Hexagonal array of subunits in tight junctions separated from isolated rat liver plasma membranes.

Solubilization of isolated rat liver plasma membranes in 1% deoxycholate and centrifugation yielded a fraction (pellet) that consisted mainly of tight junctions (zonulae occludentes). An hexagonal array of subunits similar to that previously found in a number of the unfractionated plasma membranes was demonstrated in all the membrane sheets of these preparations by negative staining. It is concluded that the hexagonal subunit pattern is present in the tight junctions, and that this structural differentiation may be related to the intercellular diffusion afforded by the junctional membrane.

Distribution and antibody-induced redistribution of a mammary tumor virus-induced and a normal antigen on the surface of mouse leukemia cells.

By means of the indirect membrane immunofluorescence test, the distribution and antibody-induced redistribution (patching and capping) of a mammary tumor virus-induced (MLr) and a normal (Thy 1.2) cell-surface antigen were compared on mouse thymocytes and leukemia cells (GRSL2). At 0 degrees C Thy 1.2 fluorescence was ringlike and more intense on GRSL2 cells than on thymocytes, whereas MLr fluorescence on GRLS2 cells at this temperature was patchlike and brighter than Thy 1.2 fluorescence. At 20 or 37 degrees C, capping of Thy 1.2 on both cell types was readily achieved but MLr capping occurred only in a few GRSL2 cells and was less pronounced. However, after addition of the secondary antibodies, MLr capping was markedly increased by gradual cooling of cells to about 17 degrees C. Conversely, after addition of antibodies at 0 degrees C, gradual warming of cells under the fluorescence microscope resulted in extensive capping both of MLr and Thy 1.2 at approximately 13-14 degrees C. Rapid cooling or rapid warming led to almost instantaneous capping. These results may be explained by the occurrence of phase transitions or phase separations in the particular temperature range. Another difference between capping of Thy 1.2 and MLr was that the former caps were small and eventually were endocytosed, whereas the MLr caps were large and were exfoliated from the cells.

Multi-hit kinetics of tumor formation, with special reference to experimental liver and human lung carcinogenesis and some gneral conclusions.

On the basis of the multi-hit concept of cancer formation, the relationships between tumor incidence and dose and time of administration of carcinogen have been analyzed. Simple mathematics have been used, since the available data, in our opinion, hardly justify more sophisticated formularization. The exponential relationship between the cumulative tumor incidence and the dose and time of administration of carcinogen can be described as l(d,t) approximately dmtr. With use of Druckrey's formula, dtn=k, it was derived that the exponent of time, r, is equal to m-n, in which m is the power of the dose dependency of tumor formation [l(d) approximately dm measured at a fixed time] and n is the autonomous time factor featured in the former formula. The factor m is interpretable in terms of the number of discrete events (hits) required for tumor formation, whereas the factor n is mainly determined by the rate of proliferation of intermediate cell populations participating in the carcinogenic process. Since r and n can be experimentally determined, the formula allows the calculation of the exponent (m) of the dose dependency of tumor formation. Analysis of malignant liver tumor formation in the rat by continuous administration of diethylnitrosamine yeilded m = 7, from which it was concluded that seven hits were instrumental in the induction of these liver-cell tumors. Analysis of the formation of less malignant liver tumors after one pulse exposure to the same carcinogen suggested that the process was initiated by at most two concomitant hits in a liver cell and brought to completion by three spontaneous events (changes). The view was advanced that tumor formation in general may result from hits inflicted by the carcinogen applied and from "background" hits (i.e., spontaneous changes and/or hits by carcinogenic stimuli from the environment or present endogenously) and that the relative contribution of these two types of hits to the end effect may depend on dose level and potency of the carcinogen under consideration. It was pointed out that the direct measurement of the dose-response relation (l(d) approximately dm) yields only the number of hits contributed by the carcinogen applied anose rate is low or very low, the contribution of background process becomes significant, and these hits contribute to the power of time, r, of the incidence-time relation. Under these conditions, the formula m-n=r becomes (mex + mb)n=r, where mex and mb denote the number of hits scored by extrinsic carcinogen and background processes, respectively. It is argued that the epidemiological data on lung cancer caused by smoking [l(d) approximately d with respect to smoke dose, mex= 1; l(t) approximately t5 with respect to duration of smoking] are not compatible unless at least one additional background hit (mb greater than or equal to 1) is postulated...

Kinetics of induction and growth of enzyme-deficient islands involved in hepatocarcinogenesis.

A single application of various hepatocarcinogens to rats results in the formation of islands of enzyme-deficient liver cells, which are mainly irreversible and very probably represent the first cellular stage involved in the process of liver cancer formation. Comparison of the island-size distributions obtained for different carcinogens indicated that proliferation is a common property of islands that is independent of the inducing carcinogen and does not need any further presence of carcinogen or other stimulating factors. Toxic doses resulted in all cases in an enhanced island size. The number of islands induced by a single dose of carcinogen was enhanced by a prior partial hepatectomy only in the case of the dialkylnitrosamines, dimethylnitrosamine, and diethylnitrosamine. Dose-response relationships measured with diethylnitrosamine, the carcinogen with the lowest toxicity as compared with the carcinogenic action, indicated that island formation is due to a one-hit process, i.e., that one specific alteration in the target cell is responsible for the precancerous transformation. These kinetics and the low probability of transformation might indicate that the crucial hit is scored at the genetic level. The irreversible action of carcinogen (memory effect) and the influence of time on cancer formation (time effect) are discussed in terms of induction and proliferation of irreversible cell populations serving as precursor of the cancer cell. The number of specific alterations (hits) involved in the development of the malignant cancer cell is also briefly discussed.

Influence of mouse major histocompatibility complex (H-2) on N-ethyl-N-nitrosourea-induced tumor formation in various organs.

The influence of the major histocompatibility complex (MHC) of the mouse (H-2) on carcinogen-induced tumorigenesis was investigated. Mice of five H-2 congenic strains on the C57BL/10 background were treated with the direct-acting carcinogen N-ethyl-N-nitrosourea at the age of 15 days, and examined for tumors when moribund. Significant differences between strains in susceptibility to N-ethyl-N-nitrosourea-induced tumors in lung, small intestine, and liver were found. For lung tumors the strains B10.A and 2R were most susceptible, the strains 4R and B10 were relatively resistant. The strain 5R was intermediate. Susceptibility to small intestine tumors was highest in the strain 2R, intermediate in the strain B10.A, the strains 4R, 5R, and B10 were relatively resistant. The location of the tumors in the intestine was also affected by H-2. In the strain 2R most tumors are located in the proximal part, in 4R in the distal part. Tumorigenesis in the liver was highest in the strain 2R, intermediate in the strains B10.A, 4R, and B10, and lowest in the strain 5R. We conclude that susceptibility to carcinogen-induced tumors in the lung, small intestine, and liver in congenic strains on the C57BL/10 background is H-2 haplotype dependent. Susceptibility to tumors in the lung and intestine has a similar strain distribution, but differs from that for liver tumors. Males were more susceptible than females in the strain B10 (lung tumors) and 4R (small intestine and liver tumors). This indicates haplotype- and organ-specific, sex-related influences on tumor development. The possible mechanism(s) of H-2 effects on chemically induced tumorigenesis are discussed. Apart from the well-known immunological functions of the MHC, the involvement of hormonally related effects of the MHC is considered as well.

Quantitation of virus-induced (MLr) and normal (Thy.1.2) cell surface antigens in isolated plasma membranes and the extracellular ascites fluid of mouse leukemia cells.

Plasma membranes were isolated by two methods from mouse leukemia cells containing mammary tumor virus-induced (MLr) and normal (Thy.1.2) antigens on their surfaces. A number of chemical components, enzymic activities, and the antigenic contents were determined in subcellular fractions and found to be specifically concentrated in the plasma membrane fractions. The major part of the cellular MLr, in contrast to Thy.1.2, was present in the 105,000 X gmax supernatant of the cell homogenate. This and other results indicate an easy release of the antigen from the plasma membrane. A considerable amount of MLr was also present in the ascites fluid, partly free and partly bound, supposedly in an immune complex that allowed the isolation of three components of similar molecular weights as mammary tumor virus components. The extracellular presence of MLr may illustrate that, by shedding of antigen, the tumor may protect itself against the immunological defense of the host.

Separation of intact rat hepatocytes and rat liver nuclei into ploidy classes by velocity sedimentation at unit gravity.

A system is described which permits the separation of isolated hepatocytes and isolated rat liver nuclei belonging to different ploidy classes by velocity sedimentation at unit gravity. The problem of obtaining single cells suspensions is discussed and preparations were obtained that contained 96% single hepatocytes. By improving the sedimentation method, it took 2.5 h to separate rat liver nuclei on sucrose gradients into diploid and tetraploid ploidy classes. Recoveries were generally over 95%. The diploid band was 99% pure. DNA and protein content of the ploidy classes were measured. After partial hepatectomy and [3H]thymidine injection it was found that the label moved largely into the tetraploid compartment. Isolated hepatocytes were fractionated in 1 h on Ficoll gradients. Erythrocytes were separated from small nucleated cells and the population of hepatocytes was clearly separated from these two cell populations. Diploid hepatocytes were 80% and tetraploid hepatocytes were 99% pure. Viability was about 80% after fractionation. The gene dosage of NADPH cytochrome c reductase, succinate dehydrogenase and lactate dehydrogenase was estimated in diploid and tetraploid hepatocytes. Gene dosage was equal in diploid and tetraploid hepatocytes for succinate dehydrogenase and NADPH cytochrome c reductase. It is suggested, after correcting for non-viable tetraploid hepatocytes, that the gene dosage of lactate dehydrogenase was significantly lower in diploid than in tetraploid hepatocytes.

Fluorescence polarization measurements on normal and tumour cells and their corresponding plasma membranes.

Using 1,6-diphenyl-1,3,5-hexatriene as a probe, the degree of fluorescence polarization (P) at 25 degrees C of intact and disrupted cells and isolated plasma membranes were compared for a variety of systems. 1. Human erythrocytes, mouse thymocyte and leukemia cells, rat liver and hepatoma cells, and human and mouse milk fat globules displayed P values ranging from 0.300 to 0.120. 2. P values or probe labelling rates of intact and disrupted cells were similar. 3. As compared with whole or disrupted cells, the higher to much higher P values of plasma membranes isolated from the corresponding cells showed only a limited mutual variation. 4. delta P values, being the difference in P values between plasma membranes and whole cells were attributed to the extent to which endomembranes and non-membrane lipids contributed. Among these, triglycerides had the greatest relative effect. 5. Though a particular isolation procedure for plasma membranes may select for more rigid fragments, this effect is by far not sufficient to account for the observed delta P values. It is concluded that the fluorescence polarization technique with a lipophilic probe applied to whole cells represents a measure of the average fluidity of all lipids being present in a cell and thus does not exclusively monitor the cell surface membrane.

Comparative lipid analysis of purified plasma membranes and shed extracellular membrane vesicles from normal murine thymocytes and leukemic GRSL cells.

The lipid fluidity in purified plasma membranes (PM) of murine leukemic GRSL cells, as measured by fluorescence polarization, is much higher than in PM of normal thymocytes. This was found to be due to relatively low contents of cholesterol and sphingomyelin and a high amount of unsaturated fatty acyl chains, especially linoleic acid, in the phospholipids. PM from GRSL cells contain markedly more phosphatidylethanolamine than those from thymocytes. For both GRSL cells and thymocytes the detailed lipid composition of isolated PM was compared with that of the corresponding shed extracellular membranes (ECM), which were isolated from the ascites fluid and from thymus cell suspensions, respectively. The somewhat decreased lipid fluidity of thymocyte ECM as compared to their PM, can be ascribed to the increased cholesterol/phospholipid molar ratio (0.88 vs. 0.74). No other major differences were found between the lipid composition of these membranes. In contrast, significant differences were found between PM and ECM from GRSL cells. In this system a much lower lipid fluidity of the shed ECM was found, due to the much increased cholesterol/phospholipid molar ratio (3.5-fold) and sphingomyelin (9-fold) content, as compared to the PM. Further, the ECM contain relatively more lysophosphatidylethanolamine and less phosphatidylcholine and -inositol. ECM contain a higher amount of polyunsaturated fatty acids, especially in the phosphatidylethanolamine and lysophosphatidylethanolamine classes. On the other hand, the fatty acids of phosphatidylcholine and lysophosphatidylcholine are more saturated than in PM. In particular, ECM of GRSL cells contain less oleic and linoleic acid residues and more arachidonic acid and 22:polyunsaturated fatty acid residues than PM. The possible relevance of these differences with respect to the mechanism of shedding of vesicles from the cell surface, is discussed.

Elongation factor 2 as the target of the reaction product between sodium selenite and glutathione (GSSeSG) in the inhibiting of amino acid incorporation in vitro.

The product of the reaction between sodium selenite and glutathione, designated as selenodiglutathione (GSSeSG), nearly completely inhibits amino acid incorporation from [14C]leucyl-tRNA by free polyribosomes isolated from rat liver. The mechanism of this inhibition was studied on the basis of the following three findings. Glutathione decomposes GSSeSG to harmless products; GSSeSG acts instantaneously on some component of the complete incubation system during preparation of the incubation vessels (at 0 degrees C); once GSSeSG has reacted its inhibitory effect cannot be reversed by glutathione. Accordingly, the effect of GSSeSG on the various steps of the amino acid incorporation process was studied by varying the sequence of additions of the reaction components, GSSeSG and GSH. The results of these and other experiments showed elongation factor 2 to be target of GSSeSG. The GSSeSG-B blocked factor could be regenerated by reduction with glutathione reductase and NADPH.

Studies on plasma membranes. XXII. Fatty acid profiles of lipid classes in plasma membranes of rat and mouse livers and hepatomas.

Plasma membranes were isolated from rat and mouse livers, a transplanted rat hepatoma, two transplanted mouse hepatomas and spontaneous mouse hepatomas. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophosphatidylcholine, free fatty acids and triglycerides were separated and their fatty acid profiles determined. The various lipid classes of rat and mouse liver plasma membranes each demonstrated more or less specific fatty acid profiles. The number of double bonds decreased in the order: phosphatidylserine greater than or equal to phosphatidylethanolamine greater than phosphatidylinositol greater than phosphatidylcholine greater than or equal to sphingomyelin and lysophosphatidylcholine. Small species differences were noted in most lipid classes. A marked sex difference was observed in sphingomyelin of mouse liver membranes but in none of the phospholipids of rat liver membranes. The increased cholesterol content of all hepatoma versus liver plasma membranes was accompanied by a decrease of fatty acyl poly-unsaturation in most lipid classes of the rat hepatoma but not of the mouse hepatoma membranes. The fatty acid profiles of the mouse hepatoma membranes deviated much less from those of mouse liver than did the pattern of rat hepatoma versus rat liver. The results were discussed in relation to lipid fluidity of which fatty acyl unsaturation and cholesterol are the main parameters.

Formation by diethylnitrosamine and persistence of O4-ethylthymidine in rat liver DNA in vivo.

The formation by diethylnitrosamine (DEN) and persistence of O4-ethylthymidine in rat liver DNA in vivo has been studied using enzymic hydrolysis of DNA, cation exchange column chromatography and thin-layer chromatography (TLC). The amount of O4-ethylthymidine represented about 1% of the total ethylation; its half-life in vivo was 19 (range 16--24) days, the same value as obtained for O2-ethylthymidine. The persistence of O2- and O4-ethylthymidine, rather than the rapid removal of O6-ethylguanine, favours the former miscoding base adducts as relevant molecular lesions in rat liver carcinogenesis.

Stem cell carcinoma in the small intestine of mice treated transplacentally with N-ethyl-N-nitrosourea: some quantitative and histological aspects.

A high incidence of tumours of the small intestine occurred in mice of the B10.A/SgSnA strain treated transplacentally with N-ethyl-N-nitrosourea (ENU). In these adenocarcinomas, histologically different tumour cells which resembled the 4 cell types of the normal intestinal epithelium were present. Since normal intestinal epithelial cells are thought to originate from common stem cells, the tumours seem to be derived from these stem cells.

Phospholipid unsaturation and plasma membrane organization.

A comparison has been made between the unsaturation of plasma-membrane phospholipids,present in the human erythrocyte, rat liver, mouse liver and a rapidly growing rat hepatoma. Of the double bonds present in the hydrocarbon chains of the membrane phospholipids,onethird is contributed by sphingomyelin plus phosphatidyl choline and the remainder by phosphatidyl serine, ethanolamine and inositol. Assuming that the phospholipids are asymmetrically distributed in the two leaflets of the bilayer in general, the consequences of this asymmetry in combination with cholesterol content and fatty acid distribution on plasma membrane organization and function are discussed. It is suggested, that the organizational disposition of plasma membrane components other than phospholipids is at least related if not dependent upon the latter's asymmetric distribution in the bilayer.

Studies on plasma membranes. XXIII. Hormone-like action of concanavalin A on liver plasma membranes: inhibition of (Na+-K+)ATPase.

Concanavalin A inhibits the (Na+-K+)-ATPase activity of isolated rat-liver plasma membranes, while leaving the Mg2+-ATPase unaffected. Glucagon and cyclic AMP act supplementary to the lectin in the inhibition. The lectin effect is counteracted by insulin and L-epinephrine, and is completely abolished by the beta-adrenergic blocking agent propranolol. Results are discussed on the basis of the known interactions of concanavalin A with plasma membrane components, including its hormone-like action.

Studies on lung tumours. III. Oxidative metabolism of dimethylnitrosamine by rodent and human lung tissue.

The oxidative metabolism of the carcinogen dimethylnitrosamine (DMN) was studied in mouse, rat, hamster and human respiratory tissue. [14C]DMN was purified by Dowex-1-bisulfite column chromatography to remove a contaminant (probably [14C]formaldehyde) interfering with the enzyme assay. Since formaldehyde and methyl carbonium ions - yielding methanol with water - are considered to be the primary products of DMN metabolism, tissue slices were assayed for the production of [14C]CO2 from 14C-labelled methanol, formaldehyde, formate, and DMN. Oxidation of formaldehyde to formate was not, but oxidation of formate to CO2 was very much rate-limiting. This rate-limiting step was circumvented by introducing quantitative chemical oxidation of formate to CO2 by mercury(II)chloride following the enzymic reaction. Since oxidation of methanol to CO2 proved to be insignificant, production of CO2 from DMN by lung tissue enzymes and HgCl2 may serve as a parameter for N-demethylating activity and the production of the suspected carcinogenically active methyl carbonium ions. The DMN-N-demethylating activities of lung tissue slices of two mouse strains with widely different susceptibilities to formation of lung adenomas by DMN differed significantly, but the difference seemed too small to explain the divergence in tumourigenic response. The enzymatic activities decreased in hamster bronchus, hamster trachea, hamster lung, GRS/A mouse lung, C3Hf/A mouse lung, human lung, Sprague-Dawley rat lung, in that order. The reported resistance of the hamster respiratory system to tumour induction by DMN may therefore not be due to poor DMN-N-demethylating capacity.

Studies on lung tumours. IV. Correlation between [3H]thymidine labelling of lung and liver cells and tumour formation in GRS/A and C3Hf/A male mice following administration of dimethylnitrosamine.

Male mice of the inbred strain GRS/A are highly susceptible to lung tumour but refractory to liver tumour formation, whereas the opposite relation holds for C3Hf/A male mice. Liver and lung cells of these 2 mouse strains were studied autoradiographically after intraperitoneal injection of [3H]dimethylnitrosamine (DMN) and of [3H]thymidine at days 1--14 after administration of unlabelled DMN. Corresponding cell types in the lungs or livers of these 2 mouse strains bound similar amount of [3H]DMN. Among the various types of lung cells only the alveolar Type II cells, from which the lung adenomas derive, showed a strain-specific difference in [3H]thymidine labelling indices, much more cells becoming labelled in the case of the GRS/A than of the C3Hf/A strain at days 3--7 after carcinogen administration. Opposite thymidine labelling indices were exhibited by the parenchymal liver cells of the 2 strains, with C3Hf/A now showing a greater response than did GRS/A males. Thus thymidine-labelling and tumourigenic responses of target lung and liver cells to carcinogen in the 2 strains coincided. Sulphur dioxide and carbon tetrachloride mimicked the effects of DMN on the thymidine labelling indices of, respectively, the lung alveolar Type II and the thymidine labelling indices of, respectively, the lung alveolar Type II and the liver parenchymal cells of the 2 strains. The nature of the differential effect of carcinogen on the [3H]thymidine labelling of the cells and the correlation of these patterns with susceptibility to tumour formation, are briefly discussed.