Species-specific differences in the toxicity of puromycin towards cultured human and Chinese hamster cells

The toxicity of the protein synthesis inhibitor puromycin towards a number of human and Chinese hamster cell lines has been examined. In comparison to cells of human origin, Chinese hamster cells exhibited about 25-fold higher resistance towards puromycin. These differences appeared to be species related as all the cell lines from any one species showed similar sensitivity towards puromycin. The incorporation of [3H]leucine in the hamster cell lines was accordingly found to be more resistant to the inhibitory effects of puromycin as compared to human cells. Studies on the cellular uptake of [3H]puromycin showed that in comparison to human cells, the drug uptake/binding in the hamster cell lines was greatly reduced. However, protein synthesis in the extracts of hamster and human cells showed no significant differences in sensitivity towards puromycin. These results show that the observed species related differences in cellular toxicity to puromycin are due to differences in the cellular uptake/binding of the drug.     

Is protein synthesis necessary for prostaglandin production by guinea-pig endometrium?

The outputs of prostaglandin (PG) F-2 alpha, PGE-2 and 6-keto-PGF-1 alpha from Day-7 and Day-15 guinea-pig endometrium in culture were reduced by the inclusion of actinomycin D, cycloheximide and puromycin in the culture medium, with the output of PGF-2 alpha from Day-15 endometrium being particularly affected during the first 6 h of culture. The intrauterine administration of actinomycin D on Day 10 decreased the outputs of PGF-2 alpha and PGE-2, but not of 6-keto-PGF-1 alpha, from Day-15 endometrium in culture without affecting PG output from Day-15 myometrium in culture. Actinomycin D, cycloheximide and puromycin did not reduce PG output when superfused over the Day-7 and Day-15 guinea-pig uterus in vitro for 20 min, indicating that these compounds do not have a rapid inhibitory effect on endometrial PG synthesis. In fact, they tended to stimulate PG output during this 20-min period, with cycloheximide having a pronounced effect on PGE-2 output. The synthesis of secreted proteins, but not of cellular proteins, was greater by Day-15 than by Day-7 endometrium in culture. Actinomycin D, cycloheximide and puromycin inhibited the synthesis of secreted and cellular proteins by Day-7 and Day-15 endometrium in culture. Protein synthesis and PG synthesis in the endometrium were both inhibited to a greater extent by cycloheximide and puromycin than by actinomycin D. The intrauterine administration of actinomycin D on Day 10 reduced the syntheses of secreted and cellular proteins by Day-15 endometrium in culture. These findings indicate that the endometrial synthesis of PGs, particularly of PGF-2 alpha towards the end of the oestrous cycle, is dependent upon endometrial protein synthesis.     

Effects of metabolic inhibitors on spontaneous and interferon-boosted human natural killer cell activity

Human natural killer (NK) cell activity can be augmented by pretreatment with partially purified preparations of human interferon (IF). Studies have now been performed to determine the metabolic processes required for and involved in spontaneous NK activity and augmentation of cytotoxicity. A 4-hr 51Cr release cellular cytotoxicity assay was used to measure the NK activity, and peripheral blood leukocyte cells (PBL) were treated with: a) x-ray or mitomycin C; b) actinomycin D; or c) emetine, cycloheximide, pactamyhcin, or puromycin to assess the roles of DNA, RNA, and protein synthesis, respectively, in spontaneous NK activity and in boosting by IF. Prolonged incubation (18 hr) of PBL after blockage of synthesis of DNA almost completely abrogated NK activity; however, NK activity could be partially or totally restored to these populations by incubation of the effector cells for 1 hr at 37 degrees C with IF. Blockage of DNA synthesis for 1 hr had no effect on spontaneous NK activity or on boosting by IF. Inhibition of RNA synthesis also had no effect on spontaneous NK activity. Treatment of PBL with actinomycin before exposure to IF prevented boosting, but treatment with the RNA synthesis inhibitor after boosting with IF for 5 to 6 hr no longer had an appreciable effect on cytotoxicity. The effect of protein synthesis inhibitors on spontaneous NK activity was dependent on the inhibitor selected. Emetine and puromycin totally abrogated spontaneous NK activity at concentrations of inhibitor that blocked 3H-leucine incorporation 90% or more. In contrast, cycloheximide and pactamycin had only minimal effects on spontaneous NK activity but totally abrogated the boosting of IF.     

Studies on the amoebo-flagellate transformation inPhysarum polycephalum

Flagellar formation in the true slime mold,Physarum polycephalum, involves a sequence of events during which amoebae are changed into flagellate cells. In the present study a series of inhibitors thought to inhibit RNA and protein synthesis and microtubule assembly were added in an attempt to characterize the metabolic processes associated with this amoebo-flagellate transformation. Proflavin (inhibitor of cellular RNA synthesis), puromycin, cycloheximide and streptomycin (inhibitors of protein synthesis), blocked the transformation; however, actinomycin D (inhibitor of DNA-dependent RNA synthesis) did not block this transformation. On the other hand, 2-mercaptoethanol and dithiothreitol did block flagella formation, but even high concentrations of colchicine failed to have such an effect. Flagellate formation was more strongly inhibited by inhibitors of oxidative phosphorylation than by other respiratory inhibitors; this suggests that oxidative phosphorylation takes part in the energy metabolism of this transformation.     

Effect of the Inhibition of Protein Synthesis on the Establishment of Transformation by Polyoma Virus

Puromycin was used to study the effect of the inhibition of protein synthesis on transformation of hamster cells (BHK21) by polyoma virus. The drug was used at a concentration (10(-4)m) which caused in these cells a drastic but fully reversible inhibition of protein synthesis. A two- to threefold enhancement of transformation rate was obtained when the cells were exposed to puromycin for a period of 5 hr that started at the end of the virus adsorption period. No further enhancement was produced by prolonging puromycin treatment up to 13 hr after infection. The possibility that the observed effect on transformation rate could be mainly attributed to cell selection by puromycin was excluded. In addition, the relevance of a number of possible secondary effects of puromycin (inhibition of cell division, inhibition of deoxyribonucleic acid synthesis, etc.) was also ruled out. The effect of puromycin on transformation appeared to be dependent on the time (relative to infection) of addition of the drug. In fact, no transformation enhancement was observed when the cells were exposed to puromycin prior to infection or beyond the 10th hr after infection. Since another drug known to affect protein synthesis (p-fluorophenylalanine) was also shown to produce similar effects, it is suggested that transformation enhancement results from the inhibition of protein synthesis during a sensitive period closely following adsorption of the virus.     

Gibberellin-induced Yeast Sporulation in Relation to RNA and Protein Metabolism

Gibberellic acid (GA) promoted sporulation in yeast when added to the sporulation medium. When added together with GA, metabolic inhibitors of RNA synthesis such as 8-azaguanine, 2-thiouracil, and actinomycin D suppressed selectively the promoting effect of GA on sporulation. The effect of 8-azaguanine and 2-thiouracil was alleviated by simultaneous addition of guanine and uracil, respectively. The promoting effect of GA on sporulation was also suppressed by inhibitors of protein synthesis such as ethionine, chloramphenicol, and puromycin. Methionine eliminated the inhibitory effect of ethionine on the GA action.     

Further studies on the effects of blocking agents on protein synthesis in goldfish brain

Abstract— In further experiments on the effects of antibiotic agents on protein synthesis in the goldfish brain, doses of intracranially-injected puromycin or acetoxycycloheximide higher than those previously employed did not hasten the onset of inhibition of incorporation of intraperitoneally-injected [³H]leucine into brain protein. The antibiotic-resistant incorporation was not due to the presence of labelled blood protein in the brain. After the intracranial injection of labelled puromycin, the appearance of radioactivity in the acid-soluble fraction of brain was blocked by acetoxycycloheximide. Repeated daily intracranial or intraperitoneal injections of puromycin were lethal, and acetoxycycloheximide was not protective, indicating that peptidyl-puromycin was present in the brain but did not account for the lethality of puromycin. Behavioural experiments argued against but did not totally exclude the possibility that peptidyl-puromycin was responsible for the amnestic effect of puromycin. Puromycin aminonucleoside, O-methyl tyrosine and 5-guanylyl methylenediphosphonate had little or no effect on protein synthesis in brain, but gougerotin was slightly inhibitory.     

Response of Intracellular Proteolysis to Alteration of Bacterial Protein and the Implications in Metabolic Regulation

An assessment has been made of the extent to which the breakdown of microbial cellular proteins is regulated by their metabolic state or function. For this purpose, a number of agents and conditions that alter the synthesis, structure, or utility of cellular protein were examined for the effect on their lability. In Escherichia coli, 5-fluorouracil, p-fluorophenylalanine, norleucine, canavanine, thienylalanine, and puromycin, which engender nonfunctional cellular protein en masse, and ultraviolet irradiation increase the breakdown rate of proteins synthesized in their presence as much as two- to threefold without altering the general capacity for proteolysis. The effects are complicated by, but experimentally distinguishable from, secondary changes in proteolysis that accompany growth inhibition. In contrast, no potentiation of proteolysis is elicited by the presence of suppressor genes, by the administration of heat, or by the biosynthetic alterations attending large changes in the conditions of cultivation or by those attending bacteriophage infection. Thus, although mass perturbations in protein conformation are catabolically distinguishable, the more individual and limited conformational modifications that might occur in disuse do not appear to be the primary determinants of the protein turnover rate. In Bacillus subtilis, turnover synthesis of protein during starvation is as susceptible to treatment with actinomycin D as that during growth. Treatment alters neither the rate of intracellular proteolysis nor the catabolic pattern of the modicum of proteins that are still synthesized. It is concluded that there is no correlation between metabolic stability of protein and the stability of its messenger ribonucleic acid.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. II. Inhibition by inhibitors of protein synthesis.

The effect of inhibitors of protein synthesis on the killing of tumor cells by in vitro activated macrophages was determined. Cytotoxicity was inhibited by concentrations of puromycin, pactamycin, and actinomycin D that almost completely inhibited protein synthesis by guinea pig macrophages, but not by concentrations of drug that inhibited protein synthesis by only ± 50%. Cytotoxicity was inhibited when the effector macrophages were pretreated with the metabolic inhibitors, but not when the drugs were added 30 to 60 min after the initiation of the reaction. Pretreatment with puromycin or pactamycin also markedly inhibited the binding of tumor cells by mediator activated macrophages. These results are consistent with the hypothesis that one possible mechanism by which inhibitors of protein synthesis inhibit macrophage mediated cytotoxicity is by inhibiting close contact between effector and target cells. The finding that pretreatment of activated macrophages with trypsin also inhibits tumor cell killing suggests that protein synthesis may be necessary to maintain an adequate number of “recognition structures” on the macrophage membrane, structures that mediate the initial contact between the activated macrophage and the target tumor.     

Repair Replication of HeLa Cell Deoxyribonucleic Acid in Cells Infected with Newcastle Disease Virus or Mengovirus or Treated with Puromycin

We examined repair replication of HeLa cell deoxyribonucleic acid (DNA) in cells infected with mengovirus or Newcastle disease virus or treated with puromycin. Cellular DNA was damaged by ultraviolet light and then pulse-labeled with (3)H-thymidine. Autoradiographic analysis of non-S-phase DNA synthesis (repair replication) showed that there was no inhibition of this process at a time when overall cellular DNA synthesis was severely inhibited by either virus infection or puromycin treatment.     

Inhibition of hepatocytic protein degradation by methylaminopurines and inhibitors of protein synthesis

Nutritional control of protein degradation in isolated rat hepatocytes can take place in the absence of protein synthesis. Suppression of degradation by amino acids (step-up) is unaffected and the enhanced degradation seen upon amino acid deprivation (step-down) is only partially inhibited by cycloheximide at a concentration (10^?3 M) which inhibits protein synthesis virtually completely. Protein degradation per se is, however, inhibited by cycloheximide as well as by puromycin, apparently at least in part by mechanisms additional or unrelated to their effect on protein synthesis. Several puromycin analogues (methylaminopurines) are stronger inhibitors of protein degradation than of protein synthesis, most notably puromycin aminonucleoside and 6-dimethylaminopurine riboside (N⁶, N⁶-dimethyladenosine). The latter compounds appear to specifically inhibit cellular autophagy, since neither the degradation of endocytosed protein (asialofetuin) nor the extralysosoma (amino acid-, propylamine- and leupeptin-resistant) degradation are affected.     

A comparison of the effects of metabolic inhibitors upon direct and antibody-dependent lymphocyte mediated cytotoxicity

The effect of metabolic inhibitors upon the lysis of allogeneic lymphocyte target cells by cytotoxic T lymphocytes and nonimmune lymphocyte K cells were studied by using identical culture conditions. Inhibition of lysis in both systems was induced by cycloheximide, puromycin, actinomycin D, diisopropylfluorophosphate, 2-deoxyglucose, antimycin A, oligomycin, and cytochalasins A and B, whereas ouabain and mitomycin C did not diminish lysis in either system. The strikingly parallel responses to a variety of agents that alter cellular processes suggest that both forms of lysis utilize similar mechanisms.     

Effects of ATP and cell development on the metabolism of high molecular weight aggregates of abnormal proteins in rabbit reticulocytes and cell-free extracts

Abnormal proteins synthesized in rabbit reticulocytes in response to (i) the lysine analogue aminoethylcysteine and (ii) puromycin, form high molecular weight aggregates prior to degradation. Inhibitors of ATP synthesis partially inhibit catabolism of the aminoethylcysteine-induced abnormal protein; degradation of puromycin peptides synthesized after incubation with 25 μg/ml puromycin was not inhibited. Catabolism of the analogue-induced high molecular weight aggregate of abnormal protein in cell-free extracts was markedly stimulated by ATP, whereas proteolysis of the aggregated puromycin-peptides was ATP-independent. The ability of the reticulocytes to degrade the puromycin-peptide aggregates was found to decrease with cellular maturity. It is suggested that the energy-dependency for proteolysis is in some way related to the chain length of the abnormal protein synthesized.     

The metabolic requirements for transcellular movement and secretion of collagen.

Cultures of chick tendon fibroblasts were capable of normal ATP production and protein synthetic activity even though the normally high rate of glycolysis was markedly reduced by substitution of pyruvate for glucose. Iodoacetate and 2-deoxyglucose reduced ATP levels and protein synthesis even in the presence of pyruvate. Under these conditions, both inhibitors were shown to have effects on the energy metabolism of cells which were apparently unrelated to an inhibition of glycolysis. Selective inhibition of either glycolysis, by incubation in glucose-free medium, or of oxidative phosphorylation, by incubation with an uncoupler, was shown to have little effect on cellular ATP levels or intracellular transport and secretion of collagen. However, inhibition of both glycolysis and oxidative phosphorylation resulted in decreased cellular ATP levels and an inhibition of collagen secretion. This effect was not due to a requirement for continued protein synthesis, since inhibition of protein synthesis with cycloheximide or puromycin had little effect on collagen secretion. The ATP requirement for intracellular transport and secretion is discussed in relation to the secretory pathway for collagen.     

Effect of puromycin on sugar transport in isolated rat adipocytes

This study was performed to determine whether puromycin effects sugar transport into isolated rat adipocytes. Time-course and kinetic studies demonstrated that puromycin competitively inhibited 3-O-methylglucose transport. The data indicate that there is an acute effect of puromycin on sugar transport via competitive inhibition that is independent of the inhibition of protein synthesis.     

Effect of puromycin on protein and glycerolipid biosynthesis in isolated mucosal cells

The effect of puromycin on phosphatidylcholine and triacylglycerol synthesis was studied in isolated cells of rat intestinal mucosa using radioactive palmitate, glycerol, 2-hexadecylglycerol, and lysophosphatidylcholine as markers. Puromycin caused a 60–65% inhibition of phosphatidylcholine biosynthesis but did not affect the formation of triacylglycerols. Under comparable conditions protein synthesis was inhibited 90–95% and glycoprotein synthesis 60–70%. The utilization of the various lipid precursors indicated that puromycin inhibited the biosynthesis of phosphatidylcholine via both the CDP-choline and the lysophosphatidylcholine pathways, without interfering with triacylglycerol synthesis from either phosphatidic acid or monoacylglycerol precursors. Since both phosphatidylcholines and proteins are involved in the assembly of chylomicrons, it is suggested that the effect of puromycin on chylomicron formation could be due to an inhibition of the biosynthesis of any one or all three of the membrane components: proteins, glycoproteins, and phosphatidylcholines.     

Relationship between cell-mediated and humoral immune attack on tumor cells. I. Drug and hormone effects on susceptibility to killing and macromolecular synthesis

The effect of metabolic inhibitors and hormones on the susceptibility of P815 murine mastocytoma cells to antibody-C and cell-mediated killing, and on the ability of the cells to synthesize DNA, RNA, protein, carbohydrate, and lipid was tested. Pretreatment of the cells with adriamycin, actinomycin D, and puromycin increased the sensitivity of the cells to killing by rabbit anti-P815 antibody plus guinea pig C, but not by allogeneic P815-sensitized spleen cells. Conversely, mitomycin C treatment enhanced the cells' sensitivity to cell-mediated, but not antibody-C, killing. Insulin and hydrocortisone, but not epinephrine, were effective in decreasing the susceptibility of the cells to killing by both antibody-C and cell-mediated attack systems. The kinetics of the drug-induced increase and the hormone-induced decrease in susceptibility of the cells to antibody-C killing correlated with a decrease and increase, respectively, in the ability of the cells to incorporate fatty acid into complex cellular lipid. No such correlation was found between cellular lipid synthesis and tumor cell susceptibility to cell-mediated killing. The ability of the cells to resist either form of immune attack was not dependent on the ability of the cells to synthesize DNA, RNA, protein, or complex carbohydrate. These results suggest that the susceptibility of these tumor cells to antibody-C vs cell-mediated killing may be linked to different metabolic properties of the cells, and may reflect differences in the mechanisms of humoral vs cellular immune attack.     

The Relation Between Ion Absorption and Protein Synthesis in Beet Disks

Disks of red beet storage tissue were incubated under asepticconditions permitting the development of various metabolic processescommonly associated with aged disks, and the effects of chloramphenicoland puromycin on protein synthesis, on the development of invertaseactivity and ion absorption capacity, and on ion absorptionper se were determined. Low concentrations of chloramphenicoland puromycin inhibit the development of ion absorption capacitybut stimulate invertase development and protein synthesis, whilehigher concentrations inhibit all three processes. In contrastion absorption itself is unaffected by puromycin, but is sensitiveto quite low concentrations of chloramphenicol The D-threo andL-threo isomers of chloramphenicol have sharply contrasted effectson the development, as distinct from the utilization, of ionabsorption capacity. The D isomer inhibits the development ofion absorption capacity more effectively than the L isomer whichin turn inhibits absorption more effectively than the D isomer. A reappraisal is made of the hypothesis that ion absorptionis directly linked with protein turnover and to account forthe results a model is proposed in which D-threo-chloramphenicolis active both as an uncoupler of oxidative phosphoryalationand as an inhibitor of protein synthesis, while L-threo-chloramphenicolacts only in the former capacity and puromycin only in the latter.It is concluded that the inhibition of ion uptake by chloramphenicolcannot be attributed to a contemporaneous effect on proteinsynthesis. However, the results are consistent with the involvementof ATPase proteins in ion uptake.     

Effect of protein-synthesis inhibitors on testosterone production in rat testis interstitial tissue and Leydig-cell preparations.

Luteinizing-hormone-stimulated testosterone biosynthesis was inhibited by cycloheximide during incubation of rat testis intersitial tissue in vitro and also by puromycin and cycloheximide during incubation of Leydig-cell preparations, but not by chloramphenicol. These results suggest that a protein regualtor(s) formed by cytoplasmic protein synthesis is involved in steroidogenesis in the rat testis. The specific effect of cycloheximide and puromycin on protein synthesis rather than on other non-specific processes is suggested by the inhibition of protein synthesis and steroidogenesis with different doses of the inhibitors and the lack of effect of cycloheximide on luteinizing-hormone-induced adenosine 3':5'-cyclic monophosphate production. Stimulation of testosterone production by luteinizing hormone during superfusion of interstitial tissue was detectable within 10-20 min and reached a maximum of 120 min, and thereafter slowly decreased. Cycloheximide added at maximum steroid production caused a rapid decrease in testosterone synthesis which followed first-order kinetics (half-life 13 min), thus indicating that the protein regulator(s) has a short half-life. No effect of cycloheximide, puromycin or chloramphenicol on testosterone production in the absence of added luteinizing hormone was found, suggesting that the basal production of testosterone is independent of protein synthesis.     

Effect of different protein synthesis inhibitors on the exocrine pancreatocytic autophagocytosis in vivo.

The translational inhibitor cycloheximide is also used as an inhibitor of cellular autophagy and intracellular degradation of endogenous cellular proteins. Some evidence for a similar effect of other inhibitors of protein biosynthesis is also available (largely from in vitro systems). In the present study, the in vivo effects of cycloheximide, emetine and puromycin on autophagy in murine exocrine pancreatic and liver cells were tested using electron microscopic morphometry. The experiments were based on the fact that when the formation of autophagosomes is inhibited, a regression of the autophagolysosomal compartment can be measured, provided intralysosomal degradation in the pre-existing autophagic vacuoles continues at an unchanged rate. To make the measurements easier, autophagolysosomal compartment of the cells was enlarged by administering vinblastine (10 mg/kg b.wt.) for 2 h when the inhibitors were given for an additional 30 min. During this time cycloheximide (0.2 mg/g b.wt.), emetine (0.12 mg/g b.wt.) and puromycin (0.2 mg/g b.wt.), respectively caused 35, 25 and 52% regression of the pancreatocytic autophagolysosomal compartment. Since all the above translational inhibitors inhibited autophagocytosis as well, the possibility of a coupling between the regulation of synthesis and inhibition of proteins arises.