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P O Seglen

Publications and source records attributed to P O Seglen.

At least 91 records · Page 5Linked to original sources
PubMed1986

Use of a hydrolysable probe, [14C]lactose, to distinguish between pre-lysosomal and lysosomal steps in the autophagic pathway.

[14C]Lactose, introduced into the cytosol of isolated rat hepatocytes by means of electropermeabilization, is sequestered autophagically in the same way as the established sequestration probe, [14C]sucrose. However, unlike the inert sucrose molecule, lactose is rapidly hydrolysed in the lysosomes, and can therefore be used to probe the last step of the autophagic pathway (i.e. fusion with the lysosome). During autophagy lactose is present only at a low, steady-state level in pre-lysosomal vacuoles (probably autophagosomes), serving as a useful marker for these organelles. If autophagosome-lysosome fusion is blocked with vinblastine (Kovács et al., Exp cell res 137 (1982) 191), [14C]lactose will accumulate continuously as a function of the sequestration rate, and reach a high level in the pre-lysosomal vacuoles. Density gradient analysis, using chloroquine (CLQ) to alter lysosomal density, suggests that these organelles have a broad density distribution (1.08-1.13 g/ml), thus differing significantly from the distribution of lysosomes.

Adenine↗
PubMed1986

Increase in cis-dichlorodiammineplatinum (II) cytotoxicity upon reversible electropermeabilization of the plasma membrane in cultured human NHIK 3025 cells.

A series of brief electrical high-voltage discharges were given to cultured NHIK 3025 cells to render the plasma membrane transiently permeable to drugs. Using [14C]sucrose as an inert marker which normally does not cross plasma membranes, increased permeability could be demonstrated for no longer than 10 min following electrical treatment, indicating that the permeabilization was entirely reversible. The reversibility of the treatment was further demonstrated by a lack of effect on cell growth and colony-forming ability. When cells were given electrical discharges immediately before or during exposure to cis-dichlorodiammineplatinum(II)(cis-DDP) the cytotoxic drug effect increased. By using electrical discharges during a 2 hr drug treatment period the cytotoxicity was enhanced to an extent corresponding to at least a 3-fold increase in drug uptake relative to unpermeabilized cells. This increase in drug uptake was confirmed by direct measurements of the amount of cell-associated Pt by atomic absorbtion spectroscopy. The results suggest that uptake across the plasma membrane may be the rate-limiting factor in the cytotoxic effect of cis-DDP. Furthermore, the methodology applied in the present study may prove useful in assessing the influence of membrane permeability on the effect of other cytotoxic drugs.

Cell Division↗
PubMed1986

Characterization of hepatocytes from carcinogen-treated rats by two-parametric flow cytometry.

Hepatocytes in the adult rat are normally mostly tetraploid, but sequential carcinogen treatment (diethylnitrosamine/2-acetylaminofluorene) induces the development of a predominantly diploid hepatocyte population. By two-parametric flow cytometry (simultaneous measurement of DNA and protein content within the same cell) it could be shown that diploid hepatocytes from carcinogen-treated rats had only half the size (protein content) of the tetraploid cells from the same liver, but the same size as diploid hepatocytes from normal rats.

2-Acetylaminofluorene↗
PubMed1986

Changes in cellular ploidy and autophagic responsiveness during rat liver carcinogenesis.

Liver carcinogenesis was initiated in young rats by diethylnitrosamine/partial hepatectomy and promoted by dietary 2-acetylaminofluorene (for 4 weeks). Eight weeks after initiation, hepatocytes were isolated by means of collagenase perfusion and analyzed by means of flow cytometry. Whereas cells and cell nuclei from normal or hepatectomized livers were predominantly tetraploid, most of the hepatocytes/nuclei from carcinogen-treated rats were diploid. Neoplastic liver nodules and hepatocellular carcinomas also contained almost exclusively diploid nuclei, suggesting that diploidization may be an essential feature of liver carcinogenesis. Two-parametric analysis (simultaneous flow cytometric determination of DNA and protein content within the same cell) revealed that the diploid cells were only half as big as the tetraploid cells. They could therefore be separated from the latter by centrifugal elutriation. Normal, isolated hepatocytes responded to amino acid deprivation by increasing their rates of autophagic sequestration (measured with electroinjected (14C)sucrose as a probe) and endogenous protein degradation, the resulting protein loss eventually leading to cell death. Hepatocytes from carcinogen-treated rats were much less responsive to amino acid deprivation, preserved their protein better, and survived for longer periods of time in culture than did normal cells. The reduced autophagic responsiveness may conceivably give carcinogen-altered cells a survival advantage even in vivo, that could contribute to their outgrowth during carcinogenesis.

2-Acetylaminofluorene↗
PubMed1986

Isolation of carcinogen-induced diploid rat hepatocytes by centrifugal elutriation.

The majority of hepatocytes isolated from rats treated with carcinogens (diethylnitrosamine plus 2-acetylaminofluorene) were found to be diploid, whereas most of the hepatocytes from normal rats are tetraploid. The carcinogen-induced diploid hepatocytes were only one-half the size (protein content) of the tetraploid hepatocytes, and could therefore be separated from the latter by centrifugal elutriation. The elutriation technique thus makes it possible to isolate a relatively pure fraction of carcinogen-induced cells. The diploid cells had the same liver-specific enzymatic and functional properties as the tetraploid cells and were thus undoubtedly of hepatocytic origin.

Animals↗
PubMed1986

Use of electrical methods in the study of hepatocytic autophagy.

Electropermeabilisation--i.e. subjecting cells to a short series of high-voltage pulses- has been used to make isolated rat hepatocytes transiently permeable to small molecules. The permeabilised cells can be loaded with [14C]sucrose at 0 degrees C and resealed by a brief incubation at 37 degrees C. The electrical treatment does not impair cellular function, since autophagic sequestration of sucrose occurs at the same rate as in untreated cells. The latter are permeable to sucrose immediately after isolation, due to the presence of hyperpermeable surface blebs similar to those formed in response to electrical treatment. In order to separate [14C]sucrose-sequestering organelles from cytosol, the cells are broken up by electrodisruption--i.e. a single high-voltage pulse given in a non-ionic medium (isotonic sucrose)--and the resulting cell corpses are sedimented through a dense metrizamide/sucrose cushion to form a cytosol-free pellet containing intact lysosomes. Since mitochondria also take up sucrose, the radioactivity in autophagosomes/lysosomes must be selectively extracted with a low concentration of digitonin. The resulting procedure provides a specific bioassay of autophagic sequestration. The sequestration is virtually completely inhibited by the autophagy inhibitor 3-methyladenine, and at temperatures below 20 degrees C. The sequestration step is energy-dependent, and can probably account for the known energy requirement of autophagic-lysosomal protein degradation.

Animals↗
PubMed1986

Radiolabelled sugars as probes of hepatocytic autophagy.

The sugars [14C]sucrose, [14C]lactose and [3H]raffinose have been loaded into isolated rat hepatocytes by means of electropermeabilisation, and used as probes of autophagic-lysosomal function. All three sugars are sequestered autophagically; in addition sucrose and lactose are taken up by mitochondria, and lactose is hydrolysed intralysosomally. Autophagically sequestered radioactivity accumulates in relatively large vacuoles (not in microsomes). These vacuoles initially have a density which, on average, is lower than that of the lysosomes. With time, the lysosomes become lighter and coincident with the radioactive vacuoles in density gradients, a phenomenon that is partially prevented by the autophagy inhibitor 3-methyladenine (3MA). This may indicate that resting, dense lysosomes become active (light) by engaging in autophagy as well as in other processes (endocytosis?). To assess the extent of convergence of the autophagic and endocytic pathways, the effect of endocytosed invertase on autophagically sequestered sucrose has been investigated. Practically all of the sucrose is eventually degraded the enzyme, indicating a complete intermixing of autophagic and endocytic pathways at the level of the lysosome. The density of lysosomes can be increased by treatment of cells with leupeptin. Most of the vaculoes containing sequestered radioactivity also increase their density under these conditions, even in the presence of vinblastine, an inhibitor of autophagosome-lysosome fusion. This indicates that a substantial fraction of the autophagosomes may be physically associated with lysosomes prior to fusion, a hypothesis supported by electron microscopic observations. Both autophagic sequestration (of raffinose) and autophagic-lysosomal degradation (of lactose, measured by an HPLC method) is completely inhibited by 3MA, whereas endocytic-lysosomal degradation (of asialofetuin) is unaffected. Since endogenous protein degradation is only partially (70%) suppressed by 3MA, it is clear that the remaining degradation must be non-autophagic (non-sequestrational), and most likely non-lysosomal.

Animals↗
PubMed1985

Use of digitonin extraction to distinguish between autophagic-lysosomal sequestration and mitochondrial uptake of [14C]sucrose in hepatocytes.

In isolated rat hepatocytes, electroinjected [14C]sucrose is sequestered both by mitochondria and by autophagosomes/lysosomes. Radioactivity can be selectively extracted from the latter organelles by low concentrations of digitonin, thereby providing a specific bioassay for autophagic sequestration. By including a digitonin extraction step in the assay procedure, autophagic [14C]sucrose sequestration could be shown to be virtually completely (greater than 90%) suppressed by the autophagy inhibitor 3-methyladenine (10 mM), whereas mitochondrial sugar uptake was unaffected. An amino acid mixture likewise suppressed autophagic sequestration very strongly, while having no detectable effect on the mitochondria.

Adenine↗
PubMed1985

Autophagic-lysosomal and mitochondrial sequestration of [14C]sucrose. Density gradient distribution of sequestered radioactivity.

[14C]Sucrose, introduced into the cytosol of isolated rat hepatocytes by means of electropermeabilization, was sequestered by sedimentable subcellular particles during incubation of the cells at 37 degrees C. The sedimentation characteristics of particle-associated [14C]sucrose were different from the lysosomal marker enzyme acid phosphatase, suggesting an involvement of organelles of greater size than the average lysosome. Isopycnic banding in isotonic metrizamide/sucrose density gradients resolved two major peaks of radioactivity: a light peak (1.08-1.10 g/ml) coinciding with lysosomal marker enzymes, and a dense peak (1.15 g/ml), coinciding with a mitochondrial marker enzyme. The dense peak was preferentially associated with large-size particles having the sedimentation properties of mitochondria, and it was resistant to the detergent digitonin at a concentration which extracted all of the radioactivity in the light peak. Similarly the autophagy inhibitor 3-methyladenine prevented accumulation of [14C]sucrose in the light peak, while the radioactivity in the dense peak was unaffected. We therefore tentatively conclude that the light peak represents autophagic sequestration of [14C]sucrose into lysosomes (and probably autophagosomes) while the dense peak represents a mitochondrial uptake unrelated to autophagy.

Acid Phosphatase↗
PubMed1985

Cell cycle traverse and protein metabolism in human NHIK 3025 cells: the role of anchorage.

We have studied the effect of cell anchorage on the human cell line NHIK 3025 in vitro, to see whether the growth regulating effect of cell anchorage primarily affected DNA division cycle or mass growth cycle. It was found that cell to cell anchorage had the same effect on cell cycle progression as anchorage to a solid surface, which indicates that it is anchorage per se and not cell shape that is important for growth control in NHIK 3025 cells. When NHIK 3025 cells were grown without attachment to a solid surface, both G1 and cell cycle duration was prolonged by 6 h, which means that the prolonged cell cycle was due to a prolonged G1. During the first part of the cell cycle the rate of protein synthesis and degradation was constant, and at the same level in cells grown with and without attachment. This means that the prolonged G1 was not due to a reduced protein accumulation or mass growth. Towards the end of the cell cycle protein accumulation was reduced. This effect was either due to a size control before cell division or a secondary effect of the prolonged G1. We therefore conclude that cell anchorage as a growth regulator primarily affects the DNA/cell division cycle.

Cell Adhesion↗
PubMed1985

Conversion of dense lysosomes into light lysosomes during hepatocytic autophagy.

Incubation of isolated rat hepatocytes under conditions which support maximal autophagy (amino acid-free medium) caused a marked alteration in the density distribution of lysosomes in continuous metrizamide gradients (mean peak density reduced from 1.14 to 1.09 g/ml). The autophagic sequestration inhibitor 3-methyladenine (3MA) partially prevented the density shift, presumably by stopping the formation of light autophagosomes which otherwise fuse with dense lysosomes and thereby alter the lysosomal density.

Adenine↗
PubMed1985

Reduced autophagic activity, improved protein balance and enhanced in vitro survival of hepatocytes isolated from carcinogen-treated rats.

Sequential carcinogen treatment (diethylnitrosamine/partial hepatectomy followed by 2-acetylaminofluorene (2-AAF] induced multiple hepatocarcinomas in rats with 100% certainty within a year. Enzyme-altered lesions, i.e. gamma-glutamyltranspeptidase (GGT)-positive and/or ATPase-negative cell foci, were numerous already at 8 weeks, and suspensions of purified hepatocytes isolated (by collagenase perfusion) at this time contained 30-40% GGT-positive cells. These hepatocyte suspensions were markedly deficient with respect to autophagic protein degradation (in comparison with cell suspensions from normal rats), and the cells lost less protein and survived much better than normal hepatocytes in culture under conditions of amino acid deprivation (which activates the autophagic mechanism). The anabolic advantage of reduced autophagy may possibly contribute to the selective outgrowth of preneoplastic cells during the earliest stage of liver carcinogenesis. Inclusion of the autophagy inhibitor 3-methyladenine in the culture medium elevated the survival of normal hepatocytes up to the level seen with hepatocytes from carcinogen-treated animals, suggesting that protection of normal cells by autophagy suppression may be a potentially interesting therapeutic principle.

2-Acetylaminofluorene↗
PubMed1985

Autophagic sequestration of [14C]sucrose introduced into isolated rat hepatocytes by electrical and non-electrical methods.

Isolated rat hepatocytes were found to become permeable to [14C]sucrose at 0 degree C under three different conditions: Immediately following their liberation from the collagenase-perfused liver. Following a short incubation under hypoxic conditions. After electropermeabilisation. All three conditions were characterised by the formation of small protuberances (blebs) indicative of localised cell surface damage, and it is possible that the stretched plasma membrane of such blebs acted as a high-permeability region. Disappearance of blebs and restoration of normal plasma membrane impermeability could be achieved by a short (15 min) incubation at 37 degrees C. It could be shown that [14C]sucrose introduced into rat hepatocytes by non-electrical means was autophagically sequestered at the same rate as [14C]sucrose introduced electrically. In both cases the sequestration was inhibited by the specific autophagy inhibitor 3-methyladenine to a similar extent. The subcellular distribution of sequestered isotope in metrizamide/sucrose density gradients was found to be independent of the conditions of its introduction into cells.

Adenine↗
PubMed1984

Trapping of electro-injected [14C]sucrose by hepatocyte mitochondria: a mechanism for cellular autofiltration?

[14C]Sucrose was introduced, by means of electropermeabilization, into the cytosol of isolated rat hepatocytes to serve as an inert marker of the intracellular fluid. Radioactivity accumulated in mitochondria (as well as in lysosomes) at a rate of 2-3%/h for 4 h of incubation at 37 degrees C, as shown by subcellular fractionation in isotonic density gradients. This unprecedented mitochondrial trapping of a molecule which does not cross most biological membranes points to the possibility that mitochondria participate in some kind of autofiltration of cellular fluid.

Acyltransferases↗
PubMed1984

Amino acid control of protein degradation in normal and leukemic human lymphocytes.

Lymphocytes from normal human subjects and from patients with chronic lymphocytic leukemia were found to degrade their endogenous protein at similar rates (2.5-3.0%/h) when incubated in an amino acid-free buffer. Protein degradation was inhibited 20-35% by inhibitors of autophagic sequestration (amino acids, 3-methyladenine) and by inhibitors of intra-lysosomal proteolysis (leupeptin, propylamine), the extent of inhibition being similar in normal and leukemic lymphocytes. The inhibitor effects, together with the electronmicroscopic demonstration of autophagosomes in the lymphocyte cytoplasm, is taken as evidence for the existence of an autophagic-lysosomal pathway in human lymphocytes, potentially responsible for as much as one-third of their overall protein degradation.

Adenine↗