Disorganisation of cytoskeleton and differential cell adhesion in cells resistant to Photodynamic Therapy.

CASAS, ADRIANA; SANS-RODRIGUEZ, FRANCISCO; DI VENOSA, GABRIELA; BATLLE, ALCIRA; STOCKER, JUAN CARLOS; JUARRANZ, ANGELES

Bath, Inglaterra

Congreso; 12th Congress European Society for Photobiology – ESP 2007; 2007

European Society for Photobiology

The appearance of cells resistant to Photodynamic Therapy (PDT) is crucial for the outcome of the therapy. In the present work we analysed the differential expression and distribution of different cytoskeleton and adhesion proteins in cells resistant to 5-aminolevulinic acid (ALA)-PDT. For that purpose we employed the LM3 murine adenocarcinoma cell line and the sublines Cl4 and Cl8, isolated from the former after successive rounds of ALAPDT. We performed direct and indirect immunofluorescent staining as well as Westernblot assays to evaluate distribution the above mentioned proteins. Whereas actin stress fibers were observed in the parental LM3 cells, this organisation becomes perturbed in Cl4 cells and completely loss organisation is observed for Cl8 cells. Sequential focal planes recorded from the basal to the apical cell regions show that whereas at the middle cell plane, a rim of cortical actin is conserved in Cl4 cells, the cell-substrate interface was altered. In Cl8 cells, F-actin is completely disorganised from the apical to the basal planes.In LM3 and Cl4 cells, E-cadherin and beta-catenin are located at the plasma membrane connecting neighbouring cells. However, both proteins become disorganised in Cl8 cells, with interdigitations appearing in the cell to cell contacts. A nuclear distribution of beta-catenin was also observed in a low percentage of Cl8 cells.In addition, whereas vinculin distribution is confined to the focal adhesion points in LM3 cells, a diffuse cytoplasmatic pattern is observed in Cl4 and more markedly in Cl8 cells. However, Westernblot assays did not show differential expression of actin, E-cadherin, vinculin or Beta-catenin. We also evaluated adhesion to extracellular matrix proteins. Whereas the 3 lines adhered equally to fibronectin, Cl4 adheres 1.3-fold and Cl8 2- fold to fibronectin compared to LM3.In the scratchwoundhealingassay, the migratory ability of the cells was: Cl8 >Cl4 >LM3, and this probably correlates with the impaired cell to cell adhesion in the resistant clones. To sum up,ALA-PDT affects cell to cell adhesion, adhesion to substrate, migration and cytoskeleton, and this could result in resistant cell lines with a different metastasic phenotype.