The remote effects of radiation after hypofractionated irradiation with protons of Ehrlich ascites carcinoma in mice

This work focuses on the study of remote effects (duration of remission, recurrence rate, and average lifespan) in mice with Ehrlich ascites carcinoma exposed to oligofractionated irradiation with a pencil scanning beam of protons depending on the volume of the tissue being irradiated and the interval between dose fractions. The results show higher antitumor efficacy and a considerable increase in the average life span of mice after hypofractionated irradiation with a pencil scanning beam of protons at a total dose of 60 Gy of the gross tumor volume compared with the planning target volume.     

APOPTOSIS OF EHRLICH MOUSE ASCITES CELLS INDUCED BY LONG-TERM EXPOSURE OF WEAK ELECTROMAGNETIC FIELDS IN VIVO

To study the possibility of apoptosis of tumor cells induced by weak electromagnetic fields (EMFs) in vivo, mice were inoculated with Ehrlich ascites cells and exposed to a long-term electromagnetic field (1 mT, 700 KHz). During the treatment, growth curves of mice were measured and compared between exposed and sham-exposed mice. The results show that the growth curves of healthy controls agree well with the ideal curve of logistic growth, but the growth curves of cancer mice deviate from the ideal curve. There is no difference in growth curves between exposed, and sham-exposed healthy mice, and they both agree with the ideal curve. However, a notable difference in growth curves between exposed and sham-exposed cancer mice was obtained. Moreover, the curves of sham-exposed mice deviate even more than those of the exposed mice; in other words, the growth curves of Ehrlich ascites mice deviate from the ideal curve of healthy mice but are shifted toward it by the EMF treatments. After the treatment, apoptosis of Ehrlich ascites cells from inoculated mice was analyzed by several methods, including flow cytometry, fluorescence microscopy, and DNA gel electrophoresis. Statistical analysis from flow cytometry shows that the apoptotic ratio of cells from exposed Ehrlich ascites mice was significantly higher than that from sham-exposed treated mice. Microscopic observation of Ehrlich ascites cells stained with acridine orange (AO) and propidium iodide (PI) showed typical apoptotic changes in exposed animals whose cell nuclei were highly condensed or fragmented and uniformly stained green by the AO, whereas cell nuclei from sham-exposed mice were stained green and showed a fine reticular pattern. Agarose gel electrophoresis of DNA from exposed mice showed that the chromatin DNA exhibited ladders, a characteristic feature of internucleosomal degradation of DNA by EMF treatments. For interactions between external electromagnetic fields and DNA, the mechanism of apoptosis of tumor cells induced by weak EMFs is discussed.     

Tumor-Specific Transplantation Resistance in Mice after Treatment of Initial Tumors with Streptococcus thermophilus

Antitumor activity observed by treatment with Streptococcus thermophilus was further investigated. The mice cured from fibrosarcoma by treatment with heat-killed preparation of S. thermophilus, when challenged with fibrosarcoma failed to take up the tumor. However, these cured mice when challenged with sarcoma-180 or Ehrlich ascites carcinoma, did not show significant changes in tumor take and/or survival compared to their respective controls. Similarly, mice cured from sarcoma-180 were challenged with fibrosarcoma, sarcoma-180 or Ehrlich ascites carcinoma. Though there was no change in the mean survival time (MST) of the dying mice regarding sarcoma-180 or Ehrlich ascites carcinoma, there was 50 and 30% increase in the number of mice that showed total regression respectively over controls. However, there was no difference in the growth rate of fibrosarcoma. Similar observations were made with mice cured from Ehrlich ascites carcinoma, challenged with these tumors. These findings thus suggest that the antitumor response was tumor-specific and that tumor-associated antigens may have a role in imparting this specificity. Bacterial treatment non-specifically augmented this primary response.     

Hypofractionated irradiation of the solid form of Ehrlich ascites carcinoma in mice by a thin scanning proton beam

The dynamics of the growth of Ehrlich ascites carcinoma in mice of the SHK line exposed to hypofractionated high-dose irradiation by a thin scanning proton beam has been analyzed for different irradiation volumes and different time intervals (from 4 to 24 hours) between two 30-Gy fractions. Irradiation of the gross tumor volume and the planned target volume was performed within the Bragg peak; the energy of protons at the outlet of the accelerator ranged from 85 to 100 MeV. Hypofractionated irradiation of the gross tumor volume of Ehrlich ascites carcinoma resulted in a more pronounced antitumor effect than the irradiation of the planned target volume. The effect did not depend on the interval between the irradiation episodes.     

The effect of ascitic fluid on the growth of Ehrlich tumor and Lewis carcinoma]

In the study of the effect of ascitic fluid and dialysate of Ehrlich ascites tumor cells (m.m. less than 15 kDa) on the growth of Ehrlich and Lewis carcinoma it was found that the ascitic fluid significantly decreased the size of Ehrlich tumor (by more than 50% on day 9-25 after the tumor cell inoculation). It also reduced Lewis carcinoma tumor volume by more than 30% during 3 weeks after the tumor cells inoculation. Dialysate of Ehrlich tumor cells significantly inhibited the growth of Ehrlich tumor too. It is suggested that this test-system simulates inhibition of a small tumor by a big tumor in vivo.     

The effect of proteinase inhibitors on the level of degradation and unscheduled synthesis of DNA in ultraviolet- and gamma-irradiated cellsof mouse ascitic carcinoma

Proteinase inhibitors (e.g. antipain, pepstatin A, and phenylmethane sulfonyl fluoride) were shown to decrease the rate of incision DNA degradation and the level of unscheduled synthesis of DNA in Ehrlich ascites tumor cells exposed to UV-light and gamma-radiation. The results obtained indicate that nuclear proteinases are involved in regulation of DNA repair process.     

The role of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion

The biochemical and biophysical roles of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion were studied. (1) Various kinds of cell, such as Ehrlich ascites tumor cells, mouse melanoma cells (B16-CW1 cells) and human epidermoid carcinoma cells (KB cells), could fuse in Ca2+-free medium containing a cheletor, glycoletherdiaminetetraacetic acid, in the same way as in Ca2+-containing medium. (2) The ATP content in Ehrlich ascites tumor cells decreased rapidly when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (3) Intracellular adenine nucleotides leaked out into the reaction medium when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (4) On addition of the virus, O2 consumption of Ehrlich ascites tumor cells decreased in Ca2+-free medium, but not in Ca2+-containing medium. (5) HVJ (Sendai virus) did not affect production of lactate by Ehrlich ascites tumor cells in both Ca2+-free medium and Ca2+-containing medium. These observations suggest that the role of extracellular Ca2+ in virus-induced cell fusion is to maintain the ATP and other intracellular metabolite contents at normal levels instead of triggering the fusion reaction itself.     

Effectiveness of a fast neutron beam to induce DNA damage in Ehrlich ascites carcinoma cells irradiated in a aqueous phantom

The study of damages to DNA molecules of Ehrlich ascites tumor cells exposed in water phantom to fast neutron beam (mean energy of 6 MeV) showed that changes in the extent of DNA injury did not correlate with the absorbed dose distribution at the phantom depth of 8-12 cm, and also that the dose-response function in the phantom was different from that obtained upon irradiation of cells in the air.     

Effect of subtherapeutic doses of docetaxel (taxotere) on the efficacy of radiotherapy and pro-oxidant-antioxidant balance in rats with Guerin's carcinoma

In the experiments at Wistar male rats the effect of subtherapeutic doses of docetaxel (5 and 10 mg/kg) on the radiotherapy efficacy (20 Gy of single-dose X-rays) namely growth rate of Guerin's tumor and prooxidant-antioxidant balance in liver and blood of animals bearing tumors was investigated. It has been demonstrated that docetaxel at dose 5 mg/kg given 18 hours before irradiation resulted in significant tumor growth delay (2.3-2.7-fold) in comparison with group of rats that received only irradiation. After application of higher dose of docetaxel there was no statistically significant change of tumor size along the whole experiment (14 and 21 days after tumor implantation). Content of lipid peroxidation products was revealed to be considerably increased after chemotherapy and concurrent irradiation when docetaxel was used in a dose of 10 mg/kg. At the same time glutatione peroxidase activity and antioxidative activity of blood plasma were reduced. In the rat liver chemoradiotherapy led to decrease of glutathion peroxidase and glutathione-S-transferase activity to greater degree at docetaxel dose of 10 mg/kg. The obtained results allow to conclude that higher dose of docetaxel and concurrent irradiation resulted in the most effective Guerin's carcinoma growth delay and considerable deviation of antioxidant-prooxidant balance of tissues in the direction of the last.     

Regulation of cyclic AMP level and lactic acid production in Ehrlich ascites tumor cells.

1. Quercetin (3.3',4',5,7-pentahydroxy flavone) at the concentration of 10(-4) M, as well as 2-10(-2) M theophylline and 1.5 - 10(-4) M prostaglandin E2 caused maximal rise of cyclic AMP in Ehrlich ascites tumor cells. 2. No additional increase of cyclic AMP level in these cells was found when both quercetin (10(-4) M) and theophylline (2-10(-2) M) were present in the incubation medium, while combination of quercetin (10(-4) M) and prostaglandin E2 (1.5 - 10(-4) M) has a synergistic effect on the level of cyclic AMP. 3. Degradation of cyclic AMP by homogenate of Ehrlich ascites tumor cells was inhibited by both quercetin and theophylline. 4. Quercetin, and to a smaller but significant extent theophylline, inhibited the lactic acid production in Ehrlich ascites tumor cells while prostaglandin E2 did not change the glycolytic rate in these cells. No synergistic inhibitory effect on lactic acid production was found when combinations of quercetin and prostaglandin E2, quercetin and theophylline or prostaglandin E2 and theophylline were tested. 5. Treatment of Ehrlich ascites tumor cells with dextran sulfate abolished the inhibitory effect of quercetin on lactic acid production, while the effect of the bioflavonoid on cyclic AMP levels was not altered.     

Comparative effects of Yoshi-864 and busulfan on certain transplantable murine tumors.

Yoshi-864 extends markedly the survival times of mice bearing L1210 leukemia or Ehrlich ascites carcinoma. Busulfan, with methanesulfonate leaving groups identical with those of Yoshi-864, is without effect. Tumor cells from mice bearing the Ehrlich tumor and treated with Yoshi-864 have a persistent reduction in ability to synthesize DNA. Synthesis of DNA in cells from mice treated with busulfan is moderately suppresed at 48 hr after treatment, but returns virtually to the control value at 72 hr.     

The antitumor effect of bleomycin combined with bestatin against Ehrlich ascites carcinoma in mice

The effect of bleomycin against Ehrlich ascites carcinoma transplanted subcutaneously to mice used in combination with bestatin was investigated. Male Balb/c mice weighting approximately 20 g and bred in our laboratories were used in this study. Each mouse was injected in its left lateral abdominal region subcutaneously with 7 X 10(6) tumor cells in 0.2 ml of ascites fluid. The mice were divided into four groups: control, bestatin alone (5 mg/kg intraperitoneally on Days 9-14), bleomycin alone (10 mg/kg intraperitoneally on Days 7 and 8), and bestatin plus bleomycin. Our results show that bestatin enhances the antitumor effect of bleomycin against Ehrlich ascites carcinoma as measured by the increased survival rates. Being an agent of very low toxicity, bestatin should be considered as a part of the chemoimmunotherapy protocol.     

The interphase death of dividing cells. The effect of oxygen and the pH of the medium on the interphase death of Ehrlich ascites carcinoma cells under the action of ionizing radiation

Interphase death of in vitro irradiated (200 Gy) Ehrlich ascites tumor cells was studied as a function of oxygenation level and medium pH. The presence of oxygen both during and after irradiation as well as the increase in pH from 7.4 to 8.1 were shown to increase interphase death rate. The pronounced threshold dependence of interphase death of cells upon their concentration may be attributed to hypoxia occurring in a pericellular medium when cells concentration exceeds the threshold.     

The effect of nonspecific immune stimulation with Corynebacterium parvum and polidin on the Ehrlich ascites tumor growth

Investigations were performed: a) to compare the effect of two nonspecific immunostimulants, Polidin and Corynebacterium parvum, on the development of Ehrlich ascites carcinoma in mice; b) to determine whether the effects are dependent on the tumor cell dose inoculated into the animals. C. parvum and Polidin administered prior to Ehrlich ascites tumor inoculation have a protective effect evidenced by a delay in tumor development, a retardation in tumor growth and a prolonged survival of the tumor host. The effect of immunostimulants was highly dependent on the tumor cell dose inoculated into mice and was more marked with C. parvum.     

The modelling of the growth of Ehrlich carcinoma and teratoma T-36 with ascitic fluid and tumor-cell dialysate]

The study of the effect of ascitic fluid and dialysate of Ehrlich ascites tumor cells (M.m. less than 15 kDa) on the growth of Ehrlich carcinoma and teratoma T-36 has shown that both the ascitic fluid and dialysate can protect tumor cells in vivo. The number of animals with tumors increased from 0% in control animals to 60 and 20%, respectively, in experimental ones after transplantation i.m. of 20 x 10(3) Ehrlich tumor cells into mice. Compared to control, ascitic fluid and dialysate of Ehrlich ascites tumor cells increased the rate of tumor growth to 195 and 153%, respectively. It is suggested that this test-system simulates the effect of tumor humoral factors in vivo.     

Suppression of the initiation of DNA synthesis as an index of cellular radiosensitivity

A gamma-radiation dose (Di) suppressing DNA synthesis initiation by 35% in primary suspension cultures of mammalian cells, is nearly the same as D0 for survival of clonogenic cells of the same lines and tissues. The extent of DNA synthesis suppression is assessed by impulse 3H-thymidine incorporation in the acid-insoluble fraction of irradiated cells. The values of Di determined in this way for HeLa cells, Ehrlich ascites tumor cells, mouse bone marrow and thymus cells are 2.0, 1.5, 1.5, and 1.0 Gy, respectively; as determined by clonogenic capacity of these cells, Di = 1.9, 2.0, 1.3, and 1.0 Gy, respectively.     

The effect of cancer chemotherapy on lysosomal enzymic activities (acid phosphatase) of Ehrlich ascites carcinoma cells.

The distribution of acid phosphatase in Ehrlich ascites carcinoma cells was studied by histochemical techniques in mice treated with antitumoral agents (Tio-Tepa, Cosmegen). The results proved the accuracy of the histoenzymatic demonstration of APh activity for the study of early changes induced in Ehrlich ascites tumor by antitumoral drugs : these changes precede those revealed by routine histological methods and are related to the degree of sensitivity of the tumor to the drugs.     

Reaction of 3-ethoxy-2-oxobutyraldehyde bis(thiosemicarbazonato) Cu(II) with Ehrlich cells. Binding of copper to metallothionein and its relationship to zinc metabolism and cell proliferation

The copper complex of 3-ethoxy-2-oxobutyraldehyde bis(thiosemicarbazone) or CuKTS is reduced and dissociated upon reaction with Ehrlich cells. Titration of the cells with the complex leads to the specific binding of copper to metallothionein with 1 to 1 displacement of its complement of zinc. Under conditions of complete titration of metallothionein, 1.25-2.5 nmol CuKTS/10(7) cells, cellular DNA synthesis is rapidly inhibited but no long term effects on cell proliferation are observed. The kinetics of redistribution of Cu and Zn in Ehrlich cells in culture and in animals were studied after pulse reaction of CuKTS with cells. After exposure of cells to the noncytotoxic concentration of 2.5 nmol of CuKTS/10(7) cells, nonmetallothionein bound copper is lost rapidly from the cells, after which copper in metallothionein decays. New zinc metallothionein is made as soon as exposed cells are placed in culture. New synthesis stops when the level of zinc in metallothionein reaches control levels. A second pulse treatment of cells with CuKTS to displace zinc from metallothionein again stimulates new synthesis of the protein to restore its normal concentration. The kinetics of metal metabolism in Ehrlich cells exposed to 5.5 nmol of CuKTS/10(7) cells, which inhibits cell proliferation, are qualitatively similar except there is a pronounced lag before new zinc metallothionein is synthesized. The Ehrlich ascites tumor in mice responds to CuKTS similarly to cells in culture. It is also shown that cultured Ehrlich cells do not make extra zinc metallothionein in the presence of high levels of ZnCl2, and fail to accumulate copper in the presence of large concentrations of CuCl2.     

Effect of tumor growth and preliminary irradiation on the state of DNA degradation processes in the liver cell nuclei

This study is devoted to the analysis of DNase activity and DNA fragmentation level in liver cells nuclei of rats with transplanted Guerin's carcinoma on irradiation background. Was shown, that in an organism of previously irradiated rat with tumor the dominance of neoplasm development over the consequences of the irradiation is observed on the initial stages of the experiment. The alignment of gamma-irradiation influencing already takes place from the first stages after Guerin's carcinoma transplantation, which is testified by the decrease of DNase activity of liver cells nuclei and the decrease of a degree of nuclear DNA fragmentation. On the stationary stages of tumor growth the general action of the factors causes differences in the parameters of the investigated animal groups. It shows that together with the constant reduced level of DNase activity the processes of DNA fragmentation strengthen and their intensity reaches maximum during this period. The nature of the fragmented DNA accumulation is similar to the irradiated organism on the first days after the radiation factor removal. Thus, the radiation-induced signal in the organism with tumor is leveled under the conditions of active neoplasm growth; nevertheless, the consequents of its operating do not disappear, and only move away in time.     

The effect of polycations on cell membrane stability and transport processes

Summary The interaction of poly-l-lysines of different molecular weights (PL) with Ehrlich ascites tumor cells was studied experimentally with respect to cell surface binding, cell electrophoresis, cytotoxicity and membrane permeability. Although they decrease the net negative charge of Ehrlich ascites cells similarly at low PL concentrations, low molecular weight PL was less cytotoxic and less damaging to the potassium transport mechanism than was high molecular weight PL. At certain PL concentrations, membrane damage was reversible on reincubation in PL-free media. The amount of bound polylysine as determined with fluorescent labeled polylysine was compared by electrophoresis to the amount of polylysine expressed on the electrokinetic surface. The results indicated that only a small fraction of polylysine bound to Ehrlich ascites tumor cells was electrokinetically detectable. The adsorption of polylysine to Ehrlich ascites tumor cells was not describable by the usual adsorption isotherms. It is suggested that the same number of monomeric lysine units of high and low molecular weight PL are adsorbed at the cell electrokinetic surface, but cytotoxicity is dependent on molecular weight. Although the negative charge of human red blood cells could be reversed at low PL concentrations, no such effect could be observed for ELD (a subline of Ehrlich ascites carcinoma) cells even at high PL concentrations. The relationship of PL binding to the stimulation of macromolecular uptake is discussed.