EEA1 CARRYING VESICLES ARE NOT AUTOPHAGOSOMES IN SERUM-DEPRIVED HeLa CELLS.

According to current model, stimulation of EGF-receptor endocytosis results in recruitment onto early endosomes cytosolic tether protein EEA1 necessary for their further fusions. However, EEA1-positive vesicles are found in the cells not treated with growth factor, that were incubated in serum-free conditions. It is known also that prolonged serum deprivation induces autophagosomes formation, the process possibly involving endocytic compartments. To check whether EEA1-positive vesicles seen in serum-deprived HeLa cells are autophagosomes, we here evaluated colocalization of EEA1 and autophagosome marker LC3 and studied dynamics of the EEA1- and LC3-vesicles' number and size during 12­36 h cell cultivation in serum-free medium. It was found that the number of autophagosomes per cell is significantly less than the number of EEA1-vesicles. We show that serum starvation results in increase of only mean autophagosomes' size, while the number and size of EEA1-vesicles did not changed. Colocalization of EEA1 and LC3 in serum-free cells was very low during first 12­18 h of starvation and increased insignificantly only by 36 h. Biosynthetic pathway inhibition by Golgi apparatus disruption by brefeldin A, decreased the number and increased the size of EEA1-vesicles. LC3-vesicles also demonstrated an increase of mean size and growth of colocalization with EEA1. Thus, we conclude that the majority of EEA1-vesicles in serum-starved cells are not autophagosomes. More pronounced effect of brefeldin A indicates that blockade of biosynthetic pathway is more strong stress factor comparing to serum deprivation in HeLa cells. This also suggests that this pathway is involved in EEA1-vesicles biogenesis.

[Effect of polychromatic visible light combined with infrared radiation on tumorigenicity of murine hepatoma cells and their sensitivity to lytic activity of natural killers].

Tumorigenicity of murine hepatoma cells (MH22a) and their sensitivity to lysis by natural killers (NKs) have been studied after exposure to polychromatic visible and infrared light (VIS-IR, 480-3400 nm, 40 mW/cm2), similar to the terrestrial solar spectrum without its minor UV component, in order to elucidate the involvement of this important environmental and physiotherapeutic factor in regulation of the anti-tumor defense system. The MH22 cells were in vitro exposed to VIS-IR light and their sensitivity to lytic activity of NKs was evaluated. We found that sensitivity of MH22a cells to lysis by NKs after exposure to VIS-IR light at a dose of 4.8 J/cm2 increased 1.5-2 times, while it did not change after exposure to a dose of 9.6 J/cm2 at all ratios (1 : 5-1 : 50) of the number of NKs (effectors) to that of hepatoma cells (targets). The increase in the sensitivity of hepatoma cells to NKs was accompanied by structural changes of cell surface: the capability of supramembraneous glycoproteins (glycocalix) to sorb the vital dye alcian blue (AB) was significantly lower as compared with the unexposed cells of control group. However, no changes in AB sorption was revealed in hepatoma cells exposed to the light at a dose 9.6 J/cm2. Tumorigenicity of photo-irradiated MH22a cells has been studied in the in vivo experiments. Light-exposed (4.8 and 9.6 J/cm2) and intact hepatoma cells were transplanted into syngenic mice C3HA. Tumor volumes 25 days after transplantation proved to be smaller after exposure to the light at both doses than in the control group (4-4.5 times and 2.5-4 times, respectively), which correlated with the increase in the sensitivity to lisys by NKs and decrease in the AB sorption only after light exposure at dose 4.8 J/cm2. Using the flow cytometry method we could show that VIS-IR light at the applied doses did not interfere with the distribution of hepatoma cells over the cycle phases and thus deceleration of the tumor growth was not associated with cytostatic effect of VIS-IR light. To evaluate effect of polychromatic light on the growth of the preformed tumors, the 5-day course of daily light exposures of tumor bearing mice C3HA was carried out in 10 days after subcutaneous transplantation of 2 x 10(5) cells of syngene hepatoma when the tumors had developed in 100% animals. Like in the case of transplantation of the light-exposed cells, irradiation of the tumor bearing mice at doses 4.8-9.6 J/cm2 resulted in deceleration of tumor growth (2.1-2.9 and 2.2 times respectively) for 4 weeks as compared with non-irradiated mice.