Suppression of Ehrlich ascites tumors in mice by minute virus of mice.

As a model system to study parvoviral oncosuppression, Ehrlich ascites (EA) cells were injected into the peritoneal cavities of ICR mice, and the effect of im injection of minute virus of mice (MVM) on EA tumor growth was examined. Coinjection with MVM resulted in a dramatic inhibition of EA tumor formation. Tumor suppression required viable, infectious virus. Mice that had survived one EA-MVM coinjection acquired long-term resistance to additional injections of EA cells.

Inter- and intramolecular interactions of highly purified Rous sarcoma virus-transforming protein, pp60v-src.

We have purified intact pp60v-src, the product of the Rous sarcoma virus src gene, over 2400-fold, based on the phosphorylation of tumor-bearing rabbit IgG. The purification procedure involved detergent extraction of the particulate fraction of the cells and sequential chromatography on hydroxylapatite, butyl agarose, DEAE-Sephacel, ADP-agarose, and Sephacryl S-200. Analysis of the preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single silver-stained band with an apparent molecular weight of 60,000. Our results show that the activities of this preparation were qualitatively similar to those described previously for partially purified pp60v-src. Upon analysis by two-dimensional gel electrophoresis, the purified pp60v-src yielded one major species which migrated to the same position as the least acidic of the three major species detectable in cellular lysates, suggesting that the pp60v-src had been dephosphorylated during the purification procedure. We found that pp60v-src was very prone to aggregation; to maintain it as a monomer both Nonidet P-40 and KCl were required. Under conditions which maintained pp60v-src as a monomer, the rate of autophosphorylation was independent of its concentration and thus proceeded via an intramolecular process. Preincubation of pp60v-src with ATP or GTP as well as nonphosphorylating analogs of ATP or GTP preserved its phosphorylating activity toward alpha-casein whereas its activity was reduced 80% upon preincubation in the absence of nucleotides. We suggest that protection with nucleotides rather than autophosphorylation accounts for the apparent increase in the activity of pp60v-src after incubation of the enzyme with ATP.

cAMP-independent protein kinase activity is correlated with growth of rat mammary tumors.

An important portion of the protein kinase activity in the 7,12-dimethylbenz(a)anthracene (DMBA) induced rat mammary tumour is inhibited by the bioflavonoid quercetin (10(-4) M). By partial purification on a DEAE Cellulose column it was shown that the quercetin-inhibitable enzyme activity can be eluted in a separate peak which contains markedly reduced cAMP-dependent protein kinase activity. Since quercetin does not affect the cAMP-dependent protein kinase activity, this drug becomes a potent tool for the quantitation of this special activity in the tumor. By hormonal manipulation, namely ovariectomy and estrogen treatment, it was shown that changes in the growth rate of the tumour were closely correlated with the magnitude of these special protein kinase activities. These results suggest a possible cause-and-effect relationship between cyclic AMP-independent protein kinase activity and tumour malignancy in this chemically induced tumor. These data are similar to recent findings in viral-induced malignant transformation.

The effect of quercetin on the phosphorylation activity of the Rous sarcoma virus transforming gene product in vitro and in vivo.

The phosphotransferase activity of the Rous sarcoma virus src gene product, pp60src, was inhibited both in vitro and in vivo by the bioflavonoid quercetin. The Ki for the inhibitory effect was in the range of 6-11 microM under conditions in vitro. The inhibitory effect of quercetin was competitive towards the nucleotides ATP and GTP as substrates for pp60src and was non-competitive towards alpha-casein as the protein substrate of this kinase activity. In contrast, studies in vitro of the phosphotransferase activity of the catalytic subunit of the cAMP-dependent protein kinase showed that this flavonoid did not inhibit the phosphorylation of physiological substrates of this enzyme. In cultured cells the half-maximal inhibition of tyrosine phosphorylation of pp60src as well as the phosphorylation of the Mr = 34000 protein, a physiological substrate of pp60src, was in the range 0.06-0.08 mM.

Characterization of the Rous sarcoma virus transforming gene product.

This report describes quantitative results of in vitro phosphorylation of the Rous sarcoma virus transforming gene product, pp60src, using ATP or GTP as phosphate donors. The Km values for the phosphorylation of pp60src by ATP and GTP were similar (10-36 and 25-36 microM, respectively) and the Vmax values were different (5-7 and 1.5-1.7 nmol min-1 mg-1 of pp60src, respectively). The radiolabeling of pp60src by [gamma-32P] ATP was inhibited by ADP and dATP at 20-fold higher concentrations by 75 and 83%, respectively. Other nucleotides served as weaker inhibitors under the same conditions. The radiolabeling of pp60src by [gamma-32P]GTP had lower specificity for this nucleotide, since ATP, dATP, ADP, dGTP, GDP, and TTP had at least a 50% inhibitory effect. The phosphorylated products of approximate Mr = 60,000 that were produced with ATP or GTP were shown to be the same protein molecule since they both could be immunoprecipitated with antibody raised against p60src produced by bacterial recombinants. Structural analysis revealed that the use of GTP resulted in phosphorylation of a tyrosine residue on the COOH-terminal region of pp60src, apparently the same site which contains the tyrosine phosphorylated in infected cells. In contrast, the use of ATP resulted in phosphorylation of several additional tyrosine residues on the NH2-terminal region of the molecule. In thermolability studies, the t1/2 values for the phosphorylation of pp66src in preparations from wild type virus-infected chicken cells were 5.1 min for both ATP and GTP, whereas the t1/2 values for the phosphorylation of pp60src in preparations from temperature-sensitive transformation mutant-infected cells were 1.1 min for both phosphate donors. Similar observations were found with alpha-casein as substrate.

Evidence that the Rous sarcoma virus transforming gene product is associated with glycerol kinase activity.

This communication provides biochemical, immunological, and genetic evidence that pp60src, the Rous sarcoma virus transforming gene product, is associated with glycerol kinase activity. Our investigations demonstrated that the compound phosphorylated by pp60src or by glycerol kinase (EC 2.7.1.30) from Candida mycoderma share the same electrophoretic and chromatographic mobilities. The glycerol kinase and protein kinase activities of pp60src were inhibited similarly by preincubation with immune IgG. Both activities were reduced 6-9-fold in pp60src preparations derived by immunoaffinity chromatography from cells which were infected with NY68, a temperature-sensitive transformation mutant of Rous sarcoma virus. The thermolability at 41 degrees C of the glycerol kinase activity of pp60src from the mutant virus-infected cells was greater (t/2 = 1.3 min) than the same activity in pp60src preparations from wild type virus-infected cells (t/2 = 4.8 min).

Protein kinase activity in liver of heat-acclimated hamsters.

Protein kinase activity in incubated liver slices from 35 degrees C heat-acclimated (HA) hamsters was 70% higher than in similar slices from 23 degrees C control (C) hamsters. Adding glucagon to the incubation medium increased protein kinase activity by 65% in slices from C animals, but by only 30% in slices from HA animals. Binding of [3H]cAMP to proteins of a low-speed supernatant fraction of incubated and homogenized slices was 30% lower for HA than for C hamsters. For each acclimation group this binding was reduced 30% by incubation of the slices with glucagon. The activities of phosphorylase kinase, phosphorylase phosphatase, and phosphorylase alpha in slices incubated with or without glucagon did not differ between groups. Addition of glucagon increased phosphorylase kinase by 30% and phosphorylase alpha by 40% but caused no change in phosphorylase phosphatase activity. These results suggest that heat acclimation of the hamster increases the amount of a species of liver protein kinase that is different from the one that mediates the effect of glucagon on glycogenolysis.

Regulation of protein kinases activity by quercetin in Ehrlich ascites tumor cells.

Cyclic AMP-independent protein kinase activities from Ehrlich ascites tumor cells, partially purified by DEAE-cellulose and phosphocellulose chromatography were inhibited by quercetin. The cyclic AMP in the tumor ascites cells and the cyclic AMP-dependent protein activity from this tumor and from bovine and mouse tissues were unaffected by this drug. Since we reported that quercetin elevates cyclic AMP level in Ehrlich ascites tumor cells, this bioflavonoid may have a dual effect on the protein kinase activities in these cells, thus, increasing the cyclic AMP-dependent and decreasing the cyclic AMP-independent protein kinase activities.

The effect of glucagon and prostaglandin E1 on cyclic AMP levels in liver of heat exposed hamsters.

Cyclic AMP levels in liver slices of hamsters exposed to 35 degrees C for 21 days and controls maintained at 22 degrees C was found to be similar in basal conditions. Glucagon (10 microgram/ml) caused 3.5 times elevation of cyclic AMP levels in control hamsters and 9 times elevation in 35 degrees C exposed hamsters, thus a difference of 150% of the nucleotide concentration was found between the two experimental groups. When 10(-2)M theophylline was added, the cyclic AMP levels were 80% higher in 35 degrees C exposed hamsters both in the presence and absence of 10 microgram/ml glucagon. The difference between controls and heat exposed animals was found to be the same when various concentrations of both glucagon or prostaglandin E1 were added to the liver slices. Adenylate cyclase activity was similar in both experimental groups, while low Km phosphodiesterase was significantly less active in the liver of 35 degrees C exposed animals when compared to the controls.

Effect of liposome-encapsulated quercetin on DNA synthesis, lactate production, and cyclic adenosine 3':5'-monophosphate level in Ehrlich ascites tumor cells.

Quercetin (3,3',4',5,7-pentahydroxyflavone) entrapped within phospholipid vesicles inhibited [3H]thymidine incorporation and lactate production in Ehrlich ascites tumor cells. The presence of serum proteins in concentrations of up to 40 mg/ml did not alter the extent of the inhibitory effect of the liposome-entrapped flavone. At the same concentrations, serum proteins eliminated almost completely the inhibitory effects of the free quercetin on these metabolic processes. Prolonged sonication and different lipid compositions of the liposomes did not increase the inhibition of [3H]thymidine incorporation by the entrapped quercetin in the presence of serum albumin. On the other hand, serum albumin eliminated the elevation of the cyclic adenosine 3':5'-monophosphate level in these cells produced by both free and liposome-entrapped flavone. Attachment between Ehrlich ascites tumor cells and quercetin-loaded liposomes is demonstrated in negatively stained electron micrographs. The liposomes ranged in size from 50 to 100 nm. After 10 min of incubation, these liposomes were distributed randomly on the cell surface at distances of 100 to 200 nm.

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.

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.

A rapid effect of kinetin on rehydration of tobacco leaf tissue.

Partly dehydrated tobacco leaf tissue (Nicotiana rustica), stripped of the lower epidermis, was used to study the effect of kinetin on the rate of rehydration. Depending on the rate of rehydration in untreated tissue, kinetin either increased or decreased rehydration rates. The response to kinetin was very rapid and could be discerned in less than 2 minutes. On extensive dehydration, the tissue lost the capacity to respond to kinetin. Salinity stress, which decreases the endogenous level of cytokinins in the plant, conditions the leaf to stimulation of rehydration by kinetin.It is suggested that cytokinins may play a role in controlling water permeability of the leaf tissue.

Water permeability of bilayer lipid membranes: Sterol-lipid interaction.

Bilayer lipid membranes were generated in an aqueous medium from synthetic, egg or plant phosphatidyl choline (PC) or from plant monogalactosyl diglyceride (MG). The water permeability of the black membranes was determined by measuring the net volume flux produced by a NaCl gradient. The osmotic permeability coefficient,P os, was markedly affected by the number of double bonds in the fatty acid conjugates of the lipids: the greater the degree of unsaturation, the higher the value ofP os. The temperature dependence ofP os of the lipid membranes was studied over a range of 29 to 40°C. The experimental activation energy,E a , estimated from the linear plots of log (P os)versus 1/T, was significantly higher for MG membranes (17 kcal/mole) than for the various PC membranes (11 to 13 kcal/mole), probably owing to hydrogen bonding between MG and water molecules. In comparison with PC membranes, the membranes generated from PC and cholesterol (1∶1 molar ratio) had lowerP os but similarE a values. Likewise, either stigmasterol or ß-sitosterol decreasedP os of MG membranes, whileE a was not affected by the sterols. MG-cholesterol membranes were specifically characterized by a unique value ofE a (-36 kcal/mole) thus indicating temperature dependent structural changes.