Effect of removal of serum from the culture medium and sublethal roentgen irradiation on microvilli and invaginations of the cell membrane of Ehrlich ascites tumor cells in monolayer cultures

In order to find out modifications of microvilli and invaginations, the cellular surfaces of Ehrlich ascites tumor cells in monolayer culture (basal medium of Eagle + 10% fetal calf serum) were investigated with the aid of electron-microscopic cross-sections. The tumor cells had been cultured without serum 24 hours prior to investigation or irradiated with 2 Gy. Morphometric evaluation after cell culture in a serum-free medium showed a reduced number ob microvilli and a diminution of sections of microvilli. As already described before, a reduction of cell proliferation, of the microtubule-microfilament system, and of the endocytosis activity occurs under these serum-free conditions. The number of invaginations (related to a constant membrane part) was reduced by nearly 50% after serum extraction. Similarly to serum extraction, sublethal X-ray irradiation reduced the sections of microvilli, whereas the number of microvilli increased slightly. Contrary to the effect of serum extraction, the irradiated cells showed twice as many invaginations as the non-irradiated control cells. These differences in the surface structures are interpreted as a result of modified growth stimulations (+/- serum) and radiogenic reparation processes.     

Electron microscopy studies on the pinocytosis activity of Ehrlich ascites tumor cells in monolayer culture]

During the phase of exponential growth ferritin was added to the culture medium of monolayer cell cultures from the Ehrlich ascites tumor in order to observe the pinocytosis phenomenon in these cells by means of electron microscopy. After a phase of adsorption, the ferritin arrived in vesicles and vacuoles of the cytoplasm - probably by ligature of coat invaginations. Within larger-sized vesicles, which might have been formed by fusion of smaller ones, less electron-dense ferritin-free internal compartments are resulting possibly from interaction with primary lysosomes; this is interpreted as a sign of interior digestive phenomena. Sometimes, this internal compartment was coated with a structure similar to a membrane. In the further course, concentration of the electron-dense zones containing ferritin occurred in the marginal zone of the vacuoles. Some vacuoles comprised two or three of the less electron-dense compartments. Vacuoles containing ferritin were also observed comprising residual membranes. The formation of these transition stages to residual bodies is regarded as an interaction and fusion of heterophagocytic or autophagocytic vacuoles with primary lysosomes.     

Ultrastructural studies of the effect of x-rays and quinacrine (Atebrin) or chloroquine (Resochin)--alone or in combination--on Harding-Passey melanoma cells in monolayer culture

Monolayer cells of a Harding-Passey melanoma (HPM 73 cells) which were irradiated during the phase of exponential growth with an X-ray dose of 4 Gy of 8 Gy did not show any ultrastructural changes four days after 4 Gy, whereas cells irradiated with 8 Gy showed slight damages such as swollen mitochondria and vacuoles. As shown by the electron microscope, a sole addition of a sublethal quantity (6 X 10(-6) M) of quinacrine (Atebrin) or chloroquine (Resochin) did not lead to significant cell modifications. Those melanoma cells with were pre-irradiated with 8 Gy and then incubated during four days with 6 X 10(-6) M of quinacrine (Atebrin) or 6 X 10(-6) M of chloroquine (Resochin) showed severe damages. There was an increased rate of vacuoles and segregational structures in cytoplasm. The mitochondria were increased and swollen and the cellular surfaces had less microvilli. However, microtubules and microfilaments seemed more distinct. The melanin concentration increased under this treatment. The cell nuclei were increased in volume and seemed to be rather void of chromatin. These reactions of cells on quinacrine (Atebrin) and chloroquine (Resochin) are explained by the known inhibition effect exerted by these substances on DNA synthesis, especially as far as the processes of DNA reparation are concerned. The changes of the microtubule-microfilament system could be due to a correlation with the increase of digestive intracellular processes connected with the catabolism of radiation-damaged structures.     

Effect of 2-deoxy-D-glucose on DNA double strand break repair, cell survival and energy metabolism in euoxic Ehrlich ascites tumour cells.

Effects of 2-deoxy-D-glucose (2-DG) on DNA double strand break (dsb) repair, cell survival and on the energy metabolism were investigated in exponentially growing Ehrlich ascites tumour (EAT) cells. Cells in suspension were exposed to 40 Gy of X-rays and allowed to repair (up to 4 h) with or without 2-DG at 37 degrees C. DNA dsb rejoining was measured by means of clamped homogeneous electric field (CHEF), a pulsed field gel electrophoresis technique. The fraction of activity released (FAR) during electrophoresis (DNA associated 14C-thymidine) was used as a parameter to determine the number of dsb present in the DNA. Biphasic kinetics for dsb repair were observed. The presence of 2-DG significantly inhibited the slow component of dsb repair. The presence of 2-DG also enhanced radiation-induced cell killing. ATP content of cells was measured by a bioluminescence method. ATP content in exponentially growing cells was about 4 pg per cell. The level of ATP was reduced by 50% in presence of 2-DG (C2-DG/CG = 1.0).