Disorganisation of cytoskeleton in cells resistant to photodynamic treatment with decreased metastatic phenotype.

The appearance of cells resistant to photodynamic therapy (PDT) is crucial for the outcome of this antitumoral treatment. We had previously isolated two sublines resistant to PDT derived from the mammary adenocarcinoma LM3 [A. Casas, C. Perotti, B. Ortel, G. Di Venosa, M. Saccoliti, A. Batlle, T. Hasan, Induction of murine tumour cell lines resistant to ALA-mediated Photodynamic Therapy, Int. J. Oncol. 29 (2006) 397-405.]. These clones have severely impaired its metastatic potential in vivo together with decreased general anchorage-dependent adhesion and invasion. In the present work we analyzed the differential expression and distribution of cytoskeleton and adhesion proteins in these cell lines. In both resistant clones, loss of actin stress fibers and disorganization of the actin cortical rim was observed. E-cadherin and beta-catenin and vinculin distribution was also disorganized. However, Western blot assays did not show differential expression of actin, E-cadherin, vinculin or beta-catenin. It was demonstrated that PDT strongly affects cell-cell and cell-substrate adhesion through the reorganization of some cytoskeletal and adhesion proteins. Changes in the metastasis phenotypes previously found are likely to be ascribed to these differences.

A mechanism for the fluorogenic reaction of amino groups with fluorescamine and MDPF.

Fluorescamine and 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) react with primary amines at alkaline pH to yield highly fluorescent products, claimed to be diaryl-2-hydroxy-pyrrolinones. However, it seems to have been overlooked that these products appear as the pseudobase of coplanar and cationic diarylpyrrolones. Horse blood smears subjected to fluorogenic MDPF and fluorescamine reactions showed eosinophil granules with bright blue-white fluorescence, which required washing at neutral pH and was dependent on the presence of amino groups. The fluorescence of MDPF-butylamine product was abolished at alkaline pH and by bisulphite, suggesting that nucleophilic attacks to the pyrrolone ring (with formation of carbinol (pseudobase) and sulfonate derivatives, respectively), destroy the planarity and conjugation of the whole molecule, thus abolishing the emission at long wavelength. Analysis of the correlation between the largest conjugated fragment (LCF) values and the emission peaks of several fluorophores (fluorescamine- and MDPF-butylamino products, non-rigid fluorochromes) showed the best-fit when the cationic pyrrolones were considered. Although the pseudobases of fluorescamine- and MDPF-amino derivatives are formed at alkaline pH, a full conjugated, coplanar and cationic molecule is suggested to be the true fluorescent product.