Barbara A. Booth

Folate metabolism in methotrexate-sensitive and -resistant Ehrlich ascites cells☆

Abstract A methotrexate-resistant subline of the Ehrlich ascites carcinoma contained approximately 14-fold more dihydrofolate reductase activity than did the drug-sensitive parent neoplasm. The activities of a number of tetrahydrofolate-dependent enzymes (i.e., of N 5 -formyltetrahydrofolate synthetase, N 5 , N 10 -methylenetetrahydrofolate dehydrogenase, N 5 -formyltetrahydrofolate isomerase, and serine hydroxymethylase) in cells of the resistant subline were similar to those of the sensitive neoplasm; thus, the increase in dihydrofolate reductase activity in the methotrexate-resistant cells was specific. Several properties of partially purified dihydrofolate reductase preparations from each cell line were similar, suggesting that no quantitative changes in the enzyme accompanied the transition to the resistant state. The elevated dihydrofolate reductase activity in the drug-resistant variant appeared to be sufficient to account for the refractoriness of this neoplasm, since considerable enzyme activity could be detected in these cells after large amounts of methotrexate. The problems of relating in vitro enzyme assays to events occurring in vivo are discussed.

Metabolic effects of zinc in intact cells—Comparative studies of zinc chloride and the zinc chelate of kethoxal bis(thiosemicarbazone)

Abstract Kethoxal bis(thiosemicarbazone)Zinc(II) [Zn(II)KTS] and zinc chloride were both cytotoxic to sarcoma 180 ascites cells; equivalent quantities of zinc administered in chelate form were considerably more toxic to the neoplastic cells. This correlated with the finding of greater accumulation of zinc by cells treated with Zn(II)KTS, as compared to those exposed to ZnCl 2 . In general, however, the metabolic consequences of ZnCl 2 appeared to be greater than those of Zn(II)KTS. The biosynthesis of DNA as measured by the incorporation of [ 3 H]thymidine and [8- 14 C]adenine into DNA was markedly sensitive to both agents; although thymidine was converted to the triphosphate level at a rate comparable to controls in cells in which the biosynthesis of DNA was initially inhibited by Zn(II)KTS or ZnCl 2 , at later times after drug treatment the degree of phosphorylation of thymidine was markedly depressed. Shortly after exposure of cells to Zn(II)KTS, the incorporation of [6- 14 C]orotic acid into RNA was strongly inhibited, while the utilization of [5,6- 3 H]uridine and [U- 14 C]aspartic acid for the biosynthesis of these macromolecules was unaffected. In contrast, ZnCl 2 initially caused inhibition of the incorporation of [ 3 H]uridine into RNA, concomitant with pronounced stimulation of the conversion of [6- 14 C]orotic acid and [ 14 C]aspartic acid to RNA. Inhibition of the incorporation of all labeled precursors into RNA accompanied prolonged exposure to either of these agents. Significant blockage of equal magnitude of the incorporation of [1- 14 C]leucine into protein and of glycolysis was produced by Zn(II)KTS or ZnCl 2 ; however, Zn(II)KTS caused greater inhibition than ZnCl 2 of the respiration of sarcoma 180 cells.

Alterations in purine nucleotide biosynthesis induced by 2-amino-6-chloropurine☆

Abstract Exposure of sarcoma 180 ascites cells to 2-amino-6-chloropurine (6-C1G) in vivo resulted in several metabolic lesions in the pathways of purine nucleotide biosynthesis. Pronounced inhibition of the incorporation of glycine-2- 14 C into the guanine and adenine nucleotides of the nucleic acids, coupled with blockade of the utilization of 4-amino-5-imidazolecarboxamide-2- 14 C solely for the formation of guanine nucleotides, indicated the presence of a site of inhibition on the pathway of purine nucleotide formation de novo . The conversion of hypoxanthine-8- 14 C to nucleic acid guanine was markedly decreased by 6-CIG, whereas the utilization of this labeled precursor for the formation of nucleic acid adenine was not lowered by this agent. Under these conditions neither the incorporation of xanthine-8- 14 C nor of guanine-8- 14 C into the guanine moiety of the nucleic acids was depressed, indicating that 6-CIG or a derivative thereof inhibited the conversion of inosine 5′-phosphate to xanthosine 5′-phosphate. Blockade of this site was confirmed by the finding that 2-amino-6-chloropurine ribonucleoside 5′-phosphate inhibited the activity of inosine 5′-phosphate dehydrogenase. Molar equivalent levels of 2-amino-6-chloropurine ribonucleoside and 2-amino-6-chloropurine ribonucleoside triacetate caused a degree of inhibition of the conversion of hypoxanthine-8- 14 C to guanine nucleotides comparable to that produced by 6-C1G. 2-Amino-6-iodopurine ribonucleoside was at least as active an inhibitor of the formation of nucleic acid guanine as the chloro-derivatives.

Sarcoma 180 inhibition by combinations of 6-thioguanine and uracil mustard.

Combinations of 6-thioguanine and uracil mustard produced greater inhibition of the growth of sarcoma 180 than that equivalent to the sum of the inhibitory effects caused by the optimum levels of the individual drugs. This potentiation was accomplished without marked weight loss by the host. Some possible biochemical mechanisms responsible for the drug synergy are discussed.

Some factors affecting the tumor-inhibitory properties of combinations of azaserine and 6-chloropurine☆

Abstract The prolongation of the survival time of mice bearing sarcoma 180 ascites cells caused by combinations of 6-chloropurine and azaserine appeared to be insensitive to moderate increases in the dosage of azaserine. Maximum effectiveness of the combination of these agents occurred when either simultaneous administration of the two drugs was employed or the 6-chloropurine was given 1 hr after the azaserine; an interval of more than 3 hr caused a marked reduction in the degree of synergism. Therapy could be initiated up to 4 days after tumor implantation with some retention of antitumor properties by the combination. The ability of several purines and purine ribonucleosides to antagonize the chloropurine-induced enhancement of azaserine inhibition of sarcoma 180 was determined: guanosine prevented synergism at all levels of chloropurine tested, while adenosine, inosine, and hypoxanthine antagonized only at the lower levels of chloropurine administered. Adenine did not prevent drug enhancement. The results suggest that drug-induced blocks on the pathway to guanine nucleotides are associated with the synergism exhibited by combinations of 6-chloropurine and azaserine; however, an additional unknown site of blockade appears to be necessary for potential ion.

The effect of 4-amino-5-imidazolecarboxamide on the synergistic antineoplastic activity of 6-chloropurine and azaserine

4-Amino-5-imidazolcarboxamid hat eine antagonistische Wirkung auf die synergistische Tumorhemmung, die sich aus der gemeinsamen Anwendung von 6-Chlorpurin und Azaserin ergibt. Dies steht im Einklang mit der Fähigkeit des 4-Amino-5-imidazolcarboxamids, auch die Hemmung des Einbaues von C14-Format in das Guanin von Polynucleotiden, durch 6-Chlorpurin hervorgerufen, teilweise zu unterbinden.