The Role of the Focal Adhesion Protein PINCH1 for the Radiosensitivity of Adhesion and Suspension Cell Cultures

Focal adhesion (FA) signaling mediated by adhesion to extracellular matrix and growth factor receptors contributes to the regulation of the cellular stress response to external stimuli. Critical to focal adhesion assembly and signaling is the adapter protein PINCH1. To evaluate whether the prosurvival function of PINCH1 in radiation cell survival depends on cell adhesion, we examined PINCH1fl/fl and PINCH1−/− mouse embryonic fibroblasts and human cancer cell lines. Here, we found that the enhanced cellular radiosensitivity mediated by PINCH1 depletion observed under adhesion conditions is conserved when cells are irradiated under suspension conditions. This unsuspected finding could not be explained by the observed modification of adhesion and growth factor associated signaling involving FAK, Paxillin, p130CAS, Src, AKT, GSK3β and ERK1/2 under suspension and serum withdrawal relative to adhesion conditions with serum. Our data suggest that the adapter protein PINCH1 critically participates in the regulation of the cellular radiosensitivity of normal and malignant cells similarly under adhesion and suspension conditions

Workshop Report for Cancer Research: Defining the Shades of Gy: Utilizing the Biological Consequences of Radiotherapy in the Development of New Treatment Approaches—Meeting Viewpoint

The ability to physically target radiotherapy using image-guidance is continually improving with photons and particle therapy that include protons and heavier ions such as carbon. The unit of dose deposited is the gray (Gy); however, particle therapies produce different patterns of ionizations, and there is evidence that the biological effects of radiation depend on dose size, schedule, and type of radiation. This National Cancer Institute (NCI)–sponsored workshop addressed the potential of using radiation-induced biological perturbations in addition to physical dose, Gy, as a transformational approach to quantifying radiation

Comprehensive analysis of signal transduction in three-dimensional ECM-based tumor cell cultures

Analysis of signal transduction and protein phosphorylation is fundamental to understand physiological and pathological cell behavior as well as identification of novel therapeutic targets. Despite the fact that more physiological three-dimensional cell culture assays are increasingly used, particularly proteomics and phosphoproteomics remain challenging due to easy, robust and reproducible sample preparation. Here, we present an easy-to-perform, reliable and time-efficient method for the production of 3D cell lysates without compromising cell adhesion before cell lysis. The samples can be used for Western blotting as well as phosphoproteome array technology. This technique would be of interest for researchers working in all fields of biology and drug development

Plating efficiencies of <i>PINCH1</i>^fl/fl and <i>PINCH1</i>^−/− MEFs under indicated growth conditions.

<p>*Poly-S, polystyrene; Poly-L, poly-L-lysine; FN, Fibronectin.</p

Signal transduction modification in adherent and suspension tumor cell lines after PINCH1 knockdown.

<p>(A) Western blot on total cell lysates from PINCH1 depleted HTB43 and HTB35 cells grown under adhesion or in suspension. (B) Densitometric analysis from protein bands shown in ‘A’ after normalization to total protein or β-Actin expression and subsequently to adhesion conditions of siRNA control cells ( = 1). Co, control; P1, PINCH1.</p

Apoptosis in irradiated cells remains unchanged by PINCH1 silencing.

<p>(A) and (B) HTB43 and HTB35 were assessed for typical apoptotic nuclear morphology upon X-ray irradiation (0 or 6 Gy) and transfection with PINCH1 specific or non-specific control siRNA (mean±SD; <i>n</i> = 3).</p

Effects of PINCH1 knockout on signal transduction under adhesion and suspension conditions.

<p>(A) Western blot on total cell lysates from <i>PINCH1</i>^fl/fl and <i>PINCH1</i>^−/− MEF grown on polystyrene plus FCS (first lane), in suspension plus FCS (second lane) or in suspension without FCS (third lane). (B) Densitometric analysis from protein bands shown in ‘A’ after normalization to total protein or β-Actin expression and subsequently to adhesion plus FCS conditions of <i>PINCH1</i>^fl/fl MEF ( = 1). FCS, fetal calf serum; Susp, suspension.</p

Colony cell numbers and cell morphology remain unaltered upon PINCH1 depletion.

<p>(A) and (B) Cell numbers of 15 colonies were counted in PINCH1 knockdown and control HTB43 and HTB35 cell cultures. Results show mean±SD (<i>n</i> = 3; t-test; n.s., not significant). (C) and (D) show cellular morphology in PINCH1 depleted and control cell colonies of HTB43 and HTB35 tumor cell lines. Photographs illustrate (<i>i</i>) representative colony growth in 35-mm wells, (<i>ii</i>) a single representative colony (magnification = 10×), and (<i>iii</i>) a representative zoom (magnification = 40×) from the edge of a colony. Co, control.</p