Vergleichende Analyse von basaler und stressinduzierter Expression sowie Lokalisation der Hitzeschockproteine von 70 Kilodalton Molekulargewicht HSPA1A und HSPA1B (Hsp70) bei humanen Tumorzellen

In contrast to healthy human cells, tumor cells frequently overexpress HSPA1A and HSPA1B (Hsp70) and present the chaperones on their cell surfaces. Stress-induced, the expression of Hsp70 is generally upregulated, which can cause cytoprotective and cytotoxic effects. Also the export of soluble Hsp70 and the secretion of extracellular vesicles containing Hsp70 are intensified under stress conditions, which can provoke immunostimulatory effects. In general, the basal and stress-induced expression patterns as well as the subcellular localizations of Hsp70 can vary between different types of tumor cells. A clear correlation between expression intensity and treatment prognosis could not be established so far. Therefore, in this study, the breast adenocarcinoma cell line MCF7, the breast ductal carcinoma cell line T47D and the osteosarcoma cell line U2OS were characterized regarding basal expression intensity, progression and features of the stress response as well as localization of Hsp70. To characterize the basal and heat-shock altered expression patterns, unstressed and stressed cells were homogenized and separated into nuclear-free homogenates, cytosolic and membrane fractions by differential centrifugation. The samples were analyzed by gel electrophoresis, western blot and densitometric quantification. The results revealed that MCF7 and T47D cells expressed considerably higher quantities of Hsp70 than U2OS cells under physiological conditions. The strength of the basal expression determined the intensity of the stress response: The more Hsp70 was expressed under physiological conditions, the weaker was the stress-induced expression increase. Beside these differences, the tumor cell lines shared noticeable similarities: The temporal progression of the heat-shock induced expression fluctuations in cytosolic and membrane fractions was comparable; the ratios of cytosolic to membrane-associated Hsp70 remained constant. Furthermore, the Hsp70 expression increase reached its peak four to six hours post-stress in all tumor cell lines analyzed. Also the expression decrease rates between six and 24 hours post-stress were comparable. However, regarding the expression pattern of the constitutively expressed Hsc70, the T47D cells showed interesting particularities: Although these cells already expressed only low amounts of Hsc70 under physiological conditions, they reduced the expression in response to stress-exposure, whereas the MCF7 and the U2OS cells reinforced the synthesis. Thus, it seems likely that the maximal achievable expression levels of the chaperones are limited by cytotoxic side effects. The analysis of the subcellular localization patterns was accomplished by indirect immunofluorescence microscopy using non-permeabilized and permeabilized cells. The results indicated no (T47D, MCF7) or just slight (U2OS) differences between unstressed and stressed cells. Hsp70 remained more or less evenly distributed within the cells; the subcellular localization patterns remained essentially constant. Accordingly, it can be assumed that the stress-induced nuclear accumulation of Hsp70, observed in case of other tumor cell lines, is not a general part of the stress response. On the surfaces of the non-permeabilized cells, Hsp70, GAPDH and α-Tubulin colocalized in clusters of spherical or globular appearance with three (MCF7) to ten (T47D, U2OS) micrometer diameter. Size and shape of these clusters as well as the presence of Alix exclusively in immediate vicinity to them suggest that they may represent extracellular vesicles similar to the large oncosomes. Furthermore, also LDH, Centrin, subunits of Na+/K+-ATPases and Galectin-3 were detectable on the cell surfaces. However, the localization patterns of some of the latter proteins differed between the cells lines to a considerable degree. Therefore, it can be assumed that the composition of the proteins exposed on the vesicular structures might be specific for certain types of tumor cells. Taken together, the results of the biochemical analyses lead to the assumption that the stress resistance of tumor cells is not solely depending on the intensity of the basal Hsp70 expression. Rather, it appears that also the amount of Hsp70 which can be synthesized in addition to the basal expression level might play a role. The results of the immunofluorescence microscopic analyses suggest moreover that the shedding of large vesicles is not a special characteristic of certain tumor cell types, but a relatively widespread phenomenon. The concentration of immunoactivating molecules on these vesicles and their release could enable tumor cells to circumvent the antitumor response of the immune system.