VARIATION OF CELL KINETIC PARAMETERS IN RELATION TO THE GROWTH RATE OF THE EHRLICH ASCITES TUMOR

A multicompartmental model of the cell cycle and proliferation kinetics was used to analyse the time-course behavior of the cell cycle time, the growth fraction, and the cell loss rate during Ehrlich ascites tumor growth. The growth rate of Ehrlich ascites tumor cells as the tumor aged was significantly influenced by change in the cell cycle time.     

Mathematical description and analysis of cell cycle kinetics and the application to Ehrlich ascites tumor

The linear and nonlinear aspects of the dynamics of the cell cycle kinetics of cell populations are studied. The dynamics are represented by difference equations. The characteristics of cell population systems are analyzed by applying the model to Ehrlich ascites tumor. The model applied for the simulations of the growth of Ehrlich ascites tumor cells incorporates processes of cell division, cell death, transition of cells to resting states and clearance of dead cells. Comparison of the results obtained with the model and the experimental data suggests that the duration of the mean generation time of the proliferating EAT cells increases with aging of the tumor. An attempt is made to relate the prolongation of cell mean generation time with processes of cell death and dead cell clearance. Studying the transition of cells to the resting states, it becomes apparent that in fact transition of proliferating cells to the resting states occurs somewhere close to the end of the cell cycle and with a rate that varies with the age of the tumor. Time course behavior of the cell age, cell size, and cell DNA distribution with aging of the tumor are obtained. Variations in average size and average DNA contents are determined.     

Patterns in the combined action of irradiation and glucose loading on tumor cells

It was shown that incubation of Ehrlich ascites tumor cells with glucose or in buffer solutions of low pH decreases their viability. The cell survival rate depends on pH values irrespective of the protoxidation method and oxygenation conditions used. At the same time, radiosensitivity of Ehrlich ascites tumor cells is practically invariable with pH being decreased from 7.0 to 5.0. The effects of glucose and radiation are additive in conditions simulating the effect of hyperglycaemia in vivo.     

The reversal of effects of methotrexate on toxicity and glucose transport reduction by thymidine in Ehrlich ascites tumor bearing mice

Methotrexate (MTX) suppressed the growth of Ehrlich ascites tumor cells in vivo and reduced the cellular uptake of glucose and the density of glucose transporters on the tumor cell surface. MTX inhibition of tumor growth was partially prevented by concurrent administration of thymidine. At the same time, the rate of cellular glucose uptake, the density of glucose transporters on the cell as well as the extent of thymidine, uridine and leucine incorporation were significantly increased.     

Kinetic analysis of cell size and DNA content distributions during tumor cell proliferation: Ehrlich ascites tumor study.

In order to study the growth dynamics of proliferating and non-proliferating cells utilizing discrete-time state equations, the cell cycle was divided into a finite number of age compartments. In analysing tumor growth, the kinetic parameters associated with a retardation in the growth rate of tumors were characterized by computer simulation in which the simulated results of the growth curve, the growth fraction, and the mean generation time were adjusted to fit the experimental data. The cell age distibution during the period of growth was obtained and by a linear transformation of the state transition matrices, was employed to specify the cell size and DNA content distributions. In an application of the model, the time-course behavior of cell cycle parameters of Ehrlich ascites tumor is illustrated, and the parameters important for the transition of cells in the proliferating compartment to the non-proliferating compartment are discussed, particularly in relation to the G1-G0 and G2-G0 transitions of non-cycling cells as revealed by the variation of cell size distribution.     

Over-expression of the small heat-shock protein, hsp25, inhibits growth of Ehrlich ascites tumor cells.

hsp25 is a small, growth-related, mammalian stress protein which is highly accumulated in the stationary phase of Ehrlich ascites tumor in vivo. Ehrlich ascites cells cultivated in vitro under conditions of continuous exponential growth express hsp25 only at a low level. These cells were stably transfected with an eukaryotic expression vector carrying the coding sequence of the small heat-shock protein, hsp25, under control of the murine metallothionein promoter. The resulting cell lines (EAT II6 and EAT II8) exhibit constitutive over-expression of the small heat-shock protein, hsp25, which can be further increased by induction with cadmium. Both cell lines show increased thermoresistance. The in vitro proliferation rate of the transfected cell lines EAT II6 and EAT II8 is significantly decreased depending on the degree of cadmium-regulated over-expression of hsp25. Furthermore, a significant delay in Ehrlich ascites tumor growth in mice using the hsp25 over-expressing cells for primary inoculation could be demonstrated.     

中药麝香胶囊抑瘤作用的实验研究

目的探讨中药麝香胶囊对小鼠实验性肿瘤的疗效。方法昆明小鼠分别接种艾氏腹水癌、S-180、肝癌细胞株（hepatocellular carcinoma,HCC）三种癌细胞株,接种24h后开始给予中药麝香胶囊,每日1次,共10d。分高、中、低三个剂量给药（4、2、1g／kg以主药麝香药量计）。阳性对照以天仙丸胶囊（1g／kg）一次性灌服。阴性对照组以同体积生理盐水灌服。接种实体瘤动物于第10天处死,称瘤重,计算抑瘤率;接种腹水瘤动物,观察存活时间,计算生命延长率。结果中药麝香胶囊对实体癌有一定的抑瘤作用,但未达到药典规定的抑瘤率30％的要求;对腹水癌也有一定的抑瘤作用,但也未达到药典规定生命延长率50％的要求;统计学分析（P〉0．05）差异没有显著性。结论中药麝香胶囊抑瘤作用不明显,其配方及剂型有待进一步研究与改进。     

Effect of nonspecific immune stimulation with BCG and polidin on the Ehrlich ascites tumor growth

Investigations were performed to study: 1) the antitumor effect of BCG pretreatment on the development of Ehrlich ascites tumor in mice; 2) the effect of BCG administration in relation to the period of time before tumor inoculation and the dose levels used, and 3) the antitumor effect of an associated pretreatment of BCG and Polidin on the development of Ehrlich ascites tumor. BCG administered prior to Ehrlich ascites tumor inoculation have a protective effect evidenced by a delay in tumor development, a prolonged survival of the tumor host and, in some cases, even inhibition of tumor growth. The effect of BCG was highly dependent on 1) the dose and the time of administration of BCG and) 2 the combined pretreatment of BCG and Polidin.     

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 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.     

Increased rate of tumor cell death caused by polyamine synthesis inhibitors

This investigation was designed to determine whether cell death plays a role in the antiproliferative action exerted by polyamine synthesis inhibitors. To estimate the rate of tumor cell death, we measured the loss of 125I from mice harboring Ehrlich ascites tumor cells in which DNA was labeled with 5-125I-iodo-2'-deoxyuridine. DL-alpha-difluoromethylornithine (0.85 mumoles/g body weight/6 h), and enzyme-activated irreversible inhibitor of ornithine decarboxylase, and methylglyoxal-bis(guanylhydrazone) (45 nmoles/g body weight/6 h), an inhibitor of S-adenosylmethionine decarboxylase, were both found to increase the rate of 125I excretion. Our data suggest that these polyamine synthesis inhibitors provoke an increase in the rate of tumor cell death beyond that normally occurring during growth, methylglyoxal-bis(guanylhydrazone) being considerably more potent than DL-alpha-difluoromethylornithine. These in vivo data were corroborated by a study where the host-mediated responses did not have to be considered. Thus, Ehrlich ascites tumor cells were adapted for suspension growth in culture and treated with methylglyoxal-bis(guanylhydrazone) or DL-alpha-difluoromethylornithine. The growth kinetics and the colony forming efficiency of the drug-treated cells clearly show that polyamine synthesis inhibitors not only slow the growth rate but also cause an increase in tumor cell death.     

KINETIC ANALYSIS OF CELL SIZE AND DNA CONTENT DISTRIBUTIONS DURING TUMOR CELL PROLIFERATION: EHRLICH ASCITES TUMOR STUDY

In order to study the growth dynamics of proliferating and non-proliferating cells utilizing discrete-time state equations, the cell cycle was divided into a finite number of age compartments. In analysing tumor growth, the kinetic parameters associated with a retardation in the growth rate of tumors were characterized by computer simulation in which the simulated results of the growth curve, the growth fraction, and the mean generation time were adjusted to fit the experimental data. The cell age distribution during the period of growth was obtained and by a linear transformation of the state transition matrices, was employed to specify the cell size and DNA content distributions. In an application of the model, the time-course behavior of cell cycle parameters of Ehrlich ascites tumor is illustrated, and the parameters important for the transition of cells in the proliferating compartment to the non-proliferating compartment are discussed, particularly in relation to the G₁-G₀ and G₂-G₀ transitions of non-cycling cells as revealed by the variation of cell size distribution.     

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.     

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.     

Osmotic properties of ehrlich ascites tumor cells during the cell cycle

Ehrlich ascites tumor cells were grown and maintained in continuous spinner culture. The population of dividing cells was synchronized by a double thymidine block technique. Cell cycle phases were determined graphically by plotting mitotic index, cell number, and DNA synthesis against time. Changes in the osmotic properties of Ehrlich ascites tumor cells during the cell cycle are described. Permeability to water is highest at the initiation of S and progressively decreases to its lowest value just after mitosis. Heats of activation for water permeability vary during the cell cycle, ranging from 9–14 kcal/mole. Results may imply changes in the state of water in the membrane during the cycle. The volume of osmotically active cell water is highest during S and early G2 and decreases during the mitotic phase, as cells undergo division. Total water content remains stable at 82% (w/w) during the cycle. Total concentration of the three major ions (Na, K, Cl), expressed as mEq/liter total cell volume, does not change. The fraction of total cell water which is osmotically active (Ponder's R) decreased gradually from 0.75 at S to about 0.56 following mitosis. Findings suggest that a fraction of the total water within the cell exists in a “bound” form and is, therefore, incapable of being shifted under the driving force of osmotic pressure. This fraction of bound water increases during the cell cycle. Possible alterations in membrane fluidity and the state of water in the cell are discussed.     

Analysis of cell flow and cell loss following X-irradiation using sequential investigation of the total number of cells in the various parts of the cell cycle

The cell flow and cell loss of an in vivo growing Ehrlich ascites tumour were calculated by sequential estimation of changes in the total number of cells in the cell cycle compartments. Normal growth was compared with the grossly disturbed cell flow evident after a 5 Gy X-irradiation. The doubling time of normal, exponentially growing cells was 24 hr. The generation time was 21 hr based on double-isotope labelling studies and the potential doubling time was 21 hr. Thus, the growth fraction was 1.0 and the cell loss rate about 0.5%/hr. Following irradiation, a transiently increased relative outflow rate from all cell cycle compartments was found at about 3 and 40 hr, and from S phase at 24 hr after irradiation. Minimum flow rates from all compartments were found up to 20 hr. Cell loss as calculated from the cell flow was compared with non-viable cells determined by Percoll density separation. Increase in cell loss as well as non-viable cells was observed at 24 hr after irradiation at the time of release of the irradiation-induced G2 blockage. Up to 50 hr, about 70% of the initial total number of cells were lost. The experiments show the applicability and limitations of cell flow and cell loss calculations by sequential analysis of the total number of cells in the various parts of the cell cycle.     

Localization of 25-hydroxyvitamin D3-1 alpha-hydroxylase activity in the mammalian kidney

The tRNA from Ehrlich ascites tumor cells is deficient in the modified nucleoside Q (queuosine). Continuous infusion of Q base (queuine) to tumor-bearing mice reverses the deficiency of Q in Ehrlich ascites tRNA, and coincidently, causes an inhibition of tumor growth.     

Analysis of Cell Flow and Cell Loss Following X-Irradiation Using Sequential Investigation of the Total Number of Cells In the Various Parts of the Cell Cycle

The cell flow and cell loss of an in vivo growing Ehrlich ascites tumour were calculated by sequential estimation of changes in the total number of cells in the cell cycle compartments. Normal growth was compared with the grossly disturbed cell flow evident after a 5 Gy X-irradiation. The doubling time of normal, exponentially growing cells was 24 hr. the generation time was 21 hr based on double-isotope labelling studies and the potential doubling time was 21 hr. Thus, the growth fraction was 1.0 and the cell loss rate about 0.5%/hr. Following irradiation, a transiently increased relative outflow rate from all cell cycle compartments was found at about 3 and 40 hr, and from S phase at 24 hr after irradiation. Minimum flow rates from all compartments were found up to 20 hr. Cell loss as calculated from the cell flow was compared with non-viable cells determined by Percoll density separation. Increase in cell loss as well as non-viable cells was observed at 24 hr after irradiation at the time of release of the irradiation-induced G₂ blockage. Up to 50 hr, about 70% of the initial total number of cells were lost. the experiments show the applicability and limitations of cell flow and cell loss calculations by sequential analysis of the total number of cells in the various parts of the cell cycle.     

Anaerobiosis and oxygen recovery: changes in cell cycle distribution of Ehrlich ascites tumor cells grown in vitro.

The cell cycle distribution of in vitro cultured Ehrlich ascites tumor (EAT) cells was analysed by pulse-cytophotometry to characterize the growth cessation observed under anaerobic conditions. DNA histograms provided evidence that in the absence of oxygen EAT cells accumulate in the G1 and early S phase of the cell cycle while in the presence of oxygen an increase in G2 was observed during 24h culture period. Cellular recovery from anaerobiosis was observed soon after transfer of the cells into fresh aerobic culture medium but occurred slowly if the cells were only resupplied with air. Cell cycle analyses as well as (14C)-thymidine incorporation suggest considerable synchronization results from the introduction of anaerobiosis.     

Bioflavonoid regulation of ATPase and hexokinase activity in Ehrlich ascites cell mitochondria.

(1) The mitochondrial ATPase (EC 3.6.1.3) Ehrlich ascites cell mitochondria, was inhibited by D-glucose under physiological concentrations of ATP. The generation of ADP by the mitochondrial bound hexokinase, seems to be the reason for the D-glucose inhibitory effect. Reversal of the inhibitory effect of ADP on Ehrlich ascites cell mitochondria ATPase by an ATP-regenerating system was achieved. (2) Dissociation of mitochondrial bound hexokinase from the mitochondria eliminated the inhibitory effect of D-glucose. Rebinding of the hexokinase to the mitochondria regenerated the D-glucose inhibitory effect on Ehrlich ascites cell mitochondria ATPase. (3) Bioflavonoids such as quercetin inhibit the mitochondrial hexokinase activity, but do not change the mitochondrial ATPase activity of isolated Ehrlich ascites tumor cell mitochondria. (4) The inhibitory effect of bioflavonoids on mitochondrial bound hexokinase activity is shown to be dissociable from the ascites tumor cell mitochondria and seems to be associated with regulatory rather than catalitic sites of the enzyme.