Cultured Ehrlich Ascites Tumor Cells Research Articles

Catalytic enzyme histochemistry and biochemical analysis of dihydroorotate dehydrogenase/oxidase and succinate dehydrogenase in mammalian tissues, cells and mitochondria.

Dihydroorotate dehydrogenase (EC 1.3.3.1 or EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. In eukaryotes it is located in the inner mitochondrial membrane, with ubiquinone as the proximal and cytochrome oxidase as the ultimate electron transfer system, whereas the rest of pyrimidine biosynthesis takes place in the cytosol. Here, the distribution of dihydroorotate dehydrogenase activity in cryostat sections of various rat tissues, and tissue samples of human skin and kidney, was visualized by light microscopy using the nitroblue tetrazolium technique. In addition, a hydrogen peroxide-producing oxidase side-reactivity of dihydroorotate dehydrogenase could be visualized by trapping the peroxide with cerium-diaminobenzidine. The pattern of activity was similar to that of succinate dehydrogenase, but revealed a less intensive staining. High activities of dihydroorotate dehydrogenase were found in tissues with known proliferative, regenerative, absorptive or excretory activities, e.g., mucosal cells of the ileum and colon crypts in the gastrointestinal tract, cultured Ehrlich ascites tumor cells, and proximal tubules of the kidney cortex, whilst lower activities were present in the periportal area of the liver, testis and spermatozoa, prostate and other glands, and skeletal muscle. Dihydroorotate dehydrogenase and succinate dehydrogenase activity in Ehrlich ascites tumor cells grown in suspension culture were quantified by application of nitroblue tetrazolium or cyanotolyl tetrazolium and subsequent extraction of the insoluble formazans with organic solvents. The ratio of dihydroorotate dehydrogenase to succinate dehydrogenase activity was 1:4. This was in accordance with that of 1:5 obtained from oxygen consumption measurement of isolated mitochondria on addition of dihydroorotate or succinate. The ratio determined with mitochondria from animal tissues was up to 1:15 (rat liver, bovine heart). The application of the enzyme inhibitors brequinar sodium and toltrazuril verified the specificity of the histochemical and biochemical methods applied.

Wild-type and cultured Ehrlich ascites tumour cells differ in tumorigenicity, lectin binding patterns and binding to basement membranes.

Three different variants of the Ehrlich ascites tumour (EAT) cell were derived and the lectin surface reactivities, as well as the malignant characteristics of each variant, were studied. Wild-type cells (EAT-wt) were selected for growth on basement membranes and tissue culture plastic to give EAT-c cells. The EAT-c were passaged in mice by i.p. injection, giving rise to a third variant (EAT-c/m). Each of these three cell variants was characterized for: (i) specific lectin agglutinability patterns; (ii) the ability to produce ascites tumours in mice; (iii) the ability to produce solid tumours; and (iv) the attachment to and growth on basement membranes and purified extracellular matrix molecules. Analysis of the total protein and carbohydrate content of each cell line showed that there was an increase in the glycosylation of the EAT-c cells compared to EAT-wt cells. After repeated passage of the EAT-c/m cells in mice, the glycosylation level of the EAT-c/m cells returned to that of the EAT-wt cell line. In addition, the EAT-c cells displayed an increase in the number of terminal non-reducing sugars which could indicate either an increase degree of branching or the presence of additional N- and/or O-linked oligosaccharide chains of the cellular glycoproteins. This phenotype was retained by the EAT-c/m cells which had been passaged repeatedly in mice. The most significant increase was in the content of sialic acid-containing glycoproteins found in the EAT-c cells. The sialic acid-binding lectin Maackia amurensis leukoagglutinin (MAL) agglutinated all three EAT cell variants, while the sialic acid-binding Sambucus nigra (elderberry bark) lectin (SNA) agglutinated only the EAT-c and early-passage EAT-c/m cells. These findings indicate the presence of alpha 2,3-linked sialic acid on all three variants, but only the cultured cells and early-passage EAT-c/m cells possess the Neu5Ac alpha 2,6 linkage. The EAT-c cells attached avidly to wells coated with either laminin or fibronectin, as well as extracellular matrix produced by cultured bovine endothelial cells, but the EAT-wt and EAT-c/m cells did not. Paradoxically, the EAT-c cells were incapable of producing solid tumours when injected into a basement membrane-rich skeletal muscle bed, whereas the EAT-wt and EAT-c/m cells produced rapidly growing tumours when injected into the same environment. Lectin agglutination patterns established that ascitic tumour cells within the peritoneal cavity were derived from injected EAT-c cells.

Cultured Ehrlich Ascites Tumor Cells Show Increased N-Linked α2,6-Sialytransferase Activity

Ehrlich ascites tumor cells (EAT cells) are routinely grown in the peritoneal cavity of mice. These cells, EAT-wt, grow in suspension and exhibit a high level of α-2,3-O-linked sialyltransferase activity with benzyl-T-antigen (Galβ1,3Ga1NAc-α-O-CH2C6H5) as acceptor. These cells also contain a very low level of α-2,6-O-linked and α-2,6-N-linked sialyltransferase activity. A variant of these cells, EAT-c, has been selected to grow in cell culture, attached to the surface of culture flasks. EAT-c cells exhibit a selective increase of two- to fivefold in the activity of α-2,6-N-linked sialyltransferase activity, using asialo-α1-acid glycoprotein as acceptor. Since a similar selective increase has been previously observed in metastatic human colorectal cancer tissues, the EAT-wt/EAT-c cell system may serve as a good experimental model for the investigation of sialyltransferases and their cell surface sialylated products in relation to cancer, metastasis, and cell-cell interaction.

The “anti-pyrimidine effect” of hypoxia and brequinar sodium (NSC 368390) is of consequence for tumor cell growth

The rationale of the present study was to investigate the simultaneous effect of hypoxia and drugs with an “anti-pyrinudine effect” on tumor cell proliferation to evaluate putative changes in the sensitivity of cells to these kinds of chemotherapeutic treatment on reduced O 2 tension. Pyrimidine de novo biosynthesis, at the stage of respiratory chain-dependent dihydroorotate dehydrogenase, was found to be a biochemical target site for oxygen deficiency as well as for Brequinar Sodium TM (6-fluoro-2-(2'-nuoro-1,1'-biphenyl-4-yl)-3-methyl-4 quinoline carboxylic acid sodium salt) (Brequinar). Increasing drug concentrations (0.1–50 μM) reduced the proliferation rate of in vitro cultured Ehrlich ascites tumor cells ( ic 50 = 0.25 μM). Decreasing concentrations of O 2 reduced the proliferation rate (50% at ~3.5% O 2). Brequinar at 2.5 and 12.5 μM stimulated the incorporation of exogenous [ 14C]uridine into RNA to 140 and 190% of controls, respectively, as a result of active salvage pathways, whereas it decreased the incorporation of [ 14C]NaHCO 3 by the de novo pathway (to 20 and 5% of controls, respectively). Cells routinely grown in glucose-free, undine-supplemented medium were resistant to 12.5 μM of the drug. The complete growth pattern of the tumor cells (increase in cell number and protein, RNA and DNA content of cultures during a 24-hr culture period) was examined (i) on reducing the O 2 tension of the atmosphere stepwise from 20 to 1% O 2 (ii) on addition of 0.125 μM Brequinar; and (iii) under both conditions. The combination was found to give an additive inhibitory effect under moderate hypoxia (5–20% 0;) and a greater than additive effect if the oxygen tension was further reduced (1–5%).

Oxidative stress induced by a copper-thiosemicarbazone complex

Copper thiosemicarbazones cause considerable oxidative stress. This effect may be related to their cytotoxicity. In the present work, the chemical and cellular properties of a new ligand, pyridoxal thiosemicarbazone (H2T), and its copper(II) chelate (CuT) are assessed. CuT is toxic to cultured Ehrlich ascites tumor cells, producing nearly complete cell kill at drug/cell ratios of 2.5-4 nmol/10(5) cells in a monolayer culture over a 48-h treatment period. This concentration is at least 1 order of magnitude lower than those required for a similar degree of cytotoxicity by H2T or CuCl2. The following observations support the view that activated oxygen species are generated by interaction of CuT with Ehrlich cells: (1) Room-temperature electron spin resonance spectroscopy and atomic absorption measurements show rapid cellular uptake and CuT-thiol adduct formation. (2) Cellular thiol content is reduced. (3) High levels of DNA strand scission result from 1-h treatments of cells by concentrations of CuT that cause growth inhibition and toxicity. (4) The extent of strand scission can be increased by addition of superoxide dismutase and decreased by catalase or DMSO in the treatment medium. Catalase and DMSO do not inhibit the toxic effect of CuT. This suggests that DNA damage is not responsible for inhibition of cell proliferation by CuT.

Inhibition of Ehrlich ascites tumor in vivo by PAF-antagonists

Several lines of evidence support that PAF modulates the inflammatory and immune responses, and that tumors may inhibit both these processes. In the present study we analysed the effect of PAF antagonists on the growth of Ehrlich Ascites Tumor (EAT) in vivo. Mice were inoculated intraperitoneally with 1 × 10 3 EAT cells and the tumor growth evaluated by counting the number of peritoneal cells, 1, 6 and 10 days after tumor implantation. BN 52021 was administered intraperitoneally, intravenously or subcutaneously once or twice a day, at 1.0, 2.5, 5.0 and 20.0 mg/kg. Control animals received 0.1 ml of the vehicle in the same schedule. It was found that i.p. and i.v. administration of BN 52021 (5 mg/kg, twice a day) significantly inhibited EAT growth (80.8% and 56.0% respectively). Other routes and doses were less effective. Another PAF antagonist, SRI 63441 (5 mg/kg, i.p., twice a day) also inhibited EAT growth (80.4%). The BN 52021 added to EAT cells in culture, at concentration of 10 −3 and 10 −4 M, did not affect the viability and proliferation of tumors cells. In an attempt to understand the mechanism of this inhibition, we analyzed the peritoneal macrophages for spreading ability and H 2O 2 release. It was found that 24 h after tumor implantation there was an increase in the spreading ability of peritoneal macrophages (75%) and that, as the tumor grew, the spreading index fell to control levels (<10%). In the groups treated with BN 52021 (5 mg/kg/twice a day) the spreading remained elevated (50–60%) at all the times examined. Release of H 2O 2, measured by horseradish peroxidase-phenol red oxidation, was below detectable levels throughout tumor growth. Treatment with BN 52021 as above, significantly stimulated the release of H 2O 2, 6 and 10 days after tumor implantation (0.4 to 0.6 nmol/10 5 cells). The data presented show that PAF antagonists inhibit EAT growth in vivo and that this inhibition is concomitant with activation of peritoneal macrophages. These results suggest that macrophages can critically influence tumor growth and that PAF or PAF-like substances may modulate macrophages-tumor interactions.

Investigations on the function of creatine kinase in Ehrlich ascites tumor cells.

1. Growth and viability of in vitro cultured Ehrlich ascites tumor cells are not significantly impaired by exogenous creatine up to 40mM. Retardation of cell growth by higher concentrations depends on cell density. 2. Ehrlich cells grown in the presence of high concentrations of creatine accumulate creatine phosphate to high levels (up to 23 nmol/10(6) cells in the presence of 40mM creatine). 3. A nearly complete interruption of glycolytic ATP production or inhibition of the oxidative ATP synthesis reduces the maximal creatine to about 40-50% of controls. 4. Studies on the intracellular distribution of creatine kinase have shown, that the enzyme is only associated with the mitochondrial fraction. Titration of isolated mitochondria with digitonin revealed that the activity is located in the inter-membrane space and partly bound to the outer site of the inner membrane. 5. By growth of Ehrlich cells in creatine-free medium it is possible to obtain "creatine phosphate-depleted" cells (creatine phosphate less than 10% of controls). The growth of creatine phosphate-depleted cells as compared to controls is significantly reduced under energetic stress situations. The protein synthesis of these cells after an energetic stress (lack of glucose and oxygen) is significantly reduced as compared to creatine phosphate containing cells. 6. It is concluded that in these cells creatine kinase/creatine phosphate is a thermodynamic buffer system and not part of an energy shuttle as is postulated for muscle cells.

Effect of &lt;i&gt;L-&lt;/i&gt;and D-Lysine on the Growth of Ehrlich Ascites Tumour

Contrary to L-lysine, D-lysine showed no inhibitory effect on growth of Ehrlich ascites tumour cell in in vivo studies. The in vitro studies on the growth of cultured Ehrlich ascites tumour cells showed similar results, namely, the growth of tumour cells was inhibited when cultured in 0.02 or 0.03 M L-lysine; while 0.02 or 0.03 M D-lysine exhibited no effect.

L-histidine inhibits the growth of Ehrlich ascites tumour.

The in vitro studies on the growth of cultured Ehrlich ascites tumour cells showed similar results as the in vivo studies reported previously. The growth of tumour cells was inhibited when cultured in 0.02 or 0.03 M L-histidine. At these concentrations, L-glycine shows no significant effect.

Effects of the lipidperoxidation product 4-hydroxynonenal and related aldehydes on proliferation and viability of cultured Ehrlich ascites tumor cells.

The mechanism by which the lipid peroxidation product 4-hydroxynonenal and several other homologous, yet non biogenic aldehydes inhibit proliferation of cultured Ehrlich ascites tumor cells has been studied. Incubation of cells (5 X 10(-4)/ml) in a minimum essential medium supplemented with 10 or 20 microM 4-hydroxynonenal reduces the 36-hr cell count to 65 and 30% of the control value. The reduced growth rate is most likely due to a blockage of the DNA synthesis. Cells labelled by a [3H]-thymidine pulse prior to exposure to 4-hydroxynonenal (20 microM, 8 hr) showed no change of the specific radioactivity of the DNA, indicating that no de novo synthesis occurred in the presence of the aldehyde. In the absence of the aldehyde the specific radioactivity of the DNA decreased by 25%. A 2-hr incubation in the presence of 10 or 20 microM of 4-hydroxynonenal reduced [3H]-thymidine incorporation into the HClO4 insoluble fraction to 85 and 50% of the controls, but had no effect of the [3H]-thymidine and 86Rb uptake. Moreover, examination of the cell cultures by the Trypan Blue exclusion technique revealed that 20 microM 4-hydroxynonenal does not cause cell death. The high reactivity of 4-hydroxynonenal towards sulfhydryl groups suggests that the aldehyde inhibits DNA synthesis by interacting with a functional SH group of DNA polymerase. The specific action on DNA synthesis is abolished at an aldehyde concentration of 50 microM, which leads to 30% (6 hr exposure) and 95% (36 hr exposure) of dead cells. The cytostatic index (CI), i.e. concentration at 50% Trypan Blue positive cells/concentration at 50% inhibition of cell growth deducted from the dose effect curves is 3.0 for 4-hydroxynonenal. The other homologous 4-hydroxyalkenals with chain length of 5, 6, 7, 8, 10 and 11 carbon atoms also inhibit cell growth. The CI varied from 1.20 to 1.94, indicating that these non biogenic 4-hydroxyalkenals have a distinctively lower specific effect on proliferation than the biogenic 4-hydroxynonenal. The Michael adducts of 4-hydroxynonenal with glutathione and cysteine were nearly one order of magnitude less toxic than the free aldehyde, the CI (2.41 cysteine adduct, 2.06 glutathione adduct), however, were not improved since the growth inhibitory action was also reduced.

Growth characteristics of anaerobically treated early and late S-period of Ehrlich ascites tumor cells after reaeration.

Utilizing centrifugal elutriation, early and late S-phase cells were separated from 4, 8 and 12 h anaerobically cultured Ehrlich Ascites tumor cells strain Karzel. The cytokinetic properties of these fractions after reaeration were studied by flow cytometry and the BrdU-H 33258-technique of flow cytometry. After a 4 h period of anaerobiosis, growth of early S-phase cells is not changed, 8 h deprivation of oxygen causes a delay of cell cycle progression, while the main fraction of 12 h anaerobically treated early S-populations did not divide after reaeration within 24 h. In comparison to early S-phase cells the cell cycle progression of the main fraction of late S-period is accelerated after a 4 h exclusion of oxygen. A fraction of 8 h anaerobically pretreated late S-cells continues to cycle, but a considerable number reinitiates DNA synthesis without preceeding division. Cells with DNA content up to 8 c are detected by flow cytometry. 12 h anaerobically cultured late S-cells do not divide after reaeration, a large number of these cells starts again to synthesize DNA. A considerable part of tetraploid cells retain viability, divide and enter a new cell cycle, another part of the cells disintegrates.

Further characterization of the growth inhibitory effect of rotenone on in vitro cultured Ehrlich ascites tumour cells.

As an approach for a better understanding of the mode of action of rotenone on mammalian cells we have studied the proliferation properties, metabolism and basic cell composition of Ehrlich ascites tumour cells cultured in vitro in the presence of 2.5 microM rotenone and after removal of the inhibitor. Experiments on asynchronous cells showed a rapid cessation of cell division accompanied by increased glycolytic rate, reduced oxygen consumption, moderate increase in DNA content and a fair increase in protein and RNA content of the cultures. DNA histograms obtained by flow-cytometry revealed an accumulation of cells in the G2 and M phase of the cell cycle. Electron micrographs taken after a 24 h treatment of cells illustrated the formation of giant mitochondria and fragmented nuclei. In order to elucidate the dual effect of rotenone- inhibition of mitochondrial energy metabolism and of mitotic processes- the influence on cells of rotenone at different stages of the cell cycle was tested using Ehrlich ascites tumour cells enriched in G1, S and G2 by centrifugal elutriation. DNA histograms and [3H]thymidine labelling index curves of cells from the different fractions cultured in the presence of 2,5 microM rotenone indicated that in addition to the observed accumulation in G2 and mitotic arrest of cells, the cell cycle progression is delayed in G1 phase. This may be explained by an effect of the inhibitor on the respiratory chain. S phase cells seemed to continue the cycle for several hours at a rate comparable to that of controls. Recultivation experiments on rotenone-treated asynchronous cells in inhibitor-free medium confirmed that some cells reinitiate DNA synthesis without preceeding cell division. Thus it must be concluded that cells at all stages of the cycle are affected by rotenone, but the impairment of cellular metabolism becomes manifest and lethal as soon as the acute block at mitosis is abolished and cells reenter the cycle.

Repair of double-strand breaks (dsb) in DNA of cultured Ehrlich ascites tumour cells (EATC) and the effects of various degrees of salt-induced hypertonic shock

Repair of double-strand breaks (dsb) in DNA of cultured Ehrlich ascites tumour cells (EATC) and the effects of various degrees of salt-induced hypertonic shock

Growth Characteristics of in vitro Cultured Ehrlich Ascites Tumor Cells under Anaerobic Conditions and after Reaeration

The effects of deprivation of oxygen on proliferation kinetics of in vitro grown Ehrlich ascites tumor cells were studied by means of flow cytometry. The flow cytometric results obtained by applying the BUdR-H 33258 technique showed that 6--8 hours after establishment of anaerobiosis cells lose their capacity for proliferation and accumulate in the late G1-phase; during this time late S cells enter the G2 compartment but do not divide while cells, which are in G2+M at the beginning of anaerobiosis divide and enter G1. Cell cycle distribution remained constant up to about 20 hours, thereafter G1-cells enter the S-phase. Up to 24 hours of anaerobiosis cell number does not change and viability of the cells was not severely affected. Recultivation under aerobic conditions of 12 hours anaerobically treated cells revealed, that the cytokinetic properties of G1 cells were not impaired, while S-phase cells after reaeration did not enter G2 but began again to synthesize DNA forming cells with a DNA content greater than 4c. S-phase cells are most sensitive to deprivation of oxygen.

Circulating ceruloplasmin is an important source of copper for normal and malignant animal cells

The relative uptake of copper from ceruloplasmin and non-ceruloplasmin plasma pools, by normal and malignant cells, was investigated in vivo and in vitro, using 64Cu and 67Cu. 1. Most of the copper administered intravenously to normal and tumor-bearing rats was removed within 1 h, a substantial portion entering the liver. There were differences in the apparent avidity of individual tissues for ceruplasmin vs. ionic copper, but when calculated on the basis of actual μg absorbed, all showed a preference for ceruplasmin. 2. Appreciable amounts of copper from either source were also absorbed by the tumors, and cultured Ehrlich ascites tumor cells showed a rapid uptake and marked preference for ceruplasmin over non-ceruplasmin copper, as did primary rat muscle cell cultures. 3. Ceruplasmin protein was also absorbed by normal and neoplastic rat tissues, but less rapidly than ceruplasmin copper, as determined by administration of pure [ 3H]leucine- or [ 125I]ceruloplasmin. Copper deficiency did not accelerate this process. 4. It is concluded that, at least in rat, ceruloplasmin is the preferred plasma source of copper for normal and malignant cells, and that the copper on ceruplasmin turns over more rapidly than the protein moiety, a finding consistent with its role as a copper transport protein.

Cytochalasin B binding to Ehrlich ascites tumor cells and its relationship to glucose carrier

Cultured Ehrlich ascites tumor cells equilibrate d-glucose via a carrier mechanism with a K m and V of 14 mM and 3 μmol/s per ml cells, respectively. Cytochalasin B competitively inhibits this carrier-mediated glycose transport with an inhibition constant ( K i) of approx. 5·10 −7 M. Cytochalasin E does not inhibit this carrier function. With cytochalasin B concentrations up to 1·10 −5 M, the range where the inhibition develops to practical completion, three discrete cytochalasin B binding sites, namely L, M and H, are distinguished. The cytochalasin B binding at L site shows a dissociation constant ( K d) of approx. 1·10 -6 M, represents about 30% of the total cytochalasin B binding of the cell (8·10 6 molecules/cell), is sensitively displaced by cytochalasin E but not by d-glucose, and is located in cytosol. The cytochalasin B binding to M site shows a K d of 4–6·10 −7 M, represents approx. 60% of the total saturable binding (14·10 6 molecules/cell), is specifically displaced by d-glucose with a displacement constant of 15 mM, but not by l-glucose, and is insensitive to cytochalasin E. The sites are membrane-bound and extractable with Triton X-100 but not by EDTA in alkaline pH. The cytochalasin B binding at H site shows a K d of 2–6 · 10 −8 M, represents less than 10% of the total sites (2 · 10 6 molecules/cell), is not affected by either glucose or cytochalasin E and is of non-cytosol origin. It is concluded that the cytochalasin B binding at M site is responsible for the glucose carrier inhibition by cytochalasin B and the Ehrlich ascites cell is unique among other animal cells in its high content of this site. Approx. 16-fold purification of this site has been achieved.

Subcellular localization of daunorubicin in sensitive and resistant Ehrlich ascites tumor cells

Cellular uptake and subcellular localization of daunorubicin were studied in Ehrlich ascites tumor cells, either in vivo after inoculation of the drug to mice or in vitro after incubation of cultured Ehrlich ascites tumor cells with the drug. These cells were either sensitive (wild-type) or resistant to daunorubicin. The uptake of daunorubicin was 2–4 times less in resistant cells. About 90% of the drug which had penetrated in the cells was recovered in the nuclei. By cell fractionation, it has been established that the extranuclear daunorubicin was localized in the lysosomes. The lower uptake of the drug in resistant cells and the similar subcellular localization in both types of cells were in favour of an increased active outward transport of the drug in resistant cells.

The pH-dependence of sugar-transport and glycolysis in cultured Ehrlich ascites-tumour cells.

1. pH-dependence of glycolysis has generally been ascribed to the effects of pH on the activities of glycolytic enzymes. The present study shows that sugar transport is pH-dependent in cultured Ehrlich ascites-tumour cells. 2. The rates of glucose consumption, of 3-O-methylglucose transport, and of 2-deoxyglucose transport and phosphorylation increased as linear functions of pH, as the pH of the cell culture medium was increased from 6.1 to 8.5. Transport of glucose, as measured in ATP-depleted cells, was pH-dependent to the same extent as transport of the non-metabolizable sugars. 3. Glucose consumption rates were about 8-fold higher at pH 8.5 than at pH 6.4. About 65-85% of glucose was converted into lactate. Sugar transport rates were 2.5-fold higher at pH 8.5 than at pH 6.3. 4. pH affected both simple diffusion and facilitated diffusion. pH effect was mainly on the Vmax. of 2-deoxyglucose uptake, and on the rapid-uptake phase of 3-O-methylglucose transport. 5. It was estimated that about 70% of the pH effect on the rates of glucose consumption may be due to the effect on sugar transport and the remainder to the effect on the activities of glycolytic enzymes.

Synthesis and antitumor activity of 2-alkanesulfinyl (or alkanesulfonyl)-7-methyl-5H-1,3,4-thiadiazolo[3,2-a]pyrimidin-5-ones.

The repressing effect on the propagation of cultured Ehrlich ascites tumor cells by 1, 3, 4-thiadiazolo[3, 2-a]pyrimidine and s-triazolo[1, 5-a]pyrimidine derivatives was examined. Then the relationship between structure and activity as to a series of active compounds was discussed. The results showed that substituent groups on the 2-position of 1, 3, 4-thiadiazolo[3, 2-a]pyrimidine took an important part to reveal the cytotoxic activity. Especially, the strong activity was found among the compounds which had electrophilic substituents such as alkylsulfoxide, alkylsulfone at the 2-position.

Heterogeneity of native ribosomal 60-S subunits in Ehrlich ascites tumor cells cultured in vitro.

Native large ribosomal subunits in cultured Ehrlich ascites tumor cells analyzed by high-resolution CsCl isopycnic centrifugation consist of at least two classes of particles with densities of 1.57 g/cm3 (LI) and 1.59 g/cm3 (LII), respectively. A wash with 0.5 M KCl converts LI into particles with the density of LII particles. Incubation of derived large subunits (density 1.59 g/cm3) with 0.5 M KCl wash of reticulocyte ribosomes leads to the formation of particles with the density of LI particles. A protein with a molecular weight of 57000 present in the high-KCl wash of 60-S native subunits was virtually absent in the KCl wash of 40-S subunits or polyribosomes suggesting that specific protein factors may be present on some native 60-S subunits. Possible functions of these protein factors are discussed.