Enhancement of hyperthermic killing cultured mammalian cells by treatment with anisotonic NaCl or medium solutions

The exposure of cultured Chinese hamster cells (CHO) to anisotonic medium increased the cellular sensitivity to heat treatment at 42.3°C. A greater potentiation of heat killing is observed when the anisotonic solution consists of pure NaCl in water compared to growth medium made anisoltonic by dilution or by addition of NaCl. Hypertonic treatment caused greater heat sensitization than hypotonic treatment. Thermal tolerance observed in the control cells after 4–6 hours of heating in medium was also observed for cells exposed to anisotonic media during heating if the heating period was greater than 4 h. The exposure of cells to anisotonic media during heating if the heating period was greater than 4 h. The exposure of cells to anisotonic NaCl solutions during heating removed the shoulder from the heat survival curve, while the curves for cells heated in medium made anisotonic retained their shoulders. These studies suggest: (1) that either the plasma membrane is a primary target for heat inactivation of mammalian cells, or (2) that changes in intracellular ion concentrations enhance thermal damage occurring in critical intracellular structures.     

Induction of stress proteins in chicken embryo cells by low-level zinc contamination in amino acid-free media

It has been reported that chicken embryo cells deprived of exogenous amino acids for 4 hours synthesize stress (heat-shock) proteins. Herein, we show that amino acid deprivation is not sufficient to cause induction of stress proteins. Zinc contaminating a component of commercial cell culture medium used to prepare amino acid-free medium was an inducer in our cultures. In the absence of exogenous amino acids, the concentration of zinc ions needed for half-maximal induction of stress proteins was an order of magnitude lower than the dose required for cells in complete medium. Histidine and cystine, which have high affinities for zinc ions, were the amino acids most effective in blocking the induction of stress proteins by zinc. Problems posed by heavy metal ions in culture media and biologic fluids for searches for in vivo inducers of the cellular stress (heat shock) response are discussed.     

Alteration in the Amino Acid Content of Yeast During Growth Under Various Nutritional Conditions

Yeast cells grown under optimal and suboptimal concentrations of biotin were analyzed for the amino acid content of their soluble pool and cellular protein. Optimally grown yeast cells exhibited a maximum amino acid content after 18 hr of growth. Biotin-deficient cells were depleted of all amino acids at 26 and 43 hr, with alanine, arginine, aspartate, cysteine, glutamate, isoleucine, leucine, lysine, methionine, serine, threonine, and valine being present in less than half the concentration observed in biotin-optimal cells. At early time intervals, the amino acid pool of biotin-deficient yeast contained lower concentrations of all amino acids except alanine. After more prolonged incubation, several amino acids accumulated in the pool of biotin-deficient yeast, but citrulline and ornithine accumulated to appreciable levels. The addition of aspartate to the growth medium resulted in a decrease in the amino acid content of biotin-optimal cells but caused a marked increase in the concentration of amino acids in biotin-deficient cells. The pools of biotin-deficient yeast grown in the presence of aspartate displayed a marked reduction in every amino acid with the exception of aspartate itself. These data provide evidence that the amino acid content of yeast cells and their free amino acid pools are markedly affected by biotin deficiency as well as by supplementation with aspartate, indicating that aspartate plays a major role in the nitrogen economy of yeast under both normal as well as abnormal nutritional conditions.     

Free Amino Acids in Serine-Antagonized Cells of Tetrahymena pyriformis.

Wragg, June B. (Agricultural Research Service, Beltsville, Md.), Howard Reynolds, and Michael J. Pelczar, Jr. Free amino acids in serine-antagonized cells of Tetrahymena pyriformis. J. Bacteriol. 90:748-754. 1965.-Growth inhibition of Tetrahymena pyriformis by l-serine in a chemically defined medium was reversed by l-arginine in a manner which resembled competitive antagonism. Composition of the free amino acid pools from cells grown in either a balanced amino acid mixture or a mixture with serine concentrations which inhibited growth suggested an antagonism by serine with energy-yielding reactions. Growth in media with excess serine resulted in the accumulation of higher concentrations of free cellular amino acids and an apparent increase in the rate of conversion of arginine to ornithine, as compared with growth in the balanced medium. The results suggested that serine or a metabolic product of serine interferes with the formation of pyruvic acid. In the presence of high levels of serine, arginine appeared to be metabolized more rapidly and to be spared when alanine, aspartic acid, or glutamic acid was added to the unbalanced medium.     

The bactericidal effect of isoascorbic acid combined with mild heat

The thermal inactivation of Salmonella thompson, Escherichia coli, Staphylococcus aureus, Clostridium perfringens, Candida zeylanoides, Enterococcus faecium and E. faecalis was accelerated by the addition of sodium isoascorbate (1 mmol/l) to phosphate-buffer heating medium but not to complex food mixtures. The lethal effect of isoascorbate was nullified by heating under anaerobic conditions or by the addition of catalase. The scavengers of hydroxyl radicals, mannitol and formate were not protective whereas histidine was. Histidine may have protected by slowing the rate of isoascorbate autoxidation, a property common to other amino acids tested. Superoxide dismutase was not protective. Dehydroascorbic acid also enhanced heat killing and its action was also reversed by catalase. The bactericidal effects of mild heat plus isoascorbate or dehydroascorbic acid both apparently depend on oxidative processes but their relative effectiveness was not related to their respective rates of oxygen consumption or peroxide production. We speculate that site-specific redox reactions, involving amino-carbonyl intermediates are involved in the inactivation mechanism.     

Relationship between Auxin and Amino Acid Metabolism of Tobacco Protoplast-Derived Cells

Single amino acids were found to be highly toxic to protoplast-derived cells of tobacco (Nicotiana tabacum cv Xanthi) cultured at low density in a culture medium containing a low naphthaleneacetic acid concentration (0.05 micromolar). The cytotoxicities of alanine, aspartic acid, asparagine, glutamic acid, glutamine, glycine, lysine, proline, and valine were reduced when the naphthaleneacetic acid concentration of the culture medium was increased to 1 micromolar. This selective modification of amino acid toxicity by naphthaleneacetic acid could not be correlated with modifications of uptake rates or incorporation of these amino acids into protein or amino acid-auxin conjugates. A mutant clone resistant to high naphthaleneacetic acid concentrations and affected in root morphogenesis did not display, at the cellular level, the naphthaleneacetic acidmediated modification of amino acid cytotoxicity.     

THE EFFECTS OF PHENYLALANINE ON AMINO ACID METABOLISM AND PROTEIN SYNTHESIS IN BRAIN CELLS IN VITRO1

Incubation of brain cell suspensions with 14 mM-phenylalanine resulted in rapid alterations of amino acid metabolism and protein synthesis. Both thc rate of uptake and the final intracellular concentration of several radioactively-labelled amino acids were decreased by high concentrations oi phenylalanine. By prelabelling cells with radioactive amino acids, phenylalanine was also shown to effect a rapid loss of the labelled amino acids from brain cells. Amino acid analysis after the incubation of the cells with phenylalanine indicated that several amino acids were decreased in their intracellular concentrations with effects similar to those measured with radioisotopic experiments (large neutral > small and large basic > small neutral > acidic amino acids). Although amino acid uptake and efflux were altered by the presence of 14 mwphenylalanine, little or no alteration was detected in the resulting specific activity of the intracellular amino acids. High levels of phenylalanine did not significantly altcr cellular catabolism of either alanine, lysine, leucine or isoleucine. As determined by the isolation of labcllcd aminoacyl-tRNA from cells incubated with and without phenylalanine, there was little or no alteration in the level of this precursor for radioactive alanine and lysine. There was, however, a detectable decrease in thc labelling of aminoacyl-tRNA for leucine and isoleucine. Only aftcr correcting for the changes of the specific activity of the precursors and thcir availability to translational events, could the effects of phenylalanine on protein synthesis be established. An inhibition of the incorporation into protein for each amino acid was approximately 20%.     

THE EFFECTS OF AMINO ACIDS UPON THE DEVELOPMENT OF EXCISED FLORAL BUDS OF AQUILEGIA

Young excised floral buds of Aquilegia were grown on a chemically defined medium containing various concentrations of single amino acids or mixtures of amino acids. γ-Amino butyric acid significantly promoted floral development through the initiation and differentiation of carpels. These floral organs were generally absent on the basal medium. Alanine, glutamic acid, and aspartic acid had no effect upon floral development. All other amino acids were either ineffective at lower concentrations and inhibitory at higher concentrations or were inhibitory at all concentrations. Casein hydrolysate and a mixture of amino acids found in coconut milk were ineffective. The addition of both γ-amino butyric acid and alanine to the basal medium promoted development approaching that achieved on the coconut-milk medium. However, further growth factors appear to be required before development on coconut-milk medium is equalled or exceeded.     

DNA polymerase activity in heat killing and hyperthermic radiosensitization of mammalian cells as observed after fractionated heat treatments

Possible relations between hyperthermic inactivation of alpha and beta DNA polymerase activity and hyperthermic cell killing or hyperthermic radiosensitization were investigated. Ehrlich Ascites Tumor (EAT) cells and HeLa S3 cells were treated with fractionated doses of hyperthermia. The heating schedules were chosen such that the initial heat treatment resulted in either thermotolerance or thermosensitization (step-down heating) for the second heat treatment. The results show that for DNA polymerase activity and heat radiosensitization (cell survival) no thermotolerance or thermosensitization is observed. Thus hyperthermic cell killing and DNA polymerase activity are not correlated. The correlation of hyperthermic radiosensitization and DNA polymerase activity was substantially less than observed in previous experiments with normotolerant and thermotolerant HeLa S3 cells. We conclude that alpha and beta DNA polymerase inactivation is not always the critical cellular process responsible for hyperthermic cell killing or hyperthermic radiosensitization. Other possible cellular systems that might determine these processes are discussed.     

Substrate-dependent regulation of intracellular amino acid concentrations in cultured bovine aortic endothelial cells

Amino acid deprivation induces adaptive changes in amino acid transport and the intracellular amino acid pool in cultured cells. In this study intracellular amino acid levels were determined in cultured bovine aortic endothelial cells (EC) deprived of L-arginine or total amino acids for 1, 3, 6 and 24 h. Amino acid concentrations were analyzed by reverse phase HPLC after precolumn derivatisation. Under normal culture conditions levels of L-arginine L-citrulline, total essential and non-essential amino acids were 840 +/- 90 microM, 150 +/- 40 microM, 11.4 +/- 0.9 mM and 53.3 +/- 3.4 mM (n = 9), respectively. In EC deprived of L-arginine or all amino acids for 24 h L-arginine and L-citrulline levels were 200 microM and 50 microM, and 670 microM and 100 microM Deprivation of L-arginine or total amino acids induced rapid (1 h) decreases (30 - 50%) in the levels of other cationic (lysine, ornithine) and essential branched-chain (valine, isoleucine, leucine) and aromatic (phenylalanine, tryptophan) amino acids. L-glutamine was reduced markedly in EC deprived of total amino acids for 1 h - 6 h but actually increased 3-fold in EC deprived of L-arginine for 6 h or 24 h. Arginine deprivation resulted in a rapid decrease in the total intracellular amino acid pool, however concentrations were restored after 24 h. Increased amino acid transport and/or reduced protein synthesis may account for the restoration of amino acid levels in EC deprived of L-arginine. The sustained reduction in the free amino acid pool of EC deprived of all amino acids may reflect utilization of intracellular amino acids for protein synthesis.     

Effect of histidine on histidinol-induced heat protection in Chinese hamster ovary cells

The possible mechanism for heat protection by the protein synthesis inhibitor histidinol was investigated in CHO cells. Histidinol (HST, 5 mM), an analogue of the essential amino acid L-histidine, added for 2 hr before and during heating at 43 degrees C, protected cells from killing at 43 degrees C. Treatment with HST produced a 600-fold increase in survival from 3 x 10(-4) to 1.8 x 10(-1) after 2.5 hr at 43 degrees C. Although the cells were washed after HST treatment, substantial protective effect was still observed during heating at 43 degrees C. This protective effect gradually decreased with increased incubation time after the drug treatment. However, the protective effect was immediately reduced by treatment with histidine (HIS, 0.25-5 mM) during heating. The amount of reduction was dependent upon HIS concentration: five millimolar HIS completely inhibited HST-induced heat protection. Furthermore, protein synthesis which was inhibited by 95% by 5 mM HST, resumed immediately with 5 mM HIS treatment. In addition, when cells were labeled during or after HST treatment, neither preferential accumulation of heat shock protein families nor phosphorylation of 28 kDa protein was observed. Therefore, these results suggest that the cessation of protein synthesis itself is one of the events involved in protection.     

Amino Acid Requirements for the Growth of Aedes albopictus Clone C6/36 Cells and for the Production of Dengue and Chikungunya Viruses in the Infected Cells

Amino acid requirements for the growth of Aedes albopictus, clone C6/36, cells and for the production of dengue (DEN) and Chikungunya (CHIK) viruses were examined by growing the cells or the viruses in media which were deprived of one of the 20 amino acids. Cell growth was markedly inhibited when cystine was omitted from the medium, and to a lesser extent by arginine deprivation. On the other hand, omission of alanine, asparagine, aspartic acid, and glutamic acid at the same time did not affect cell growth. Marked accumulation of alanine was observed in the medium when the cells were grown for 8 days in complete medium, with concomitant depletion of aspartic acid and glutamic acid. The production of CHIK virus was inhibited markedly by omission of cystine from the medium after virus infection, while the production of DEN viruses was more affected by glycine deprivation, although cystine deprivation also inhibited virus production to a lesser extent. On the other hand, production of CHIK and DEN viruses was not affected when alanine, asparagine, aspartic acid, and glutamic acid were omitted from the medium at the same time.     

Amino acid concentrations in fluids from the bovine oviduct and uterus and in KSOM-based culture media.

Amino acids in bovine oviductal and uterine fluids were measured and compared with those in modified simplex optimized medium (KSOM) supplemented with either fetal calf serum or Minimum Essential Medium amino acids in addition to bovine serum albumin, fetal calf serum or polyvinyl alcohol. Concentrations of cysteine, threonine, tryptophan, alanine, aspartate, glycine, glutamate, proline, beta-alanine, and citrulline were higher in oviductal fluids than in KSOM-based culture media. Nonessential and essential amino acids were present in ratios of 5:1 and 2:1 in oviductal and uterine fluids, respectively. Concentrations of alanine (3.7 mM), glycine (14.1 mM) and glutamate (5.5 mM) were high in oviductal fluids, comprising 73% of the free amino acid pool. Of the amino acids measured in uterine fluids, alanine (3.1 mM), glycine (12.0 mM), glutamate (4.2 mM), and serine (2.7 mM) were highest in concentration, and the first three comprised 43% of the free amino acid pool. In conclusion, amino acid concentrations in the bovine reproductive tract were substantially higher than those in embryo culture media. Certain amino acids, particularly alanine, glutamate, glycine and taurine, are present in strikingly high concentrations in both oviductal and uterine fluids, suggesting that they might play important roles in early embryo development. The particular pattern of amino acid concentrations may be an important factor to be considered for the improvement of embryo culture media.     

The amino acid requirements of rabbit fibroblasts,strain RM3-56

Strain RM3-56 of rabbit fibroblasts was found to require arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine for growth in a medium containing 2 per cent dialyzed serum as the only undefined component. The requirement for serine is less specific than that of the other 13 amino acids and it is partially replaced by glycine, or alanine, or by several combinations of so called accessory amino acids. The concentrations of essential amino acids which permit maximal proliferation range from 0.005 to 0.3 mM. Cystine, glutamine, lysine, tryptophan, tyrosine, valine are toxic at concentrations of 5 mM. The rate of proliferation of RM3-56 in a medium containing all 14 essential amino acids is increased significantly by the addition of alanine and to a lesser extent by the addition of aspartic and glutamic acids and glycine. A deficiency of cystine or glutamine results in cellular degeneration within 3 to 5 days, whereas the cells remain in good condition for 2 to 3 weeks in the absence of each of the remaining 12 essential amino acids. The results obtained with RM3-56 are compared with strains HeLa, L, and U12, whose amino acid requirements have been investigated under similar conditions.     

Growth, photosynthesis, respiration, and intracellular free amino acid profiles in the unicellular alga Cyanidium caldarium. Effect of nutrient limitation and resupply

Growth, intrácellular free amino acid pools and photosynthetic and respiratory activities in nutrient sufficient cells and in N- K- and P-limited cells of Cyanidium caldarium (Tilden) Geitler, and responses to nutrient resupply were investigated. Addition of ammonium to N-limited cells and of phosphate to P-limited cells resulted in a stimulation of dark respiration and in a decrease in photosynthetic oxygen evolution. Addition of K to K-limited cells had no effect on rates of photosynthesis and respiration. Nutrient limited cells and sufficient cells exhibited different free amino acid profiles. Upon resupply of ammonium to N-limited cells levels of glutamine, citrulline, arginine, alanine, and serine increased. Also the levels of δ-aminolevulinic acid (δ-ALA) and putrescine increased notably. On adding phosphate to P-limited cells the level of glutamate decreased significantly whereas the level of alanine increased and the concentrations of other amino acids remained unaffected. On adding potassium to K-limited cells there was an increase in glutamate and citrulline concentrations, and a decrease in putrescine concentration, whereas concentrations of arginine and alanine remained at the very high levels observed already before addition. Resuspension of N- and K-limited cells in a complete growth only after 25-30h. In P-limited cells resumption of growth in complete medium occurred progressively and reached the maximum rate 30h later. P-, K- and N- limited cells resuspended into sufficient media showed different rates of ammonium and phosphate assimilation. The pattern of recovery from nutrient limitation is discussed according to the cellular role fulfilled by the nutrient which was growth rate-limiting.     

Hyperthermic killing and hyperthermic radiosensitization in Chinese hamster ovary cells: effects of pH and thermal tolerance

To quantitatively relate heat killing and heat radiosensitization, asynchronous or G1 Chinese hamster ovary (CHO) cells at pH 7.1 or 6.75 were heated and/or X-irradiated 10 min later. Since no progression of G1 cells into S phase occurred during the heat and radiation treatments, cell cycle artifacts were minimized. However, results obtained for asynchronous and G1 cells were similar. Hyperthermic radiosensitization was expressed as the thermal enhancement factor (TEF), defined as the ratio of the D0 of the radiation survival curve to that of the D0 of the radiation survival curve for heat plus radiation. The TEF increased continuously with increased heat killing at 45.5 degrees C, and for a given amount of heat killing, the amount of heat radiosensitization was the same for both pH's. When cells were heated chronically at 42.4 degrees C at pH 7.4, the TEF increased initially to 2.0-2.5 and then returned to near 1.0 during continued heating as thermal tolerance developed for both heat killing and heat radiosensitization. However, the shoulder (Dq) of the radiation survival curve for heat plus radiation did not manifest thermal tolerance; i.e., it decreased continuously with increased heat killing, independent of temperature, pH, or the development of thermotolerance. These results suggest that heat killing and heat radiosensitization have a target(s) in common (TEF results), along with either a different target(s) or a difference in the manifestation of heat damage (Dq results). For clinical considerations, the interaction between heat and radiation was expressed as (1) the thermal enhancement ratio (TER), which is the dose of X rays alone divided by the dose of X rays combined with heat to obtain an isosurvival, e.g., 10(-4), and (2) the thermal gain factor (TGF), the ratio of the TER at pH 6.75 to the TER at pH 7.4. Since low pH reduced the rate of development of thermal tolerance during heating at low temperatures, low pH enhanced heat killing more at 42-42.5 degrees C than at 45.5 degrees C where thermal tolerance did not develop. Therefore, the increase in the TGF after chronic heating at 42-42.5 degrees C was greater than after acute heating at 45.5 degrees C, due primarily to the increase in heat killing causing an even greater increase in heat radiosensitization. These findings agree with animal experiments suggesting that in the clinic, a therapeutic gain for tumor cells at low pH may be greater for temperatures of 42-42.5 degrees C than of 45.5 degrees C.     

Amino acid deprivation induces AKT activation by inducing GCN2/ATF4/REDD1 axis

Amino acid availability is sensed by various signaling molecules, including general control nonderepressible 2 (GCN2) and mechanistic target of rapamycin complex 1 (mTORC1). However, it is unclear how these sensors are associated with cancer cell survival under low amino acid availability. In the present study, we investigated AKT activation in non-small cell lung cancer (NSCLC) cells deprived of each one of 20 amino acids. Among the 20 amino acids, deprivation of glutamine, arginine, methionine, and lysine induced AKT activation. AKT activation was induced by GCN2/ATF4/REDD1 axis-mediated mTORC2 activation under amino acid deprivation. In CRISPR-Cas9-mediated REDD1-knockout cells, AKT activation was not induced by amino acid deprivation, indicating that REDD1 plays a major role in AKT activation under amino acid deprivation. Knockout of REDD1 sensitized cells cultured under glutamine deprivation conditions to radiotherapy. Taken together, GCN2/ATF4/REDD1 axis induced by amino acid deprivation promotes cell survival signal, which might be a potential target for cancer therapy.Subject terms: Cancer metabolism, Cell death     

Mechanisms of amino acid uptake in cumulus-enclosed mouse oocytes

The nutritional role of mouse granulosa cells on antral dictyate mouse oocytes has been studied by measuring the transfer of different amino acids through gap junctional channels between somatic and germ cells. When present in the incubation medium at concentrations resembling in vivo conditions, glycine, alanine, proline, serine, tyrosine, glutamic acid and lysine entered cumulus-enclosed oocytes cooperatively, while valine, leucine and phenylalanine did not. However, cooperative uptake of leucine and phenylalanine was observed at higher external precursor concentrations. We conclude that in vivo antral mouse oocytes depend on surrounding granulosa cells for amino acid uptake, with the exception of amino acids carried by the leucine exchange transport system, and propose that amino acid transfer between granulosa cells and oocytes is dependent on precursor concentrations in the coupled cells.     

Participation of blood cells in the changes of blood amino acid concentrations during maximal exercise

We determined the participation of the cellular compartment in the changes of plasma amino acid concentrations during maximal exercise test on a cycle ergometer. Following an overnight fast, male athletes were submitted to a maximal exercise test until fatigue (for 25 min approximately) to determine maximal oxygen uptake. The amino acid concentrations in total blood, plasma, and blood cells were determined before and after the maximal exercise test. Most essential amino acids were decreased significantly in the total blood concentration as a result of the maximal exercise test. However, the concentrations of most nonessential amino acids tended to be significantly increased. Amino acid concentration was increased most in plasma. Concentrations of blood cell alanine and proline were significantly increased by 26% and 15%, respectively, after the maximal exercise test. No significant differences in blood cell concentrations of other amino acids induced by the exercise test were found, although the amount of tryptophan in blood cells was increased after exhaustive exercise.     

Analysis of Simian Virus 40-Induced Transformation of Hamster Kidney Tissue in Vitro VIII. Induction of Infectious Simian Virus 40 from Virogenic Transformed Hamster Cells by Amino Acid Deprivation or Cycloheximide Treatment

Clones of virogenic simian virus 40 (SV40)-transformed hamster kidney cells were exposed to medium deficient in the essential amino acids leucine, arginine, or methionine. Infectious virus was induced after deprivation periods of from 24 to 32 hr. The highest yields of infectious SV40 were obtained from cultures deprived for 3 to 4 days. Infectious virus was also induced in cells that were treated with the metabolic inhibitor cycloheximide. Pulse labeling experiments revealed that both protein synthesis and deoxyribonucleic acid (DNA) synthesis were inhibited by concentrations of cycloheximide which were effective for virus induction. It is suggested that inhibition of protein synthesis by either amino acid deprivation or by cycloheximide was responsible for the induction of infectious virus from virogenic cells. We postulate that the inhibition of protein synthesis caused a temporary inhibition of DNA synthesis which resulted in the induction of infectious virus.