Polyamine metabolism in McCoy cells: I. Comparative studies of extracellular polyamine conjugated proteins of human fibroblast and McCoy cultures

polyamine conjugated proteins were identified in culture medium from both human skin fibroblasts and transformed mouse cells (McCoy cells). Sephadex G-100 column chromatography of medium allowed identification of two polyamine conjugated proteins from both types of cell cultures; one with Mr greater than 100,000 (MP1) and one with Mr = 60,000-70,000 (MP2). Human skin fibroblast MP1 contained putrescine and spermidine while MP1 from McCoy cultures contained putrescine, spermidine and spermine. MP2 isolated from both cultures contained all three polyamines. The relative concentration of polyamines in MP1 and MP2 for human fibroblasts and McCoy cells were different. The spermidine and spermine associated with MP1 and MP2 of McCoy cultures was covalently bound while for putrescine only 70.5% in MP1 and MP2 of McCoy cultures was covalently bound while for putrescine only 70.5% in MP1 and 74.5% in MP2 was covalently bound. The covalent nature of the polyamine protein conjugation was confirmed by autoradiography following isogel agarose isoelectric focusing. MP2 was resolved into three radiolabeled proteins with pI's between 5.25 and 5.20. Both MP1 and MP2 of McCoy cultures were heterogeneous. MP1 consisted of at least five proteins with Mr's of 180,000, 38,000, 76,000 and 68,000. The major protein (or proteins) had a pI of 5.25. MP2 consisted of at least three proteins with Mr's 72,000, 68,000 and 62,000; their pI's were between 5.20 and 5.25.     

Polyamine metabolism in McCoy cells: II. Cellular origin of excreted polyamine conjugated proteins

Incubation of McCoy cultures with medium containing 14C-putrescine resulted in the incorporation of 14C-polyamine into intracellular proteins. A greater than 100,000 dalton 14C-polyamine conjugated protein was present in the McCoy cell lysate supernatant (CLSP). CLSP was heterogeneous containing proteins with pIs ranging from 4.55 to 5.50. The major proteins had pIs of 4.55 and 5.20. Electrophoresis of solubilized McCoy cell lysate pellet revealed a 14C-polyamine conjugated protein peak with Mr approximately or equal to 70,000 (CLPP). Both CLSP and CLPP contained bound polyamine. The major CLSP polyamine was spermidine while spermine exceeded the other two polyamines (putrescine and spermidine) in CLPP. About 25% of the polyamines associated with CLSP and CLPP were covalently bound with the exception of CLSP putrescine where 62.1% was covalently bound. Results suggested the presence of a polyamine protein isopeptide bond in CLSP. Sephadex gel filtration of cultured medium resulted in the identification of two macromolecular polyamine-containing fractions (MP1, Mr greater than 100,000 and MP2, M = 60,000-70,000). Antibody raised in rabbits against a membrane-organelle preparation cross-reacted with Sephadex gel filtration derived MP1 but not with peak MP2 suggesting that MP1 but not MP2 might be a membrane constituent. Antibody raised against medium polyamine conjugated protein peak 2 (MP2) cross-reacted with the cell lysate supernatant indicating that MP2 was present in the cytosol. It did not cross-react with the cell lysate pellet preparation. Antibody against MP2 also formed a precipitation band with MP1 indicating that there might be a common antigenic site on MP1 and MP2.     

Different metabolic recycling of the lipid components of exogenous sulphatide in human fibroblasts.

Cultured human fibroblasts were fed with two differently labelled sulphatide molecules [one labelled on C-3 of the sphingosine (Sph) moiety [( Sph-3H]sulphatide), the second on C-1 of stearic acid [( stearoyl-14C]sulphatide)], and the intracellular metabolic fate of radioactivity was monitored. Incorporated radioactivity was almost all recovered in the total lipid extract, regardless of the labelling position of the added sulphatide; however, large differences in the level of incorporation occurred among labelled glycosphingolipids. For example, sphingomyelin was present as the major radiolabelled lipid after [Sph-3H]-sulphatide incubation, but was detectable only in trace amounts after [stearoyl-14C]sulphatide administration; in the latter case the radioactivity was located predominantly in glycerophospholipids. From this finding it can be inferred that the free long-chain base (sphingosine) that originates from lysosomal catabolism of sulphatide is mainly, and quite specifically, utilized for sphingomyelin biosynthesis, whereas the ceramide moiety is not; conversely the fatty acid released from ceramide is non-specifically re-utilized for phospholipid biosynthesis.     

Polyamine metabolism in Acanthamoeba: polyamine content and synthesis of ornithine, putrescine, and diaminopropane

Five polyamines which could be separated by high performance liquid chromatography were found in Acanthamoeba castellanii (strain Neff). These included in order of decreasing abundance: 1,3-diaminopropane, spermidine, spermine, norspermidine, and putrescine. Only diaminopropane and norspermidine had been found previously. Spermine was present in cultures grown in broth, but not in defined medium. Radioactive substrates were used to establish that putrescine was synthesized by decarboxylation of ornithine, ornithine was synthesized from arginine or citrulline, and diaminopropane was synthesized from spermidine. The presence of ornithine decarboxylase (EC 4.1.1.17), arginase (EC 3.5.3.1), and urease (EC 3.5.1.5) and the absence of arginine decarboxylase (EC 4.1.1.19) were established. A scheme for polyamine biosynthesis in A. castellanii is proposed.     

Subcellular metabolism of exogenous GM1 ganglioside in normal human fibroblasts

The metabolization of exogenous GM1 in normal human fibroblasts at a subcellular level is investigated in the present paper. For this a GM1 ganglioside, radiolabelled on the sphingosine moiety, was given to the cells and all the formed metabolites analyzed, in a time-course study, in enriched fractions of lysosomes, plasma membrane and microsomes. After feeding the cells, the radioactivity incorporation was relevant in the enriched lysosomal and plasma membrane subfractions whereas it was modest in the enriched microsomal fraction. The kinetic curves obtained for each enriched fraction, following a 3-day chase period, suggested a translocation of exogenous GM1 from the plasma membrane to the lysosomal apparatus and, of GM1 itself together with its metabolites, to the Golgi or endoplasmic reticulum and finally again to the plasma membrane.     

Comparative studies of glucose-fed and glucose-starved hamster cell cultures: responses in galactose metabolism.

The metabolic flow of trace amounts of D-[14C]-galactose was followed in cultures of transformed and untransformed hamster cells over a period ranging from five minutes to two hours. The results of chromatographic and enzymatic analyses of the soluble pools are described. Non-glycolytic cells(previously deprived of sugar periods of up to 24 hours) convert D-galactose to galactose-1-phosphate and uridine diphosphoglucuronic acid in 10 to 20 minutes. In the same short assay time, glycolytic cells which have been maintained for 24 hours in media containing glucose or galactose convert D-galactose to uridine diphsphogalactose and uridine diphosphoglucose (ratio 1.4:1). Long term diprivation of sugar also results in 3- to 4-fold increases in the uptake of galactose. In addition, the incorporation of galactose label into chloroformethanol soluble material appears to be influenced by the culture conditions of the untransformed cells while incorporation in the transformed cells appears unaffected. When cycloheximide is included in the maintenance medium for extended periods, the non-glycolytic cells also show increases in galactose uptake rates but the glucose-fed, glycolytic cells llose uptake ability. UDPhexose is the main galactose metabolic peak in the soluble pools of the cycloheximide-treated, glycolytic and the cycloheximide-treated, non-glycolytic cells. The results of these experiments suggests that uptake of galactose and its subsequent metabolism are under separate control.     

Cytotoxicity studies of human melanoma cells and fibroblasts.

In seven human melanoma cell lines and one human fibroblast strain some correlation of resistance to cell killing was found with two bifunctional alkylating agents (melphalan, chlorambucil) and three monofunctional agents (4(5)-(3,3-dimethyl-l-triazeno)imidazole-5(4)-carboxamide (DTIC), methylmethane sulphonate (MMS) and N-methyl-N1-nitro-N-nitrosoguanidine (MNNG), but little cross-resistance was found between these two groups of agents or with cytosine arabinoside (ara-C). In contrast to previous studies with rodent tumours, potentially synergistic (chloroquine, arginine) or antagonistic (ascorbic acid, leucine) compounds did not affect the toxicity of melphalan in a human melanoma cell line. In two melanoma lines DTIC induced patterns of DNA damage (inhibition of semi-conservative synthesis) and repair (strand breaks and repair synthesis) similar to, but not identical with, those induced by the methylating agent MMNG. These results suggest that a methylating species is derived from DTIC but has a different reactivity toward DNA compared with MNNG.     

Comparative studies on human skin fibroblasts: Life span and lipid metabolism in medium containing fetal bovine or human serum

Summary Three strains of human skin fibroblasts were cultivated in nutrient medium supplemented either with human serum or fetal bovine serum, and growth and lipid synthesis were compared. Rates of cellular growth were similar in both kinds of medium, but the replicative life spans of all three strains were curtailed significantly in human-serum medium. Incorporation of label into the major classes of neutral lipids from [¹⁴C]acetate and³H₂O was increased also in human-serum medium. Since human serum contained higher concentrations of cholesterol known to reduce endogenous cholesterol synthesis, these results were unexpected. Nonlipid factors in human serum may account for the shortened cellular life spans and increased lipogenesis and perhaps for the potential to develop atherosclerosis. Supported by grants from the Ontario Heart Foundation and Medical Research Council of Canada during the tenure of a Senior Research Fellowship from the Ontario Heart Foundation (J.T.C.) and a Scholarship from the Medical Research Council of Canada (S.G.).     

Comparative metabolic effects of fructose and glucose in human fibroblast cultures

Summary The comparative metabolic effects of fructose and glucose were determined in human fibroblast cultures. Cells were grown in four different media containing 5.5 and 27.5 mM of glucose and fructose, respectively. For these two hexoses, we compared their uptake, consumption, and conversion into¹⁴CO₂ and¹⁴C-lipids. D-Fructose was taken up in fibroblasts by an unsaturable process and its consumption was much smaller than that ofD-glucose. Whatever the experimental procedure, the glycogen content of cells grown in fructose media was significantly lower than of those grown in glucose media. Labeling of fructose and glucose with¹⁴C showed that more carbon from fructose than from glucose was incorporated into CO₂ and glycerolipids. The relative distribution of¹⁴C in the different lipid fractions was similar for both hexoses. These results indicated that the pathways of intermediary metabolism in fibroblast cultures were influenced by the nature of the carbohydrate present in the culture medium and that fructose was a better lipogenic substrate than glucose in human fibroblast cultures. This work was supported by grants for the Institut National de la Santé et de la Recherche Medicale (ATP 82-79-114).     

Studies on biotransformation of lysozyme. III. Comparative studies on biotransformation of exogenous and endogenous lysozyme in rats.

Exogenous hen lysozyme or endogenous rat lysozyme labeled with 131I was intravenously injected to rats with the same dosage, respectively, and the uptake and degradation of injected 131I-labeled rat lysozyme in liver and kidney were studied in comparison with those of 131I-labeled hen lysozyme. 1. Although the serum levels of both enzymes injected were almost indentical during the first 6 h, the liver uptake of 131I-labeled hen lysozyme was 2.2-fold more than that of 131I-labeled rat lysozyme at the peak time of 5 min after injection. The uptake and clearance of 131I-labeled rat lysozyme in the kidney were exclusively slow as compared with those of 131I-labeled hen lysozyme. 2. The intracellular distribution in the liver and kidney were examined by the differential centrifugation after injection of each lysozyme. The protein-bound radioactivity of each subcellular fraction was found to be the highest in the 12 000 X g (10 min) fraction in the liver and the 19 600 X g (20 min) fraction in the kidney. The relative specific activity of 12 000 X g fraction of the liver after injection increased with the time lapse. On the other hand, the relative specific activity of 105 000 X g (1 h) fraction of the liver attained a maximum within 5 min after injection and thereafter decreased. It was assumed that the mechanism of the uptake of injected 131I-labeled rat lysozyme in the liver and kidney was similar to that of 131I-labeled hen lysozyme. 3. The degradation of exogenous or endogenous lysozyme in subcellular particles was examined. From the effect of pH, activator and inhibitor on the degradation, the proteolytic enzyme to degrade the injected 131I-labeled hen lysozyme was indicated to be mainly cathepsin BL, with the optimal pH of about 5.0, and the injected 131I-labeled rate lysozyme was mainly degraded by cathepsin D, with the optimal pH of about 3.5 The in vitro degradation of exogenous and endogenous lysozymes showed a tendency similar to the in vivo clearance from the liver and kidney.     

Comparative metabolic studies on liver sinusoidal cells and different types of macrophages.

Sinusoidal cells of rat liver, rat peritoneal macrophages and rabbit alveolar macrophages were checked for their viability and compared with regard to cell weight, protein and DNA content. Glycogen was virtually absent from sinusoidal cells and peritoneal macrophages; alveolar macrophages contained glycogen whose level increased after activation by Freund's adjuvant and decreased during phagocytosis in the absence of glucose. Of the different nucleotides assayed, UDPglucose levels were low in the nonglycogen-forming cells, but quite high in alveolar macrophages. The capacity to metabolize galactose is much smaller in all cell types investigated than in hepatocytes.     

Comparative studies of the metabolic activation of chrysene in rodent and human skin

Metabolism and activation of chrysene was examined in mouse, rat and human skin using a short-term organ culture technique. Mouse skin released larger quantities of free dihydrodiols into the culture medium than either rat or human skin and greater quantities of chrysene metabolites became covalently bound to the DNA of mouse skin. The stereochemistry of the chrysene-1,2-diol that was formed by each skin type was examined using high-performance liquid chromatography (HPLC) with a chiral stationary phase to resolve the enantiomers. It was found that in each case the (-)-enantiomer predominated. When hydrolysates of DNA extracted from rodent or human skin that had been treated with 3H-labelled chrysene were chromatographed on Sephadex LH-20 columns, the elution profiles of the hydrocarbon-DNA adducts were found to vary between the species studied. Further examination using HPLC showed that some of the adducts formed in skin had the chromatographic characteristics of adducts formed when the anti-isomer of the 'bay-region' diol-epoxide of chrysene (r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene) reacted with DNA and that others had the characteristics of triol-epoxide adducts.     

Primary cultures of epithelial cells and long-term cultures of fibroblasts from the human umbilical cord: ultrastructural and immunocytochemical characterization

Repeated trypsinization of the full term human umbilical cord epithelium allows an homogeneous and exclusively epithelial primary culture, without fibroblastic growth. Transmission electron microscopy observations of desmosomes, cytokeratin intermediate filaments as revealed by indirect immunofluorescence and cultural aspects confirm the epithelial nature of this primary culture. Fibroblasts obtained by an explants culture method exhibit neither desmosomes nor cytokeratin intermediate filaments, which are epithelial markers. They yield characteristic long term cultures of fibroblastic aspect and growth.     

Comparative study of human chromosome replication in primary cultures of embryonic fibroblasts and in cultures of peripheral blood leucocytes

Replication of regions of chromosomes 1, 2, 3, 16, and group 4–5 was studied at the termination of the S period in primary cultures of embryonic fibroblasts (two embryos) and in cultures of peripheral blood (two women). Distinct differences were established in the pattern of late replication of the studied chromosomes in the cultures of the two types of cells. These differences consern first of all the centromeric and neighbouring regions of the chromosome. The content of late label in this region is 1.5–3 times higher in the cultures of fibroblasts than in the corresponding regions of leucocyte cultures. The difference is most pronounced in chromosomes 1, 3 and 16. It is suggested that the difference between cultures of these two types of cells in chromosome replication may be connected with the different genetic functioning of the centromeric and neighbouring regions in them. It is also possible that this difference is due to underreplication (or partial loss in an other way) of heterochromatin DNA of centromeric and neighbouring regions in leucocytes functioning for a long period without division.     

Comparative study of human chromosome replication in primary cultures of embryonic fibroblasts and in cultures of peripheral blood leucocytes

By autoradiography with ³H-thymidine and ³H-deoxycytidine it is shown that chromosomes 1 and 16 in cultures of embryonic fibroblasts at the termination of the S period synthesise AT- and GC-rich DNA at different rats: in both chromosomes the labelling of AT-bases is more intensive. In leucocyte cultures both nucleotide pairs label equally in these chromosomes. Chromosomes 2, 3, 4–5 and 21–22 are labelled equally in both cultures with respect to AT-and GC-pairs. Fibroblasts and leucocytes differ in the relative intensity of DNA synthesis at the end of the S period: chromosomes 1,16 and 21–22 contain more label in the case of fibroblasts (chromosome 1 solely due to AT-pairs) and chromosome 4–5 in the case of leucocytes. Analysis of distribution of late label along chromosome 1 showed that in fibroblast cultures the pericentromeric regions of both arms are labelled more intensively in respect to both nucleotide pairs than in leucocyte cultures. Both in fibroblast and leucocyte cultures no significant distinctions in the distribution of AT-and GC-pairs along chromosome 2 were established. In fibroblast cultures the pericentromeric regions of both arms of chromosome 3 are labelled more intensively than other regions. In leucocyte cultures the pericentromeric region of the short arm of this chromosome is labelled with the same intensively as in fibroblasts, whereas in the pericentromeric region of the long arm the intensity of incorporation of labelled synthesis precursors decreases. — Analysis of results obtained in the present study together with data of previous studied (Slesinger et al., 1974; Lozovskaya et al., 1976; Lozovskaya et al., 1977) shows that differences between the two types of cells in the intensity of late ³H-thymidine labelling in the C-heterochromatin regions of chromosomes 1 and 16 may be explained both by variation of replication time in leucocytes as compared with fibroblasts and by variation of the content of AT- rich DNA. Differences observed in other chromosomes are probably due to different times of replication of these chromosomes in leucocytes and fibroblasts. — Thus, the process of cell system differentiation involves not only differential activity of the genome (the main mechanism) that is connected with differences in the replication time of chromosomes and of their regions but also variation of the quantity of genetic material.     

Comparative study of human chromosome replication in primary cultures of embryonic fibroblasts and in cultures of peripheral blood leucocytes

Early and late replication of chromosomes in primary cultures of embryonic fibroblasts and in cultures of peripheral blood leucocytes were analysed using pulse and continuous incorporation of ³H-thymidine. In both types of cells chromosomes begin the synthesis period synchronously. The termination of the DNA synthesis of chromosomes is an asynchronous process and has peculiarities in every type of cells, chromosomes 1, 16, 21–22 are labelled relatively more intensively in fibroblast cultures than in leucocytes but the chromosomes of group 4–5, on the contrary, contain more grains in leucocyte cultures than in fibroblast cultures. It is highly probable that the discovered differences in late replication of the mentioned chromosomes in the investigated types of cells are connected with the differences in functioning of these chromosomes in the two different types of cell systems.     

Polyamine metabolism during the growth cycle of tobacco BY-2 cells.

We studied polyamine (PA) biosynthesis, oxidation and conjugation in asynchronously dividing cells of tobacco BY-2 cell suspension culture (Nicotiana tabacum L.) during 7-day growth cycle. We analyzed the levels of free and conjugated PAs and the activities of biosynthetic and catabolic enzymes during the subculture interval. The contents of free spermidine and spermine started to increase after the inoculation into the fresh medium, positively correlated with the mitotic activity of BY-2 cells and reached their maxima at the beginning of exponential phase on day 3. On the contrary, the endogenous level of free Put showed a transient decline in the lag-phase, and then increased till the end of exponential phase (day 5). The time-course of the content of PCA-soluble conjugates showed a trend similar to that of the free PAs. The inoculation of BY-2 cells into the fresh medium resulted in a sharp increase in the activities of ornithine decarboxylase (ODC, EC 4.1.1.17) and S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50). Arginine decarboxylase (ADC; EC 4.1.1.19) activity remained low during the whole subculture interval. The rise of diamine oxidase (DAO; EC 1.4.3.6) in the first day after subculture coincided with the decrease in free Put level. De novo synthesis of PAs in BY-2 cells after inoculation into the fresh medium and the participation of both PA conjugation with hydroxycinnamic acids and Put oxidative degradation in maintaining of free PA levels during the growth cycle are discussed.