HNE protein adducts and apoptosis in osteosarcoma cells

Trans-4-hydroxy-2-nonenal (HNE) is one of the major terminal products both of chemically induced and of endogenous peroxidation of n-6 unsaturated fatty acids, so that it is frequently used as an indicator of the oxidative stress 'in vivo'. HNE is considered to be not only a potent cytotoxic and genotoxic compound, as evidenced by 'in vitro' studies, but even, owing to his existence at low concentrations also in normal cells, a messenger for cell signalling in physiological conditions (1). In addition, it has been showed that the concentration of lipid peroxidation products is relatively lower in undifferentiated, highly proliferating tumor cells with respect to normal cells, and it has been hypothesized that such products may play an important role in the down-regulation of cell proliferation. The electrophilic nature of HNE explains its chemical reactivity towards thiols, aminogroups of aminoacids, proteins or DNA bases (2). Previously we showed that, after exogenous administration to a human osteosarcoma cell line, SaOS2, HNE nuclear concentration increases about threefold and that such an increase correlates with cytotoxic and cytostatic effects [3]. In this work we have developed a method to allow a good separation of the single HNE-protein adducts. The accurate quantification of such adducts provides a powerful tool for the identification of molecular targets which could mediate the aldehyde effects. The HNE-protein adducts were reduced with NaB[3H]H4 to stabilize them and allow their autoradiographic detection. The nuclear extracts were analyzed in SDS-PAGE; in the first step of analysis a 12,5 % gel and Tris-Glicine running buffer was used to obtain the separation of all proteins extracted. In the second step a 10% polyacrilamide gel was employed with MOPS and MES running buffers; each buffer produced a different migration pattern when used with the same gel. In particular we observed that to optimize the separation of proteins ranging from 21 kDa to 66 kDa it was best to use MOPS running buffer; on the other hand to resolve proteins ranging from 2.5 kDa to 36 kDa the MES running buffer was the most indicated. Finally the large molecular weight proteins (400 kDa to 55 kDa) were separated using 4-8% polyacrlamide gel with Tris Acetate SDS running buffer. After autoradiography, the bands corresponding to the protein-adducts were excised and their radioactivity was evaluated by beta-counting. The results show that the method presented here is able to separate HNE-protein adducts from distinct intracellular compartments and it could be an efficient preparatory procedure for identification of modified protein in mass spectrometry. In particular, besides nuclei, we focused on the evaluation of HNE content in mitochondria with the aim to characterize apoptotis induced by HNE in a cell line p53 and pRB mutated.