purified viral particles. We also demonstrate that inhibition of LEDGF/p75-IN interaction by specific mutants or LEDGINs precludes incorporation of LEDGF/p75 in virions, underscoring the specificity of the uptake. LEDGF/p75 depletion did however not result in altered LEDGIN potency. Conclusion: Together, these results provide evidence for an IN/Pol mediated uptake of LEDGF/p75 in viral particles and a specific cleavage by HIV protease. Understanding of the possible role of LEDGF/p75 or its cleavage fragments in the viral particle awaits further experimentation

A proteomic approach to identify early molecular targets of oxidative stress in human epithelial lens cells

Oxidative stress is one of the most relevant contributors of cataractogenesis. To identify early protein targets of oxidative stress in lens cells, we used a differential proteomics approach to CD5A human epithelial lens cells treated with 500 microM H2O2 for 30 min. This dose of H2O2 was assayed to induce efficiently a block of cellular proliferation and to activate the oxidative stress-early inducible transcription factor EGR-1 (early growth response gene product 1), previously reported as stimulated factor in a model of cataractogenesis [Nakajima, Nakajima, Fukiage, Azuma and Shearer (2002) Exp. Eye Res. 74, 231-236]. We identified nine proteins, which sensitively reacted to H2O2 treatment by using two-dimensional gel electrophoresis and matrix-assisted laserdesorption ionization-time-of-flight-MS. In addition to cytoskeletal proteins (tubulin 1alpha and vimentin) and enzymes (phosphoglycerate kinase 1, ATP synthase beta, enolase alpha, nucleophosmin and heat-shock cognate 54 kDa protein), which presented quantitative differences in expression profiles, peroxiredoxin and glyceraldehyde 3-phosphate dehydrogenase showed changes in pI as a result of overoxidation. Mass-mapping experiments demonstrated the specific modification of peroxiredoxin I active-site cysteine into cysteic acid, thus providing an explanation for the increase in negative charge measured for this protein. With respect to other global differential approaches based on gene expression analysis, our results allowed us to identify novel molecular targets of oxidative stress in lens cells. These results indicate that a combination of different approaches is required for a complete functional understanding of the biological events triggered by oxidative stress

Effects of exogenous TNC on pancreatic cancer cell growth.

<p>(A) Cells were grown for 72 hours in serum free medium containing different concentrations of TNC (0.01, 0.1, 1 and 10 µg/ml). (B) Cells were grown for 72 hours in serum free medium onto TNC coated plates (1 µg/cm²). Growth was determined with MTT assay. Data are calculated as mean +/− s.e.m. of three experiments and are expressed as percentage compared to the untreated controls (°p<0.05, *p<0.01, **p<0.001, Students two-tailed t test).</p

Cellular distribution of phospho-paxillin and vinculin proteins on coated surfaces.

<p>PANC-1 cells were let adhere to uncoated or TNC, FN and FN/TNC coated coverslips for 45 min, gently washed, fixed and stained using fluorescent secondary antibodies. Focal adhesion plaques were evidenced by co-localization of vinculin (green) and phospho-paxillin (pPAX, red). Cell nuclei were counterstained with Hoechst 33342.</p

Effects of endogenous TNC on cell proliferation and migration.

<p>PANC-1 cells were stably transfected with a vector driving the expression of <i>large</i> TNC (PANC-T2, PANC-T24 and PANC-T27 cells) and with the empty vector (PANC-C21, PANC-C23 and PANC-C27 cells). (A) Immunoblotting of precipitated cell culture medium of the transfected PANC-1 cells grown up to 80% confluence. To ensure that a comparable number of transfected cells was the source of secreted TNC, GAPDH expression in whole cell extracts was tested. PANC-T2 cells show the highest levels of secreted TNC, while lower levels are observed in PANC-T24 and PANC-T27. The control clones in comparison do not show any TNC secretion. (B) Cells were grown in complete medium. Growth was determined with MTT assay at different time points (24, 48 and 72 hours). Data are calculated as mean +/− s.e.m. of three experiments and are expressed as percentage compared to the PANC-1 cells (°p<0.05, *p<0.01, **p<0.001, Students two-tailed t test). The proliferation rate is significantly higher for PANC-T2 at all time points (p<0.001), for PANC-T24 at 24 hours (p<0.001) and at 48 hours (p = 0.022) and for PANC-T27 at 24 (p<0.001) and 48 hours (p = 0.009). (C) The proliferation of all the PANC-1 positive clones (PT) compared to the mock transfected cells (PC) is significantly higher for each tested time point (24 hours: p = 0.004, 48 hours: p = 0.012, 72 hours: p = 0.026) (D) Transfected PANC-1 cells were plated, after 24 hours medium was changed with medium containing 0.1% FBS and, after overnight incubation, the monolayer was scraped with a 10 µl pipette tip. Data are calculated as described in B. In comparison to the non transfected PANC-1 cells, migration is significantly faster for PANC-T2 (p<0.001 at both time points) and for PANC-T27 (p = 0.042 at 24 hours; p = 0.009 at 48 hours). (E) All together, the PANC-1 positive clones (PT) migrate significantly faster compared to the mock transfected cells (PC) at both time points (p<0.001).</p

Effect of TNC on the migration of pancreatic cancer cell lines.

<p>(A)Cells were plated onto uncoated plates and after 24 hours the monolayer was scraped with a 10 µl pipette tip. Cells were then incubated in serum free medium or in medium with the addition of TNC at different concentrations (0.2 µg/ml, 1 µg/ml and 5 µg/ml) up to 48 hours. (B) Cells were plated onto 24-well plates coated with three different concentrations of TNC (0.1 µg/cm², 0.5 µg/cm² and 2.5 µg/cm²) or onto uncoated plates and after 24 hours the monolayer was scraped with a 10 µl pipette tip. Cells were then incubated in serum free medium up to 48 hours. Migration of cells into wounded areas was evaluated counting migrated cells manually and by the TScratch software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021684#pone.0021684-Geback1" target="_blank">[36]</a> and a total of 2–8 fields were counted per group in each experiment. Data are calculated as mean +/− s.e.m. and expressed as fold-change compared to the non treated cells (°p<0.05, *p<0.01, **p<0.001, Students two-tailed t test).</p

HIV-1 IN/Pol recruits LEDGF/p75 into viral particles (vol 12, 16, 2015)

An amendment to this paper has been published and can be accessed via the original article.status: publishe