Palyvoda K. Modulation of apoptosis pathways by fullerenes C60 in normal and transformed T lymphocytes

Thesis is devoted to comparative study of sensitivity to cytotoxic agents (hydrogen peroxide, cytosine arabinoside, staurosporine), protective effects of fullerenes C60 and biochemical markers of apoptosis under the influence of photoexcited fullerenes C60 in normal (thymocytes from Wistar rat thymus) and transformed (human T leukemia Jurkat cell line) T- lymphocytes. Stable water colloidal solutions of pristine fullerenes C60 (purity 99,5%) were prepared at Ilmenau University (Germany). Both hydrated individual C60 molecules and C60 clusters (12 - 50 nm) were present in solution. Mercury-vapor lamp (320-600 nm) was used for fullerenes C60 photoexcitation. Caspase-3 activation, cytochrome c level in cytosol, protein tyrosine phosphorylation were analyzed using Western-blot. It was shown that after treatment with cytotoxic agents thymocytes viability was decreased and caspase-3 was activated significantly earlier as compared to Jurkat cells. Cell preincubation with fullerenes C60 was shown to prevent thymocytes death induced by cytotoxic agents, while in Jurkat T leukemia cells no protective effect of fullerene C60 was revealed. Preincubation of thymocytes with fullerenes C60 for 1 h reduced cytotoxic effects of hydrogen peroxide, cytosine arabinoside and staurosporine. By contrast, fullerenes C60 did not protect human Jurkat T leukemia cells from death induced by these agents. Investigation of T cells phosphotyrosine status demonstrated that the main immunoreactive bands detected with anti-phosphotyrosine antibodies correspond to proteins with Mr 17, 20, 30, 50, 72 and 100 kDa in thymocytes and 17, 26, 30, 55, 70, 90 and 100 kDa in Jurkat cells. In cells of both types the intensity of phosphorylation of phosphotyrosine-containing proteins was decreased after incubation with apoptosis-inducing agents studied. Preincubation of thymocytes with C60 fullerenes was followed by partial recovery of 20, 72 та 100 kDa proteins tyrosine phosphorylation, inhibited by H2O2. Fullerenes C60 were found to exhibit cytotoxic effect on transformed T lymphocytes when combined with UV/Vis irradiation. The cytotoxic effect of photoexcited fullerenes C60 in Jurkat T leukemia cells was proved to be both irradiation dose- and C60 concentration-dependent and selective - no fullerenes C60 photocytotoxicity was detected in thymocytes. It is proved that Jurkat cells death induced by C60 after UV/Vis irradiation is of apoptotic type. Only large scale DNA fragmentation was detected in control untreated Jurkat cells at 24 h and a slight increase of DNA fragmentation was observed after UV/Vis irradiation, while irradiation in the presence of C60 fullerenes was followed by internucleosomal DNA cleavage and appearance of DNA fragments of 200 and 400 bp in ladder pattern. Following the combined action of fullerenes C60 and UV/Vis irradiation caspase-3 processing was dramatically intensified - the content of 17 and 19 kDa intermediate cleavage products was increased at 6 h and reached maximum at 24 h. No substantial increase of caspase-3 activation was observed in thymocytes treated with fullerene C60 plus irradiation, while antileukemic agent cytosine arabinoside was shown to induce caspase-3 activation both in Jurkat cells and thymocytes. Jurkat T leukemia cells apoptosis photoinduction by C60 appears to be activated by mitochondrial pathway - cytochrome c release by the mitochondria to cytosol was observed 1-3 h after irradiation. Reactive oxygen species production by photoexcited fullerenes C60 inside Jurkat cells was confirmed by early (0,5 h after irradiation) activation of superoxide dismutase. The data obtained may be useful for development of photosensitizers for photodynamic therapy with selective action on leukemia cells.