The cultivation of animal cells at controlled dissolved oxygen partial pressure

A 3-liter culture vessel has been developed for the growth of animal cells in suspension at controlled pH and dissolved oxygen partial pressure (pO₂). The culture technique allows metabolically produced CO₂ to be measured; provision can be made to control the dissolved CO₂ partial pressure. In cultures containing a low serum concentration, gas sparging to control pO₂ was found to cause cell damage. This could be prevented by increasing the serum concentration to 10%, or by adding 0.02% of the surface-active polymer Pluronic F68. The growth of mouse LS cells in batch culture without pO₂ control was found to be limited by the availability of oxygen. Maximum viable cell populations were obtained when dissolved pO₂ was controlled at values within the range 40–100 mm Hg.     

Poly (ADP-ribose) polymerase activity during the growth cycle of mouse fibroblasts (LS cells).

The specific activity of poly (ADP-ribose) polymerase in isolated nuclei of mouse fibroblast cells (LS cells) was estimated throughout the growth cycle. The activity of this enzyme increased approx. 3-fold during the logarithmic phase of the cell population growth and was correlated with the increase in cell number. Upon dilution of the culture, the specific activity dropped, over 12–24 h, approx. 3-fold, to the new low level. This fluctuation in enzyme activity is unlike that of other metabolic enzymes in LS cells. It is not a result of changes in the medium. The specific enzyme activity during the growth cycle is not correlated with the DNA content of the cells. The physiological function of poly (ADP-ribose) polymerase is discussed in relation to these results.     

Lys-tRNA4 and cell division : Changes in lys-tRNA during the growth of mouse L cells

The presence of one isoaccepting tRNA species, lys-tRNA₄, has been correlated with the ability of mammalian cells to carry out cell division. The levels of this tRNA were measured during the growth of mouse L cells in culture. RPC-5 profiles of the lys-tRNA population of mouse L cells were determined at 9 points during the growth of mouse L cells in culture. The level of lys-tRNA₄ was constant at 13–14% of the total lys-tRNA population during log growth, increased to 18% when the cells became confluent and then returned to 13–14% when the cells were held at confluency. These results are in contrast to other cell lines where lys-tRNA₄ decreased at confluency. Confluent cultures of mouse L cells retained full proliferative potential since the addition of medium containing 30% serum led to renewed exponential growth. Another lys-tRNA species, lys-tRNA_5A, was present in variable amounts depending upon the nutritive state of the cells. The appearance of lys-tRNA_5A correlated with a decrease in lys-tRNA₅. These data suggest that lys-tRNA_5A is a modified lys-tRNA₅ which appears when the medium becomes depleted.     

Lys-tRNA4 levels and cell division in mouse 3T3 cells

tRNA₄^lys is an isoaccepting tRNA^lys which has been proposed as a necessary requirement for cell division in mammalian cells. We have measured the levels of this tRNA^lys during the growth cycle of mouse 3T3 fibroblasts. High levels of tRNA₄^lys were seen throughout exponential growth. However, a marked decrease in tRNA₄^lys occurred 24 h before the cells became confluent. This decrease was observed in three different 3T3 cell lines, but was not seen in a transformed 3T3 cell line. Trypsinization and replating of contact-inhibited cells returned tRNA₄^lys to the levels characteristic of exponential cells. Data from these and other cell lines show a direct relationship between the levels of tRNA₄^lys and the growth rate of cells in culture.     

The cultivation of animal cells at controlled dissolved oxygen partial pressure. Reprinted from Biotechnology and Bioengineering Vol. X, Issue 6, Pages 801-814 (1968)

A 3-liter culture vessel has been developed for the growth of animal cells in suspension at controlled pH and dissolved oxygen partial pressure (pO(2)). The culture technique allows metabolically produced CO(2) to be measured; provision can be made to control the dissolved CO(2) partial pressure. In cultures containing a low serum concentration, gas sparging to control pO(2) was found to cause cell damage. This could be prevented by increasing the serum concentration to 10%, or by adding 0.02% of the surface-active polymer Pluronic F68. The growth of mouse LS cells in batch culture without pO(2) control was found to be limited by the availability of oxygen. Maximum viable cell populations were obtained when dissolved pO(2) was controlled at values within the range 40-100 mm Hg.     

Alterations in growth requirements of kidney epithelial cells in defined medium associated with malignant transformation

The possibility has been investigated that (1) the supplements required for the growth of the Madin Darby Canine Kidney (MDCK) cell line in serum-free Medium K-l are indeed requirements for the growth of normal kidney cells in vitro, and (2) that alterations in these growth requirements are associated with malignant transformation. Consistent with the hypothesis that MDCK cells resemble normal kidney cells in culture, primary cultures of baby mouse kidney epithelial cells grow in Medium K-l and respond to the 5 components in the-medium. The growth properties of Moloney sarcoma virus (MSV)-transformed MDCK cells in defined media have been examined. Unlike MDCK cells, MSV-transformed MDCK cells form tumors in adult nude mice. Although they still respond to the 5 factors in Medium K-l, the optimal dosage for insulin is lower for the MSV transformants than for MDCK cells. The MSV transformants also have an additional requirement for growth in Medium K-l – fibronectin. Variants of MDCK cells have been isolated that have lost the PGE₁ requirement for growth in defined medium. These variant cells have acquired (1) the ability to form tumors in adult nude mice and (2) an alteration affecting cAMP metabolism, in addition to PGE₁ independence.     

Modification of glutamate- effects on neuroblastoma cells in culture by heavy metals

Sodium L-glutamate inhibited the growth (due to inhibition of cell division and cell death) of mouse neuroblastoma (NB) cells in culture in a dose dependent fashion. The sensitivity of adrenergic (NBA₂₍₁₎) and cholinergic (NBE^?) clones to L-glutamate was similar. Sodium D-glutamate, L-aspartate, α-ketoglutarate, glutamine, γ-aminobutyric acid and carbachol did not inhibit the growth of NB cells. Methylmercuric chloride (CH₃HgCl), inorganic mercury (HgCl₂), manganese chloride (MnCl₂) and lead tri-butyl acetate, by themselves inhibited the growth of NB cells in culture to a varying degree ranging from 41% to 49%. However, the combination of glutamate with CH₃HgCl, HgCl₂ and MnCl₂, produced a synergestic effect on growth inhibition of NB cells in culture. The combination of glutamate with lead tri-butyl acetate produced only an additive effect. Sodium kainate neither inhibited the growth nor potentiated the growth inhibitory effect of L-glutamate on NB cells. Neuroblastoma cells contained high levels of receptors for glutamate but not for kainate. These results show that neuroblastoma culture may be a useful model to study the mechanisms of glutamate effects and their modification by various agents.     

Coordinate regulation of glycolysis by hypoxia in mammalian cells

The impact of hypoxic exposure on the activities of all 11 glycolytic enzymes was studied in cell culture into mammalian cells—mouse lung macrophages and L8 rat skeletal muscle cells. During hypoxic exposure, the measured activity of all glycolytic enzymes increased, establishing coordinate regulation. Three nonglycolytic cytoplasmic enzymes showed no change in activity under the same conditions, suggesting a specific mechanism. Hypoxia appears to increase the activities of all glycolytic enzymes whether rate-limiting or not, presumably increasing adenosine triphosphate availability despite decreased O₂ supply.     

PGE2-induced colon cancer growth is mediated by mTORC1

The inflammatory prostaglandin E₂ (PGE₂) cytokine plays a key role in the development of colon cancer. Several studies have shown that PGE₂ directly induces the growth of colon cancer cells and furthermore promotes tumor angiogenesis by increasing the production of the vascular endothelial growth factor (VEGF). The signaling intermediaries implicated in these processes have however not been fully characterized. In this report, we show that the mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in PGE₂-induced colon cancer cell responses. Indeed, stimulation of LS174T cells with PGE₂ increased mTORC1 activity as observed by the augmentation of S6 ribosomal protein phosphorylation, a downstream effector of mTORC1. The PGE₂ EP₄ receptor was responsible for transducing the signal to mTORC1. Moreover, PGE₂ increased colon cancer cell proliferation as well as the growth of colon cancer cell colonies grown in matrigel and blocking mTORC1 by rapamycin or ATP-competitive inhibitors of mTOR abrogated these effects. Similarly, the inhibition of mTORC1 by downregulation of its component raptor using RNA interference blocked PGE₂-induced LS174T cell growth. Finally, stimulation of LS174T cells with PGE₂ increased VEGF production which was also prevented by mTORC1 inhibition. Taken together, these results show that mTORC1 is an important signaling intermediary in PGE₂ mediated colon cancer cell growth and VEGF production. They further support a role for mTORC1 in inflammation induced tumor growth.     

Further serum related alterations in the activities of some membrane marker enzymes in normal and virus transformed cells

The activities of membrane marker enzymes in normal (3T3) and simian virus transformed mouse cells (SV3T3) are affected not only by densities of cultures but also by the sera types used in the growth media. We have assayed the levels of 5'nucleotidase, monoamine oxidase and rotenone insensitive NADH ferricyanide reductase in these cells grown to sparse and confluent cultures in medium supplemented with 10% newborn calf serum (n.c.s.) or in medium supplemented with 10% foetal bovine serum (f.b.s.). It was found that in 3T3 cells grown in 10% f.b.s. the transition from sparse to confluent cultures was associated with a reduction in the activities of the marker enzymes while in those grown in 10% n.c.s., the activities of these enzymes increased. In the SV3T3 cells, the activities of all the enzymes except for monoamine oxidase decreased from sparse to confluent culture densities in cells grown in 10% n.c.s. whereas in those grown in 10% f.b.s. there were no significant change in the activities of the enzymes over the same culture densities. The results suggest that the marker enzymes are affected by sera types and culture densities.     

Enzymes of the autotrophic pathway in mating partners and transconjugants of Nocardia opaca 1 b and Rhodococcus erythropolis

Enzyme activities have been measured in the partners of a bacterial mating system consisting of the hydrogen autotroph Nocardia opaca (donor and Aut^- recipient), the heterotroph Rhodococcus erythropolis (recipient) and intra- and interspecies transconjugants after growth on fructose, pyruvate and under autotrophic conditions. Specific activities of each of the enzymes hydrogenase, phosphoribulokinase and ribulosebisphosphate carboxylase were high in autotrophically grown cells of the donor and the transconjugants: they amounted to only 10% after growth on pyruvate. The recipient cells did not grow autotrophically and the enzymes mentioned were not detectable even after growth on pyruvate. Other enzymes of the Calvin cycle were constitutively formed in all strains examined.The properties of hydrogenase (K _m for NAD, R_f in gel electrophoresis) and of ribulosebisphosphate carboxylase (K _m for RuBP and R_f) were the same in the donor and transconjugant cells. The properties of glucose-6-phosphate dehydrogenase (K _m for G-6-P and mode of inhibition by ATP and phosphoenolpyruvate) were the same in the recipient and the interspecies transconjugant cells and differed from those of the donor cells. The curves of growth under autotrophic conditions in batch culture of the donor and interspecies transconjugant were almost congruent. The specific activities of hydrogenase, phosphoribulokinase and ribulosebisphosphate carboxylase increased from 40% at the beginning to 100% at the end of the exponential growth phase; these enzymes were under coordinate control.The results are in accordance with genetic studies: the genetic information for autotrophic growth is localized on a so far unidentified genetic element and is transferred en bloc from N. opaca to Aut^- mutants of the same strain or to recipient bacteria such as R. erythropolis; expression in the wild type and transconjugant cells is the same.Abbreviations G-6-P glucose-6-phosphate - 6-PG 6-phosphogluconate - FBP fructose-1,6-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - RuBP ribulose-1,5-bisphosphate     

Changes in the cell cycle duration and karyotype of murine L cells with a shift in the mode of cultivation

A suspension subline LS of mouse fibroblasts L was adapted to the growth in monolayer (LSM subline). The duration of cell cycle and its phases was determined for both the sublines synchronized by a double thymidine block. The duration of cell cycle for LS cells is 17.0 hours, with G1, S and G2 + M being 8.8, 6.3 and 1.8 hours, resp., for LSM cells corresponding values being 31.6, 15.6, 11.0 and 4.8 hours. The karyotype of LSM cells differs from that of LS cells only slightly: there is a redistribution between the numbers of cells with 55 and 56 chromosomes, and a decrease in the number of polyploid cells from 2.0 till 0.2%. The data obtained indicate the heterogeneity of the L line culture which contains cells with long and short cycles. Depending on the mode of cultivation (suspension or monolayer), there is a certain predominance of population either with short or with long cell cycle.     

Chromatographic analysis of purine precursors in mouse L1210 leukemia

A number of antagonists of nucleotide metabolism with anti-cancer activity affect the de novo purine pathway. To determine the biochemical mechanisms of cytotoxicity of these drugs, assay procedures have been developed for measurement of the levels of intermediates proximal to IMP in the pathway for de novo purine biosynthesis in mouse L1210 leukemia cells. Purine precursors have been synthesized in vitro from [14C]glycine using enzymes from chicken liver. These 14C-labeled intermediates have been used as marker compounds to define retention times for metabolites of leukemia cells separated by HPLC and the chromatographic mobilities of these intermediates after two-dimensional thin-layer chromatography. These new chromatographic procedures have been used in combination to determine the steady-state concentrations for purine precursors in mouse L1210 leukemia cells in the exponential phase of growth: N-formylglycineamide ribotide (16 microM); N-formylglycineamidine ribotide (4.7 microM); 5-aminoimidazole ribotide (4.0 microM); 4-carboxy-5-aminoimidazole ribotide (0.46 microM); N-succino-5-aminoimidazole-4-carboxamide ribotide (11 microM); 5-aminoimidazole-4-carboxamide ribotide (16 microM); 5-formamidoimidazole-4-carboxamide ribotide (2.7 microM); and IMP (57 microM). The metabolic effects of tiazofurin (25 microM) upon mouse L1210 leukemia cells growing in culture define a "metabolic crossover point" at the reaction catalyzed by IMP dehydrogenase (EC 1.1.1.205) which confirms previous reports of inhibition of this enzyme.     

A survey of carbonic anhydrase mRNA expression in enamel cells

Enamel formation requires rigid control of pH homeostasis during all stages of development to prevent disruptions to crystal growth. The acceleration of the generation of bicarbonate by carbonic anhydrases (CA) has been suggested as one of the pathways used by ameloblasts cells to regulate extracellular pH yet only two isozymes (CA II and CA VI) have been reported to date during enamel formation. The mammalian CA family contains 16 different isoforms of which 13 are enzymatically active. We have conducted a systematic screening by RT-PCR on the expression of all known CA isoforms in mouse enamel organ epithelium (EOE) cells dissected from new born, in secretory ameloblasts derived from 7-day-old animals, and in the LS8 ameloblast cell line. Results show that all CA isoforms are expressed by EOE/ameloblast cells in vivo. The most highly expressed are the catalytic isozymes CA II, VI, IX, and XIII, and the acatalytic CA XI isoform. Only minor differences were found in CA expression levels between 1-day EOE cells and 7-day-old secretory-stage ameloblasts, whereas LS8 cells expressed fewer CA isoforms than both of these. The broad expression of CAs by ameloblasts reported here contributes to our understanding of pH homeostasis during enamel development and demonstrates its complexity. Our results also highlight the critical role that regulation of pH plays during the development of enamel.     

The effect of ethanol on arachidonic acid metabolism in the murine peritoneal macrophage

Exposure to ethanol in man has been linked to an alteration of the immune surveillance system and reduced ability of the macrophage to undergo phagocytosis. Since ethanol has been suggested to alter membrane function and inhibit the production of calcium ionophore stimulated synthesis of prostaglandins and leukotrienes by the human neutrophil and transformed murine mast cell, the dose response effect of ethanol on the biosynthesis of icosanoids by the peritoneal macrophage during zymosan phagocytosis was studied. Peritoneal macrophages from two inbred strains of mice derived from a common stock (HS) and selected for sensitivity to ethanol (shoprt sleep [SS]/long sleep [LS]) were studies. Zymosan phagocytosis was found to lead to synthesis of LTC₄ (70 ng/10⁶ cells), 6-keto-PGF_1a (5 ng/10⁶ (3 ng/10⁶ cells). For the HS macrophage, ethanol caused a dose dependent inhibition of these lipid mediators as well as inhibition of phagocytosis and release of beta-hexosaminidase. However, a difference was observed in arachidonate metabolism stimulated by phagocytosis between the LS and SS mice below 100 mM ethanol. The SS mouse had a 50% inhibition of cyclooxygenase products at 86 mM ethanol with no inhibition of lipoxygenase metabolites. The LS mice had a trend suggesting increased lipoxygenase metabolites below 100 mM ethanol. At these levels of ethanol which can be found in man, these results suggest there may be differential production of lipid mediators under genetic control.     

Expression and extinction of glial properties in glioma X neuroblastoma cell hybrids

Rat glioma cells (clone C₆TK⁻) were hybridized with mouse neuroblastoma cells (clone NA), and 18 primary and secondary hybrid clones containing one chromosome set from each parent were isolated. The hybrids were assayed for the glial marker enzymes 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and glycerol-3-phosphate dehydrogenase (GPDH). In many of the hybrid clones, the levels of CNP and GPDH were reduced to 5–20% of the activity of C₆TK⁻, as has been observed in other classes of glial X non-glial cell hybrids. In some hybrid clones, however, GPDH and CNP were expressed at high activity. Rat (glial) GPDH activity was not reduced in these clones, but mouse GPDH activity remained low, and was not “de-repressed” or “activated”. This suggests that the controls governing differentiation in neuroblastoma cells and extinction in hybrids may differ in some important details. There was a strong positive correlation between the specific activities of CNP and GPDH in the hybrid clones, suggesting that a mechanism regulates the activity of these two glial enzymes coordinately.     

Electrophoretic heterogeneity of mouse erythrocyte peptidases

Starch gel electrophoresis in conjunction with a specific staining method revealed the occurrence of five distinct peptidases in mouse red blood cells. These enzymes can be distinguished on the basis of substrate specificity and electrophoretic mobility. They have been designated peptidases A, B, C, D, and E to correspond with the nomenclature adopted for human peptidases with which the mouse enzymes appear to be homologous. Genetically determined variants of peptidase C are described. The phenotype Pep C1 occurs in C57BL/Gr mice and the phenotype Pep C2 in CBA/Gr and Strong A/Gr mice. These phenotypes and the presumed heterozygote, Pep C2-1, appear to be due to the occurrence of codominant autosomal alleles which have been designated Pep-C ¹ and Pep-C ². F₁ and F₂ crosses show segregation in the expected Mendelian ratios. F₂ embryos and their placentae show the same electrophoretic pattern for peptidase C. The occurrence of a separate locus controlling the structure of each distinct peptidase is postulated.     

Growth and activities of enzymes of primary metabolism in batch cultures of Catharanthus roseus cell suspension under different pCO2 conditions

In vitro enzyme activities of glycolysis, pentose-phosphate pathway and dark CO₂ fixation were assayed in batch cultures of heterotrophic Catharanthus roseus cells under various gassing rates and partial pressures of carbon dioxide. Detrimental effects of low pCO₂ culture conditions on the growth characteristics could be linked to marked changes in levels of enzymes of primary metabolism during growth. The enzyme levels observed during the early stages of growth were found to be more stable when a constant pCO₂ (20 mbar) was maintained and enabled exponential growth to be reached more rapidly.The importance of carbon dioxide as a conditioning factor of the culture medium is discussed.