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Purified diphtheria toxin is shown to inhibit protein synthesis in Ehrlich-Lettré ascites carcinoma cells in vitro. Protein synthesis in Ehrlich-Lettré cells is at least 10,000 times more sensitive to toxin than protein synthesis in normal mouse spleen or thymus cells. This sensitivity correlates with the observation that Ehrlich-Lettré tumors regress in mice injected with diphtheria toxin but not diphtheria toxoid. Using the criterion of inhibition of protein synthesis in vitro, we show that other mouse malignancies (lymphoma and myeloma) are also more sensitive to diphtheria toxin than normal spleen or thymus. Metastatic human breast carcinoma cells from two individuals, cells from two melanoma nodules removed at different times from a third patient, and cells from melanoma nodules from three additional individuals are shown to be more sensitive to diphtheria toxin than some normal human cells. The toxin sensitivity of protein synthesis in some of the malignant cells tested was so much greater than that of normal cells, that we have proposed that diphtheria toxin should be studied further since it might prove a useful anti-cancer agent in patients whose tumors are first shown to be highly sensitive to toxin in vitro.

L-Asparaginase (EC 3.5.1.1) inhibited respiration in sensitive, but not resistant, lines of murine lymphoma 6C3HED. Glucose, in these tumor lines, was principally converted to lactate, and very little was oxidized in the citric acid cycle or hexose monophosphate shunt. The cells derived 70-80% of their respiratory CO(2) from glutamine or glutamate. Asparaginase had no effect on the pattern of glucose utilization. The differential effect on oxygen consumption may result from the absence of asparagine synthetase in sensitive cells. Respiration may be inhibited by accumulation of the aspartate, the product of glutamate oxidation. Resistant lymphoma cells remove aspartate by converting it to asparagine. Sensitive cells, which lack asparagine synthetase, cannot make asparagine.

A selection procedure was devised for neurons and related cells that depends upon the ability of the cells to synthesize certain amine neurotransmitters. The rationale for selection is that tyrosine is an essential amino acid for most mammalian cells and that three enzymes from mammalian sources can catalyze the synthesis of tyrosine: phenylalanine hydroxylase (EC 1.14.16.1), tyrosine hydroxylase (EC 1.14.16.2), and tryptophan hydroxylase (EC 1.14.16.4). Tyrosine hydroxylase is found predominantly in adrenergic neurons and related cells that synthesize dopamine, norepinephrine, and epinephrine, and tryptophan hydroxylase in cells synthesizing serotonin or melatonin. Only 1 out of 70,000 uncloned mouse neuroblastoma cells grew well in the absence of tyrosine. Approximately 50% of the cell lines obtained by selection had tyrosine hydroxylase activity. This selection procedure thus provides a simple means of obtaining cell lines of neural origin on the basis of their ability to synthesize putative transmitters.

The metabolism of benzo (a) pyrene (BP) in normal golden hamster and BHK cells in culture was increased by treating the cells with dibutyryl cyclic AMP (dcAMP), prostaglandin E1, theophylline or aminophylline. The largest increase, 6-fold for the normal cells and 20-fold for the BHK cells, was obtained by treatment with both dcAMP and aminophylline. Treatment with aminophylline also stimulated the metabolism of 20-methylcholanthrene and 7,12-dimethylbenz (a) anthracene. The increased metabolism of these three carcinogeneic polycyclic hydrocarbons was associated with an increased cytotoxicity. Treatment with aminophylline increased the cytotoxicity of five other potent and weak carcinogenic polycyclic hydrocarbons, but not of two non-carcinogenic polycyclic hydrocarbons. The amount of BP metabolism in 27 different cell types from various mammals, including humans, ranged from less than 0.1 mug to 2.3 mug metabolized BP per 10-6 cells. Treatment of these different cell types with aminophylline gave either an increase in BP metabolism, an induction of metabolism in cells that did not metabolize without aminophylline, or no induction after treatment with aminophylline and dcAMP. The existence of responding and non-responding cell lines indicates that the regulation of the level of polycyclic hydrocarbon metabolism by dcAMP is genetically controlled. The induction of metabolism in cells that did not metabolize without aminophylline resulted in the conversion of cell resistance to cell susceptibility to the cytotoxic effect of BP. Treatment with dcAMP and aminophylline can therefore be used to increase the sensitivity of screening tests for chemical carcinogens.

Cyclic stimulation by Ca(2+) of respiration in mitochondria isolated from Ehrlich ascites-tumour cells occurs only when low phosphate concentrations (approx. 0.5mm) are also included in the incubation system. Under these circumstances the extra oxygen consumed is related stoicheiometrically to the amount of Ca(2+) taken up by the mitochondria; the values are similar to those obtained with mitochondria from rat liver in the absence of added phosphate. In contrast with liver mitochondria, up to 280nmol of Ca(2+)/mg of protein can be added to ascites mitochondria without causing any deleterious effect. Respiration in mitochondria isolated from the Yoshida ascites hepatoma (HA 130) and from the Morris hepatomas 5123C and 9618A is also stimulated by Ca(2+) in a cyclic manner. However, that in mitochondria from regenerating rat liver responds to Ca(2+) in the same way as those from normal rat liver. ADP-stimulated respiration in mitochondria from Ehrlich ascites tumour cells, but not from rat liver, is inhibited by low amounts of Ca(2+).

1. Preparations of mouse ascites-tumour cells depleted of ATP and Na(+) ions accumulated l-methionine, in the presence of cyanide and deoxyglucose, from a 1mm solution containing 80mequiv. of Na(+)/l and about 5mequiv. of K(+)/l. Valinomycin increased, from about 4 to 16, the maximum value of the ratio of the cellular to extracellular concentrations of methionine formed under these conditions without markedly affecting the distributions of Na(+) and of K(+). Similar observations were made with 2-aminoisobutyrate, glycine and l-leucine. Increasing the extracellular concentration of K(+) progressively decreased the accumulation of methionine in the presence of valinomycin. Over the physiological range of ionic gradients, the system behaved as though the absorption of methionine with Na(+) was closely coupled to the electrogenic efflux of K(+) through the ionophore. The process was insensitive to ouabain and so the sodium pump was probably not involved. 2. The amount of methionine accumulated during energy metabolism was similar to the optimal accumulation in the presence of valinomycin when ATP was lacking. It was also similarly affected by increasing the methionine concentration. 3. A mixture of nigericin and tetrachlorosalicylanilide mimicked the action of valinomycin. The anilide derivative inhibited the absorption of 2-aminoisobutyrate in the presence of valinomycin, but not in its absence. 4. Gramicidin inhibited methionine absorption and caused the preparations to absorb Na(+) and lose K(+). 5. The observations appear to verify the principle underlying the gradient hypothesis by showing that the tumour cells can efficiently couple the electrochemical gradient of Na(+) to the amino acid gradient.

Polyribosomes were isolated from a clonal line of mouse neuroblastoma grown in culture. In a heterologous in vitro system containing rat brain components, these polyribosomes were shown to direct the synthesis of neuroblastoma tubulin. Identification of the tubulin synthesized in vitro was achieved by coelectrophoresis with native neuroblastoma tubulin on sodium dodecyl sulfate polyacrylamide gels, immunoprecipitation, and demonstration of specific aggregation. Tubulin accounted for 2% of the total proteins synthesized. This in vitro protein synthesizing system offers a model for studying possible translational control mechanisms regulating the synthesis of proteins involved in nerve cell function.