L. Lee Bennett

INHIBITION OF DNA SYNTHESIS IN MAMMALIAN CELLS BY ACTIDIONE.

Abstract In mammalian cells in culture, actidione markedly inhibited the incorporation of a number of precursors into DNA under conditions that produced little or no inhibition of incorporation into RNA. In attempts to define the site of action responsible for the selective inhibition of DNA synthesis, it was found that (a) in cell-free systems actidione was without effect on DNA polymerase (deoxynucleoside-triphosphate: DNA deoxynucleotidytransferase, EC 2.7.7.7) and kinases (phosphotransferase, EC class 2.7.4) for deTMP, deCMP, deAMP, and deGMP; and (b) cell-free supernatants from cells exposed in culture to actidione contained all of the enzyme activities required for incorporation of deTMP in vitro into DNA. In addition, several types of studies failed to provide evidence for a binding of actidione to DNA in vitro. The related glutarimide antibiotics, streptovitacin A and E-73, were also without effect on DNA synthesis in vitro as measured by incorporation of deTMP. Under all experimental conditions used, actidione also profoundly depressed protein synthesis. Inhibitions of synthesis of both protein and DNA occurred so rapidly after exposure of cells to actidione that a temporal relationship between the initiation of inhibition of the two processes has not yet been established.

Metabolism and Metabolic Actions of 6-Methylpurine and 2-Fluoroadenine in Human Cells

Activation of purine nucleoside analogs by Escherichia coli purine nucleoside phosphorylase (PNP) is being evaluated as a suicide gene therapy strategy for the treatment of cancer. Because the mechanisms of action of two toxic purine bases, 6-methylpurine (MeP) and 2-fluoroadenine (F-Ade), that are generated by this approach are poorly understood, mechanistic studies were initiated to learn how these compounds differ from agents that are being used currently. The concentration of F-Ade, MeP, or 5-fluorouracil required to inhibit CEM cell growth by 50% after a 4-hr incubation was 0.15, 9, or 120 microM, respectively. F-Ade and MeP were also toxic to quiescent MRC-5, CEM, and Balb 3T3 cells. Treatment of CEM, MRC-5, or Balb 3T3 cells with either F-Ade or MeP resulted in the inhibition of protein, RNA, and DNA syntheses. CEM cells converted F-Ade and MeP to F-ATP and MeP-ribonucleoside triphosphate (MeP-R-TP), respectively. The half-life for disappearance of HeP-ribonucleoside triphosphate from CEM cells was approximately 48 hr, whereas the half-lives of F-ATP and ATP were approximately 5 hr. Both MeP and F-Ade were incorporated into the RNA and DNA of CEM cells. These studies indicated that the mechanisms of action of F-Ade and MeP were quite different from those of other anticancer agents, and suggested that the generation of these agents in tumor cells by E. coli PNP could result in significant advantages over those generated by either herpes simplex virus thymidine kinase or E. coli cytosine deaminase. These advantages include a novel mechanism of action resulting in toxicity to nonproliferating and proliferating tumor cells and the high potency of these agents during short-term treatment.

Inhibition of protein synthesis in vitro by cycloheximide and related glutarimide antibiotics

Abstract Cycloheximide (actidione) and the related glutarimide antibiotics, E-73, streptovitacin A, and streptimidone, have been studied as inhibitors of protein synthesis in vitro; mouse Adenocarcinoma 755 cells in culture were used as the source of the ribosomal and S100-pH 5 fractions. Each of these compounds markedly inhibited the incorporation of [14C]phenylalanine, [14C]lysine, and [14C]leucine in the presence of endogenous messenger RNA and the incorporation of [14C]phenylalanine in the presence of added polyuridylic acid. Inhibition occurred after the formation of aminoacyl soluble RNAs. E-73 was somewhat more effective than the other glutarimides, and the activity of the glutarimides was about the same as that of puromycin.

Activity of adenos1ne analogs against a cell culture, line resistant to 2-fluoroadenine☆

Abstract By serial transfer in the presence of 2-fluoroadenine, a subline of H.Ep. #2 cells in culture was selected for high resistance to 2-fluoroadenine. The resistance was stable when the cells were cultured in the absence of 2-fluoroadenine. The resistant cells were essentially devoid of AMP pyrophosphorylase activity, but retained IMP and GMP pyrophosphorylase and purine nucleoside kinase activities. Intact growing cells of the parent and resistant lines did not differ in capacity to incorporate into polynucleotides 14 C from adenosine-8- 14 C and hypoxanthine-8- 14 C, but, as compared to sensitive cells, the resistant cells incorporated 14 C from adenine-8- 14 C very poorly. The resistant cells were cross-resistant to adenine analogs but did not differ from the parent cells in sensitivity to a series of adenosine analogs. Many nucleosides were much more toxic to the parent cell line than were the corresponding free bases. These results suggest that a purine nucleoside kinase, presumably adenosine kinase, is the critical enzyme in the. activation of these cytotoxic analogs of adenosine.

Gene Therapy of Cancer: Activation of Nucleoside Prodrugs with E. coli Purine Nucleoside Phosphorylase

During the last few years, many gene therapy strategies have been developed for various disease targets. The development of anticancer gene therapy strategies to selectively generate cytotoxic nucleoside or nucleotide analogs is an attractive goal. One such approach involves the delivery of herpes simplex virus thymidine kinase followed by the acyclic nucleoside analog ganciclovir. We have developed another gene therapy methodology for the treatment of cancer that has several significant attributes. Specifically, our approach involves the delivery of E. coli purine nucleoside phosphorylase, followed by treatment with a relatively non-toxic nucleoside prodrug that is cleaved by the enzyme to a toxic compound. This presentation describes the concept, details our search for suitable prodrugs, and summarizes the current biological data.

FEEDBACK INHIBITION OF PURINE BIOSYNTHESIS IN H. EP. 2 CELLS BY ADENINE ANALOGS.

Abstract Fifteen analogs and derivatives of adenine have been evaluated as feedback inhibitors ofpurine synthesis in H. Ep. # 2 cells in culture. Of these compounds, only four—2-fluoroadenine, 2-fluoroadenosine, 8-aza-adenosine, and 7-deaza-adenosine (tubercidin)—were as active as adenine, which inhibited by 50% or more at a concentration of 3.7 μM. 4-Aminoimidazo (4,5- d )pyridazine also caused feedback inhibition at relatively low concentrations. 4-Aminopyrazolo(3, 4- d )pyrimidine, although toxic to H.Ep. #2 cells at about the same concentration as fluoroadenine, produced feedback inhibition only at concentrations much in excess of the toxic concentration. Substitution of adenine or fluoroadenine at the 9-position destroyed or markedly reduced the capacity for feedback inhibition. The results suggest that feedback inhibition may be a factor in the growth-inhibiting activity of some of these agents.

6-Methylthioguanylic acid, a metabolite of 6-thioguanine

Abstract Human epidermoid carcinoma (H. Ep. No. 2) cells, grown in culture in the presence of 6-thioguanine- 35 S or methionine[methyl- 14 C] plus unlabeled 6-thioguanine, contained a previously undescribed metabolite that was characterized by Chromatographic and electrophoretic migration and by chemical and enzymatic hydrolysis as 6-methylthioguanylic acid. Two other nucleotides present in these cells had the properties expected of the di- and triphosphates of 6-methylthioguanosine. In contrast to previously reported results with 6-mercaptopurine ribonucleotide and its S-methyl derivative, 6-methylthioguanylic acid was inferior to 6-thioguanylic acid as an inhibitor of PP-ribose-P amidotransferase. Thus, the methylated derivatives of 6-thioguanylic acid probably do not contribute significantly to the marked inhibition of purine synthesis de novo produced in mammalian cells by 6-thioguanine

Metabolism and Metabolic Effects of Halopurine Nucleosides in Tumor Cells in Culture

Abstract Within a series of halo derivatives of adenosine, deoxyadenosine and arabinosyladenine attempts have been made to correlate structure with cytotoxicity, substrate activity for adenosine deaminase and nucleoside kinases, and efficacy of the corresponding nucleotides against target enzymes.

The Synthesis and Biological Activity of Certain 4′-Thionucleosides

Abstract Results are presented on the synthesis and biological activity of several types of 4′-thionucleosides as potential anticancer agents. Detailed studies on the mechanism of action of 4′-thiothymidine are also presented.

Inosine analogs as substrates for adenosine kinase—Influence of ionization of the N−1 proton on the rate of phosphorylation

This study was undertaken to attempt to rationalize previously obtained and apparently conflicting findings that although adenosine kinase (EC 2.7.1.20) from H.Ep. # 2 cells did not accept inosine as a substrate, these cells became resistant to an inosine analog, 8-azainosine, only when activities of both hypoxanthine (guanine) phosphoribosyltransferase [H(G)PRTase] and adenosine kinase were lost. No evidence could be found for the presence of inosine kinase in H.Ep. # 2 cells: crude supernatants from these cells converted ring-labeled inosine to IMP, but the conversion was prevented by the addition of hypoxanthine and therefore apparently was achieved by the alternative pathway involving the action of H(G)PRTase on hypoxanthine. An investigation of inosine and inosine analogs as substrates for adenosine kinase revealed that certain inosine analogs were substrates and that the substrate activity could be correlated with the degree of ionization of the N-1 proton. Of four 6-oxo and 6-thio nucleosides studied. 8-aza-6-thioinosine had the lowest pKa (6.75) and was the only one that was a good substrate at pH 7.0; the Km was 210μM and the Vmax was about 1.5 times that of adenosine. The rate of phosphorylation of 8-aza-6-thioinosine increased markedly as the pH of the reaction mixture was increased in the pH range 6.0 to 7.0. Phosphorylation of 8-azainosine (pKa 7.45) and 6-thioinosine (pKa 7.60) was much poorer and could be demonstrated at pH 7.0 only after overnight incubation with the kinase. The fact that 8-azainosine and 8-aza-6-thioinosine are substrates whereas ionsine is not, can be rationalized by the facts that (a) the substitution of an N-atom for the C-8 atom of the nucleoside lowers the pKa so that the N-1 proton is more strongly ionized at physiological pH; and (b) the ionized form of a 6-oxo or 6-thio nucleoside resembles adenosine with respect to the bond structure at the 1- and 6-positions of the purine ring whereas the unionized form does not.