Density dependent regulation of growth in suspension cultures of L-929 cells

Suspension cultures of L-929 fibroblasts grown to densities of 6 to 10 × 10⁶ cells/ml through daily centrifugation and resuspension in fresh media, have been maintained for periods up to five months without change in viability or cell size. DNA synthesis and mitosis in these cultures is limited to 5% of the cells per day, a fraction very nearly equal to the fraction of cells rendered nonviable, most likely during the manipulations associated with medium renewal. The kinetics of the flow of cells into the S and M periods following (a) renewal of the medium and (b) dilution of the high density cultures, suggest that the large majority of the cells are in a G₀ or early G₁ phase, resuming growth readily in response to decreased cell density. This is further indicated by the sequence of the marked shifts occurring in the cell volume distribution spectrum of the high density cultures after dilution. Long term, steady state regulation of growth with retention of intact viability was thus demonstrated in the case of a long established aneuploid cell line. The fact that this occurs in suspension but not in attached cultures, supports the concept that impairment of growth control in such cells affects predominantly regulatory mechanisms located at the cell surface rather than those concerned with intracellular synthesis and metabolism.     

Density dependent regulation of growth in L-cell suspension cultures. IV. Adaptive and nonadaptive respiratory decline

Respiration as an index of oxidative energy production was investigated in a L-cell suspension culture system previously shown to exhibit density-dependent inhibition of growth. It was found that as cultures progressed from exponential growth to high density nongrowing populations (6^?10 × 10⁶ cells/ml) over a 2-week period, the respiratory rate determined from the total amount of oxygen consumed during the daily medium renewal cycle, declined from 5.4 to 1.8 fmoles O₂/cell/min. There are two components in this decrement. The first consists of a daily recurrent decline of oxygen uptake resulting from decreased availability of medium oxygen and glutamine and is readily reversed by medium supplementation. The second component which is refractory to medium supplementation and accounts for approximately 50% of the total respiratory decline, is considered to indicate an adaptive change of the respiratory capacity of the cells. This change is reversed during the lag period which precedes resumption of exponential growth upon subculture to low cell densities. The significance of these results is discussed in relation to recent reports indicating a marked depression of respiratory activity in nongrowing dense attached cultures as well.     

Purine glucosylating activity in cell suspension cultures ofSolanum tuberosum L.

Cell suspension cultures ofSolanum tuberosum L. cv. Adretta were established from leaf-derived calluses. In the search for purine glucosylating activity, the metabolism of 6-benzylaminopurine was studied. The main metabolite of BA was isolated and identified as 6-benzylaminopurine 7--d-glucopyranoside indicating the occurrence of purine glucosylating activity.Abbreviations BA 6-Benzylaminopurine - [3G]BA BA 3--d-glucopyranoside - [7G]BA BA 7--d-glucopyranoside - [9G]BA BA 9--d-glucopyranoside - RA Radioactivity - R _T Retention Time     

3H-GABA uptake and acetylcholinesterase activity in dissociated cell cultures of the medial hypothalamus.

Medial hypothalamic tissue of 1 to 4 days old rats was dissociated and cultured in vitro for 8--10 days. Neuronal perikarya were demonstrated by supravital methylene blue staining and electron microscopy. Synapses with typical vesicles and subsynaptic thickening were also observed. 3H-GABA was taken up into a small percentage of the cells in the cultures. Neuron-like perikarya and long processes accumulated the label while many neurons contained much less activity. Some astroglial and oligodendroglial cells and processes also accumulated GABA. A few neurons in these cultures contained acetylcholinesterase. It is concluded that neurons concentrating GABA and containing acetylcholinesterase are present in the hypothalamus of rats of 1 to 4 days of age and can be maintained in dissociated cell culture.     

Density dependent inhibition of cell growth in cultures of primary and established lines of cells

The effect of cell crowding on DNA synthesis (incorporation of ³HTdR and ³²PO₄) was studied by an improved method in monolayers of secondary cells and established cell lines, either normal or transformed by viruses or carcinogens. The method was based mainly on pulse labeling of cultures of cells a few hours after their seeding in equal numbers onto areas of different size in identical dishes, a condition which ensured equal physiological conditions and different degrees of crowding of cells. DNA synthesis was hardly inhibited in crowded monolayers of secondary chick, mouse and hamster embryo cells. The incorporation of radioactive thymidine and phosphate into DNA of cell lines such as BHK 21, 3T3/SV40 and L929 was strongly inhibited. An SV40-transformed line of hamster kidney cells (HKT7) synthetized DNA equally well in sparse as in crowded monolayers. In lines of human amnion (FL) and BHK 21 cells which were more extensively studied the degree of inhibition of DNA synthesis was inversely proportional to their density. Autoradiography after ³HTdR pulse-labeling indicated that the same proportion of cell nuclei were labeled in sparse and in crowded cultures. The extent of labeling (number of grains per nucleus) was lower in crowded cultures of those cells that also showed inhibition of incorporation of this label as measured by scintillation. The inhibition is thus expressed in retardation of DNA synthesis in cells in S phase rather than arresting it in a larger percentage of cells.     

Oil body formation in Euphorbia tirucalli L. cell suspension cultures

The culture of cells of Euphorbia tirucalli L. resulted in the formation of intracellular particles which osmium tetroxide staining and electron microscopy indicated to be oil bodies. Chemical analysis (TLC, HPLC, GLC and GC-MS) disclosed steroids, triterpenoids, and diterpenoids as the major oil components of the cultured cells.     

5,6-Dichlorobenzimidazole-1-beta-D-riboside, specific inhibitor of cytokinin activity in tobacco cell suspension cultures

Tobacco cell suspension cultures were supplemented with 4 to 10 microM 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DBR). The cell suspensions were cultured in the presence or without a cytokinin, kinetin (N6-furfuryladenine). The responses of the cultures to DRB and (or) kinetin were measured by the evolution of cell mass or cell counts in the cultures, as a function of time or kinetin concentration. Chlorophyll biosynthesis was also used as a test-response to cytokinin and (or) DRB activities. It was concluded that DRB behaves as a specific inhibitor of the physiological responses to cytokinins. The evolution of the inhibition ratio versus kinetin concentration shows that DRB is not a competitor of the cytokinins.     

Methyltransferase activity in Allanthm altissima cell suspension cultures

Enzymes for the methylation of 1hydroxycanthin-6-one and a series of coumarins have been isolated from Ailanthus altissima cell suspension cultures. The coumarin methyltransferases methylate aesculetin to scopoletin and isoscopoletin, but not scopoletin, to scoparone. Fraxetin was methylated to isofraxidine but not to fraxidine and only fraxidine was methylated to 6,7,8-trimethoxycoumarin. These enzymes were studied throughout the culture growth cycle with two cell lines: 1, which produced 1-methoxycanthin-6-one as the major alkaloid and 2, in which canthin-6-one was the major alkaloid.Abbreviations UV ultraviolet - DEAE diethylaminoethyl - dw dry weight - MS mass spectrometry - NMR nuclear magnetic resonance - TLC thin layer chromatography     

Induction of phenoloxidase in cell suspension cultures of Mucuna pruriens L.

The effects of limitating nitrogen-containing compounds in the medium and of adding the amino-acid analogues p-fluorophenylalanine and ethionine on both phenoloxidase activity and the accumulation of L-3,4-dihydroxyphenylalanine (L-DOPA) are reported for cell suspension cultures of Mucuna pruriens. Nitrogen limitation of the cultures, or the addition of p-fluorophenylalanine or ethionine to the culture medium resulted in an increased phenoloxidase activity. There appeared to be an inverse relationship between phenoloxidase activity and the acccumulation of L-tyrosine into L-DOPA by alginate-entrapped cells occurred at a higher rate when phenoloxidase activity was increased.Abbreviations pFPA p-fluorophenylalanine - L-DOPA L-3,4-dihydroxyphenylalanine     

Polysaccharide production in liquid cell suspension cultures of Phleum pratense L.

The production of carbohydrates by cell suspension cultures of Phleum pratense (timothy grass) is described. Extracellular polysaccharides similar in monosaccharide composition to native cell wall polymers were accumulated, together with polymers of fructose (fructans). The fructans had similar properties to the intracellular reserve polymers found in intact plants, and were found in both cells and media of young, slow-growing cultures.Production of extracellular polysaccharides differed in cultures grown on sucrose or equimolar glucose/fructose as carbon source. These differences were observed only when autoclaved media were used, and were not related to changes in either pH or osmolarity. Autoclaving medium containing radioactive glucose and fructose produced a novel, unidentified labelled compound which was absent in medium containing labelled sucrose.     

Dissimilation of methionine in cell suspension cultures from Catharanthus roseus L.

Plant cell suspension cultures from Catharanthus roseus were investigated for their capability to dissimilate methionine or its analogs in order to reutilize the sulphane group for cysteine biosynthesis. Three steps have been described as prerequisites of this process: (a) oxidative degradation by the amino-acid oxidase of methionine giving rise to methanethiol production; (b) demethylation by methyltransferases leading to homocysteine and S-methylmethionine (c) replacement of the homocysteine sulphane sulphur by alkylthiol yielding methionine and free hydrogen sulphide. A reversal of the cystathionine pathway as a source of cysteine was ruled out because the cells lack cystathionine γ-lyase. The absence of this enzyme is compensated by the S-alkyl exchange of homocysteine with methylmercaptan. Hydrogen sulphide thus liberated is used for de novo synthesis of cysteine. The complete pathway can be catalyzed by the constitutive set of enzymes present in the higher plant.     

Influence of 2-deoxy-D-glucose upon growth and invertase activity of carrot (Daucus carota L.) cell suspension cultures

Carrot (Daucus carota L.) cell suspension cultures grew well when provided with glucose, fructose, sucrose or raffinose. Galactose and melibiose supported less growth unless supplemented with glucose or fructose. In combination with ten different sugar mixtures, 2-deoxy-D-glucose (dGlc) inhibited culture growth. Inhibitory effects of dGlc were more marked with fructose, melibiose, raffinose or mixtures of these sugars in the culture medium. The presence of glucose or galactose reduced the inhibitory effects of dGlc on culture growth. Experiments with radioactive labelled sugars demonstrated that dGLc uptake was greater in the presence of fructose than glucose, and that growth inhibition of dGlc coincided with its uptake. Reduced protein content was also associated with the inhibitory effects of dGlc. Cultured cells contained lower levels of invertase (EC 3.2.1.26) activity during the active phase of culture growth (up to 25 days after subculture) than when growth had peaked and subsequently declined. Acid and alkaline invertase activities were not greatly reduced by exogenous hexoses. Invertase activity was greatest during periods of low protein content in all cultures and was inhibited by dGlc during the latter phases of the culture period. Free intracellular sugars throughout the culture period consisted mainly of glucose and fructose.     

Studies on tissue cultures of the genus Cinchona L. alkaloid production in cell suspension cultures

Initiation and culture of callus and cell suspensions of Cinchona ledgeriana and C. succirubra as well as the successful isolation and selection of a high-yielding alkaloid-forming strain derived from the leaf rachis of a C. succirubra plant are described. Results of feeding experiments with L-tryptophan using two different culture procedures are presented and discussed. Maximum alkaloid yields of up to 0.9% (based on dry weight) or 6.35 mg/l have been obtained.     

Production of 5-methoxypodophyllotoxin in cell suspension cultures of Linum flavum L.

Cell suspension cultures of Linum flavum L., routinely grown on a NAA-containing medium, accumulated low levels of the phenylpropanoid-derived lignan 5-methoxypodophyllotoxin (5-MPT), up to 0.004% on a dry weight basis. Feeding experiments with the precursor L-phenylalanine resulted in a 3–5-fold increase in 5-MPT levels, but caused the levels of PAL activity to fall. Treatment of the cultures with the elicitor Nigeran, either alone or in combination with phenylalanine, caused the 5-MPT production to cease, even though PAL activity was rapidly enhanced by these treatments. Transfer of the cultures to NAA-free medium resulted in a 40–50 fold higher level of 5-MPT accumulation, the PAL activity levels being lowered compared to the routinely grown cells. With these more differentiated cultures, phenylalanine feeding and elicitor treatment, both on its own and in combination with the precursor, had no effect on 5-MPT production, even though the PAL activity levels were higher than in the untreated cells. It can be concluded that in lignan-accumulating cultures of L. flavum, PAL activity is nearly always detectable and seems to show a reciprocal relationship with 5-MPT accumulation.Abbreviations 5-MPT 5-methoxypodophyllotoxin - PAL phenylalanine ammonia lyase (EC 4:3:1.5) - NAA naphthaleneacetic acid     

Effects of boron deficiency in cell suspension cultures of Populus alba L.

Cell suspension cultures of Populus alba L. (original cells) require at least 10 M boron for appropriate growth. Using original cells we established a cell line, T-5B, which can grow in a medium containing low levels of boron (5 M). The level of boron localized in the cell walls of T-5B cells was one-half that found in the cell walls of original cells maintained in medium containing 100 M boron, and the level of the rhamnogalacturonan II dimer, cross-linked by a borate ester, also decreased in the former. The sugar composition of whole cell walls of the T-5B cell line was similar that of the original cells, however pectic polysaccharides composed of arabinose or galacturonic acid were easily extracted from T-5B cell walls with 50 mM trans-1,2-cyclohexanediamine-N,N,N,N-tetraacetic acid. Our results suggest that boron deficiency causes a weakening of the interaction among pectic polysaccharides due to a decrease in boron-rhamnogalacturonanII cross-linkage.     

Elicitation studies in cell suspension cultures of Cannabis sativa L.

Cannabis sativa L. plants produce a diverse array of secondary metabolites. Cannabis cell cultures were treated with biotic and abiotic elicitors to evaluate their effect on secondary metabolism. Metabolic profiles analysed by ¹H NMR spectroscopy and principal component analysis (PCA) showed variations in some of the metabolite pools. However, no cannabinoids were found in either control or elicited cannabis cell cultures. Tetrahydrocannabinolic acid (THCA) synthase gene expression was monitored during a time course. Results suggest that other components in the signaling pathway can be controlling the cannabinoid pathway.     

Characterization of anthocyanic vacuolar inclusions in Vitis vinifera L. cell suspension cultures

Anthocyanic vacuolar inclusions (AVIs) are intra-vacuolar structures capable of concentrating anthocyanins and are present in over 50 of the highest anthocyanin-accumulating plant species. Presence of AVIs alters pigment intensity, total anthocyanin levels, pigment hue and causes bathochromic shifts in a spatio-temporal manner within various flowers, vegetables and fruits. A year-long study on Vitis vinifera cell suspension cultures found a strong correlation between AVI prevalence and anthocyanin content, but not the number of pigmented cells, growth rate or stilbene content. Furthermore, enhancement of the prevalence of AVIs and anthocyanins was achieved by treatment of V. vinifera cell suspension cultures with sucrose, jasmonic acid and white light. A unique autofluorescence of anthocyanins was used to demonstrate microscopically that AVIs proceed from the cytosol across the tonoplast and were able to coalesce intravacuolarly, with fewer, larger AVIs predominating as cells mature. Purification and characterisation of these bodies were performed, showing that they were dense, highly organic structures, with a lipid component indicative of membrane-encasement. These purified AVIs were also shown to comprise long-chain tannins and possessed an increased affinity for binding acylated anthocyanins, though no unique protein component was detected.