Comparison of the actions of 9-beta-D-arabinofuranosyl-2-fluoroadenine and 9-beta-D-arabinofuranosyladenine on target enzymes from mouse tumor cells.

9-beta-D-Arabinofuranosyl-2-fluoroadenine (2-F-ara-A), a derivative of 9-beta-D-arabinofuranosyladenine (ara-A) that is resistant to deamination, selectively inhibits DNA synthesis and has activity against mouse leukemia L1210 comparable to that of ara-A plus the adenosine deaminase inhibitor, 2'-deoxycoformycin. To determine if these two nucleosides have similar modes of action, comparisons were made of their effects and those of their triphosphates on enzymes known to be inhibited by ara-A or 9-beta-D-arabinofuranosyladenine 5'-triphosphate. 9-beta-D-Arabinofuranosyl-2-fluoroadenine 5'-triphosphate was more effective than 9-beta-D-arabinofuranosyladenine 5'-triphosphate in inhibiting the reduction of adenosine 5'-diphosphate and cytidine 5'-diphosphate by ribonucleotide reductase from HEp-2 cells or L1210 cells. DNA polymerase alpha from L1210 cells was equally sensitive to 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate and 9-beta-D-arabinofuranosyladenine 5'-triphosphate, and DNA polymerase beta from L1210 cells was much less sensitive to both triphosphates. S-Adenosylhomocysteine hydrolase from L1210 cells was inactivated by 2-F-ara-A and ara-A, but higher concentrations of the fluoro derivative were required. These results are consistent with 2-F-ara-A and ara-A inhibition of DNA synthesis by inhibition of ribonucleotide reductase and DNA polymerase alpha.

Effects of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine on K562 cellular metabolism and the inhibition of human ribonucleotide reductase and DNA polymerases by its 5'-triphosphate.

2-Chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine (Cl-F-ara-A) has activity against the P388 tumor in mice on several different schedules. Biochemical studies with a chronic myelogenous leukemia cell line (K562) grown in cell culture have been done in order to better understand its mechanism of action. Cl-F-ara-A was a potent inhibitor of K562 cell growth. Only 5 nM inhibited K562 cell growth by 50% after 72 h of continuous incubation. The 5'-triphosphate of Cl-F-ara-A was detected by strong anion exchange chromatography of the acid-soluble extract of K562 cells incubated with Cl-F-ara-A. Competition studies with natural nucleosides suggested that deoxycytidine kinase was the enzyme responsible for the metabolism to the monophosphate. Incubation of K562 cells for 4 h with 50 nM Cl-F-ara-A inhibited the incorporation of [3H]thymidine into the DNA by 50%. Incubation with 0.1, 1, or 10 microM Cl-F-ara-A for 4 h depressed dATP, dCTP, and dGTP pools but did not affect TTP pools. Similar inhibition of deoxyribonucleoside triphosphate pools was seen after incubation with 2-chloro-2'-deoxyadenosine. Both Cl-F-ara-ATP and Cl-dATP potently inhibited the reduction of ADP to dADP in crude extracts of K562 cells (concentration producing 50% inhibition, 65 nM). The effect of Cl-F-ara-ATP on human DNA polymerases alpha, beta, and gamma isolated from K562 cells grown in culture was determined and compared with those of Cl-dATP and 9-beta-D-arabinofuranosyl-2-fluoroadenine triphosphate (F-ara-ATP). Cl-F-ara-ATP was a potent inhibitor of DNA polymerase alpha. Inhibition of DNA polymerase alpha was competitive with respect to dATP (Ki of 1 microM). The three analogue triphosphates were incorporated into the DNA by DNA polymerase alpha as efficiently as dATP. The incorporation of Cl-F-ara-AMP inhibited the further elongation of the DNA chain, similarly to that seen after the incorporation of F-ara-AMP. Extension of the DNA chain after the incorporation of Cl-dAMP was not inhibited as much as it was with either Cl-F-ara-AMP or F-ara-AMP. Cl-F-ara-ATP was not a potent inhibitor of DNA polymerase beta, DNA polymerase gamma, or DNA primase.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbocyclic analogues of 5-fluorouracil nucleosides.

The carbocyclic analogues of 5-fluoro-2'-deoxyuridine (5-FdUrd, 1), 5-fluorouridine, and 5-fluoro-3 alpha-deoxyuridine were prepared by fluorination of the uracil nucleoside analogues with elemental fluorine. The 5-FdUrd analogue (C-5-F-2'-dUrd, 6) was enzymatically phosphorylated to the analogue of 5-FdUrd 5'-phosphate and inhibited the incorporation of 2'-deoxyuridine into DNA of murine colon 26 tumor cells and L-1210 cells in culture. Biochemical studies also indicated that C-5-F-2'-Urd (6) was a less potent inhibitor of DNA synthesis in tumor cells than was 5-FdUrd (1). C-5-F-2'-dUrd was cytotoxic (ED50 = 2.5 mcg/mL) to L-1210 cells in culture; the other two analogues were less cytotoxic. C-5-F-2'-dUrd was inactive--or, at best, borderline active--in tests against P-388 leukemia in vivo.

Nitrosoureido nucleosides as potential inhibitors of nucleotide biosynthesis.

Several nitrosoureido nucleosides (3a, 3b, 5a, 7a, 7c, and 10a) designed as inhibitors of enzymes that metabolize pyrimidine nucleotides have been prepared and their chemical and biological properties studied. The methylnitrosoureas 3a and 3b were not significantly cytotoxic to H.Ep.-2 and L1210 cells in vitro but showed moderate activity in the P388 mouse leukemia screen (79% ILS for 3a and 56% ILS for 3b). The (chloroethyl)nitrosoureas 7a and 7c inhibited proliferation of L1210 cells, were cytotoxic to H.Ep.-2 cells, and demonstrated good activity against P388 in vivo (135% ILS with one 30-day survivor for 7a and 191% ILS with two 30-day survivors for 7c). Overnight exposure of L1210 cells to 7a and 7c resulted in cell enlargement accompanied by cell lysis. Macromolecular synthesis in enlarged cells, particularly RNA and protein synthesis, was markedly increased relative to that in untreated control cells. The half-lives of each of the nitrosoureas in pH 7 buffer was determined and compared with biological activity.

Alterations in nucleotide pools induced by 3-deazaadenosine and related compounds. Role of adenylate deaminase.

3-Deazaadenine, 3-deazaadenosine, and the carbocyclic analog of 3-deazaadenosine produced similar effects on nucleotide pools of L1210 cells in culture: each caused an increase in IMP and a decrease in adenine nucleotides and had no effect on nucleotides of uracil and cytosine. Concentrations of 50-100 microM were required to produce these effects. Although 3-deazaadenosine and carbocyclic 3-deazaadenosine are known to be potent inhibitors of adenosylhomocysteine hydrolase, the effects on nucleotide pools apparently are not mediated via this inhibition because they are also produced by the base, 3-deazaadenine, and because the concentrations required are higher than those required to inhibit the hydrolase. Cells grown in the presence of 3-deazaadenine or 3-deazaadenosine contained phosphates of 3-deazaadenosine (the mono- and triphosphates were isolated); from cells grown in the presence of the carbocyclic analog of 3-deazaadenosine, the monophosphate was isolated, but evidence for the presence of the triphosphate was not obtained. A cell-free supernatant fraction from L1210 cells supplemented with ATP catalyzed the formation of monophosphates from 3-deazaadenosine or carbocyclic 3-deazaadenosine, and a cell-free supernatant fraction supplemented with 5-phosphoribosyl 1-pyrophosphate (PRPP) catalyzed the formation of 3-deaza-AMP from 3-deazaadenine. Adenosine kinase apparently was not solely responsible for the phosphorylation of the nucleosides because a cell line that lacked this enzyme converted 3-deazaadenosine to phosphates. No evidence was obtained that the effects on nucleotide pools resulted from a block of the IMP-AMP conversion, but the results could be rationalized as a consequence of increased AMP deaminase activity. This explanation is supported by two observations: (a) coformycin, an inhibitor of AMP deaminase, prevented the effects on nucleotide pools, and (b) 3-deazaadenine decreased the conversion of carbocyclic adenosine to carbocyclic ATP and increased its conversion to carbocyclic GTP. The latter conversion requires the action of AMP deaminase and the observed effects can be rationalized by a nucleoside analog-mediated increase in AMP deaminase activity. Because these effects on nucleotide pools are produced only by concentrations higher than those required to inhibit adenosylhomocysteine hydrolase, they may not contribute significantly to the biological effects of 3-deazaadenosine or carbocyclic 3-deazaadenosine.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolism and metabolic actions of 4'-thiothymidine in L1210 cells.

4'-Thiothymidine (S-dThd) is a potent inhibitor of L1210 cell growth and is active against P388 leukemia in mice. Because of these activities and its novel structure, we have begun studies of its metabolism and metabolic actions in L1210 cells in order to understand its mechanism of cytotoxicity, S-dThd inhibited the incorporation of radiolabeled precursors into DNA, but did not inhibit the incorporation of either uridine or leucine into RNA or protein, respectively, which indicated that the mechanism of its toxicity was due to its inhibition of DNA synthesis. S-dThd did not decrease the concentration of any of the natural deoxynucleoside triphosphates, which indicated that its cytotoxicity was not due to the inhibition of ribonucleotide reductase. S-dThd was readily phosphorylated and used as a substrate for DNA synthesis. Because the rate of incorporation of S-dThd into DNA was 20% that of thymidine, it is likely that the mechanism of action of S-dThd is not due to inhibition of DNA polymerases by the 5'-triphosphate of S-dThd, but instead to its incorporation into the DNA and its subsequent disruption of some function of DNA.

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.

Metabolism and metabolic effects of 2-azahypoxanthine and 2-azaadenosine.

The metabolism and metabolic effects of 2-azahypoxanthine and 2-azaadenosine were studied to elucidate the biochemical basis for their known cytotoxicities. 2-Azaadenosine is a known substrate for adenosine kinase. That 2-azahypoxanthine is a substrate for hypoxanthine (guanine) phosphoribosyltransferase is shown by the observations that, in cell-free fractions from HEp-2 cells supplemented with 5-phosphoribosyl-1-pyrophosphate, 2-azahypoxanthine inhibited the conversion of hypoxanthine to IMP but not the conversion of adenine to AMP, and hypoxanthine, but not adenine, inhibited the conversion of 2-azahypoxanthine to 2-azaIMP. [8-14C]2-Azahypoxanthine was synthesized from [8-14C]hypoxanthine via [2-14C]-4-amino-5-imidazolecarboxamide. In HEp-2 cells in culture, the principal metabolite of [8-14C]-2-azahypoxanthine was 2-azaATP; there was no detectable 14C in deoxynucleotides or in DNA or RNA fractions. 2-Azaadenosine was much more toxic than 2-azahypoxanthine, and, when used in the presence of an adenosine deaminase inhibitor, 2'-deoxycoformycin, was converted in HEp-2 cells to 2-azaATP in amounts that exceeded those of ATP in control cells. The pool of ATP was reduced by as much as 75% as 2-azaATP accumulated. In a short-term experiment (4 hr), 2-azaadenosine selectively reduced the pools of adenine nucleotides, whereas 2-azahypoxanthine reduced the pools of guanine nucleotides selectively. Both 2-azahypoxanthine and 2-azaadenosine inhibited the incorporation of formate into purine nucleotides and were without effect on the conversion of thymidine and uridine to nucleotides. 2-Azahypoxanthine inhibited the incorporation of thymidine into macro-molecules but not that of uridine or leucine; 2-azaadenosine inhibited the incorporation of all three of these precursors non-selectively. 2-AzaIMP inhibited IMP dehydrogenase competitively with IMP (Ki = 66 microM). The difference in effects of 2-azahypoxanthine and 2-azaadenosine perhaps may be due to the production, from 2-azahypoxanthine but not from 2-azaadenosine + 2'-deoxycoformycin, of 2-azaIMP, which inhibits synthesis of guanine nucleotides and thereby results in inhibition of DNA synthesis. Specific sites of action for 2-azaadenosine are yet undefined.

Metabolism of 4'-thio-beta-D-arabinofuranosylcytosine in CEM cells.

Because of the excellent in vivo activity of 4'-thio-beta-D-arabinofuranosylcytosine (T-araC) against a variety of human solid tumors, we have studied its metabolism in CEM cells to determine how the biochemical pharmacology of this compound differs from that of beta-D-arabinofuranosylcytosine (araC). Although there were many quantitative differences in the metabolism of T-araC and araC, the basic mechanism of action of T-araC was similar to that of araC: it was phosphorylated to T-araC-5'-triphosphate (T-araCTP) and inhibited DNA synthesis. The major differences between these two compounds were: (i) T-araC was phosphorylated to active metabolites at 1% the rate of araC; (ii) T-araCTP was 10- to 20-fold more potent as an inhibitor of DNA synthesis than was the 5'-triphosphate of araC (araCTP); (iii) the half-life of T-araCTP was twice that of araCTP; (iv) the catalytic efficiency of T-araC with cytidine deaminase was 10% that of araC; and (v) the 5'-monophosphate of araC was a better substrate for deoxycytidine 5'-monophosphate deaminase than was the 5'-monophosphate of T-araC. Of these differences in the metabolism of these two compounds, we propose that the prolonged retention of T-araCTP is a major factor contributing to the activity of T-araC against solid tumors. The data in this study represent another example of how relatively small structural changes in nucleoside analogs can profoundly affect the biochemical activity.

Lack of mitochondrial toxicity in CEM cells treated with carbovir.

Carbovir (CBV) is a guanine nucleoside analog with potent in vitro anti-HIV activity. A prodrug of CBV is currently being evaluated in clinical trials as a potential agent for the treatment of AIDS. The ability of CBV to inhibit mitochondrial DNA synthesis in intact CEM cells was evaluated in the present study, because most of the currently available anti-HIV nucleoside analogs have significant toxicities that result from their inhibition of mitochondrial DNA synthesis. No delayed cytotoxicity was observed in CEM cells treated with 50 microM CBV for 4 weeks. In addition, CBV at concentrations as high as 1 mM did not cause a decline in mitochondrial DNA levels and only minimally increased the concentration of lactic acid in the medium. In contrast to these results with CBV, treatment of CEM cells with 0.5 microM 2',3'-dideoxycytidine resulted in delayed cytotoxicity, a decrease in mitochondrial DNA content and increases in lactic acid levels in the medium. These results indicated that treatment of CEM cells with CBV did not result in the inhibition of mitochondrial DNA synthesis and suggested that treatment of AIDS patients with CBV, or a prodrug of CBV, would not result in some of the toxicities seen with the other anti-HIV nucleoside analogs.

Interference with HIV-1 reverse transcriptase-catalyzed DNA chain elongation by the 5'-triphosphate of the carbocyclic analog of 2'-deoxyguanosine.

In an effort to better understand features in nucleotide analogs that result in the inhibition of HIV-1 reverse transcriptase, we have evaluated this enzyme with the 5'-triphosphate of the carbocyclic analog of 2'-deoxyguanosine (CdG-TP). CdG-TP was a reasonably potent competitive inhibitor of the incorporation of dGTP into DNA by HIV-1 reverse transcriptase using either a RNA or DNA template (Ki, 1 microM). CdG-TP was a good substrate for HIV-1 reverse transcriptase on both templates, but the DNA chain was poorly extended beyond the incorporation of CdG. These results indicate that substitution of ribose with a cyclopentane ring in nucleotides is not well tolerated by HIV-1 reverse transcriptase.

The mechanism of action of 3-deazauridine in tumor cells sensitive and resistant to arabinosylcytosine.

Deazauridine inhibited growth of tumor cells in culture and in culture and in vivo; this agent was significantly more effective against L1210/AraC than against the parent sensitive line. Inhibition of growth of tumor cells in culture was prevented by uridine and cytidine and was partially alleviated by deoxycytidine, but not by deoxyuridine or thymidine. DeazaUR inhibited nucleic acid synthesis but not protein synthesis in tumor cells in culture; deoxycytidine alleviated inhibition of nucleic acid synthesis. The labeling of pyrimidine ribonucleotides by 6-14C-orotic acid was inhbited by deazaUR. DeazaUR treatment of tumor cells in culture resulted in increased uptake of cytidine-3H into RNA, whereas uridine-3H uptake into RNA was inhibited. Labelling of DNA by uridine-3H/ and cytidine-H was inhibited by deazaUR. Pools of CMP, CDP, and CTP decreased markedly during deazaUR treatment of L1210 cells in culture and in vivo. These observations in growing cells pointed to deazaUR inhibition of the synthesis of cytidylic acid. Deazauridine 5'-triphosphate was found to be an inhibitor of the synthesis of CTP from UTP catalyzed by enzyme preparations from L1210 cells. This observation is in agreement with those of McPartland et al.19 that deazaUTP inhibited CTP synthetase purified from calf liver. Deazauridine treatment of L1210 cells in culture stimulated the uptake of deoxycytidine-3H into DNA while inhibiting the uptake of 3H-labeled deoxyuridine, thymidine, deoxyadenosine, and deoxyguanosine. Intracellular pools of dCTP were decreased by deazauridine treatment in L1210 cells in culture and in vivo. Deazauridine 5'-diphosphate inhibited the enzymatic reduction of pyrimidine ribonucleoside 5'-diphosphates to the corresponding deoxyribonucleotides. These results are consistent with the view that deazauridine, after its uptake and intracellular phosphorylation, strongly inhibits the formation of CTP. This is considered to be the primary metabolic effect of the analog. A secondary effect appears to be an inhibition of dCTP formation.

Metabolism of O6-propyl and N6-propyl-carbovir in CEM cells.

The metabolism of O6-propyl-carbovir and N6-propyl-carbovir, two selective inhibitors of HIV replication, has been evaluated in CEM cells. Both compounds were phosphorylated in intact cells to carbovir-5'-triphosphate. The metabolism of these two agents was inhibited by deoxycoformycin and mycophenolic acid, but not erythro-9-(2-hydroxy-3-nonyl)adenine. No evidence of the 5'-triphosphate of either compound was detected in CEM cells.

Studies with 2,5-piperazinedione, 3,6-bis(5-chloro-2-piperidyl)-,dihydrochloride. II. Effects on macromolecular synthesis in cell culture and evidence for alkylating activity.

2,5-Piperazinedione, 3,6-bis(5-chloro-2-piperidyl)-,dihydrochloride (NSC-135758) inhibited DNA synthesis but not RNA and protein synthesis in Adenocarcinoma 755 cells in culture. The expression of such inhibition was delayed in time; it was necessary to expose tumor cells to NSC-135758 for 10-12 hours before measuring the macromolecular synthesis in order to demonstrate selective inhibition of DNA synthesis. Inhibition of DNA synthesis was demonstrated to be irreversible in human epidermoid carcinoma cells in culture. Exposure of cells in suspension culture to NSC-135758 or to melphalan for 1-4 hours, and then incubation of cells in the absence of an inhibitor for 20 hours, resulted in preferential inhibition of DNA synthesis; inhibition of RNA synthesis was observed under these conditions but was less pronounced. Chemical evidence for alkylating activity of NSC-135758 and of the bis-aziridine derived from it (NSC-201424) was obtained by demonstrating their reaction with 4-(p-nitrobenzyl)pyridine. NSC-135758 was more potent as a cytotoxic agent than was its derivative, a result which suggests that NSC-135758 is the active alkylating agent. Reaction of NSC-135758 with diethylamine was examined; the product obtained upon alkylation of diethylamine by NSC-135758 was identified from its proton magnetic resonance spectrum and by field desorption mass spectral analysis. These results support the view that NSC-135758 acts as an alkylating agent in inhibiting DNA synthesis and cell proliferation of tumor cells in culture.

Studies with 2,5-piperazinedione, 3,6-bis(5-chloro-2-piperidyl)-,dihydrochloride. III. Biochemical and therapeutic effects in L1210 leukemias sensitive and resistant to alkylating agents: comparison with melphalan, cyclophosphamide, and BCNU.

2,5-Piperazinedione, 3,6-bis(5-chloro-2-piperidyl)-,dihydrochloride (NSC-135758) selectively inhibits DNA synthesis in L1210/0 leukemia and in cyclophosphamide-resistant L1210 (L1210/CPA). Melphalan (L-PAM) inhibits nucleic-acid synthesis but not protein synthesis in L1210/0 and L1210/CPA. CPA selectively inhibits DNA synthesis in L1210/0 but does not inhibit DNA synthesis in L1210/CPA. NSC-135758 is as active in vivo against L1210/CPA and BCNU-resistant L1210 (L1210/BCNU) as it is against L1210/0. It is inactive against ic implanted L1210/0. These data clearly indicate important differences in the biologic activity of this compound compared to either CPA or BCNU. L-PAM is similar to NSC-135758 in activity against L1210/CPA and L1210/BCNU.

Comparison of the mechanism of cytotoxicity of 2-chloro-9-(2-deoxy-2- fluoro-beta-D-arabinofuranosyl)adenine, 2-chloro-9-(2-deoxy-2-fluoro- beta-D-ribofuranosyl)adenine, and 2-chloro-9-(2-deoxy-2,2-difluoro- beta-D-ribofuranosyl)adenine in CEM cells.

In an effort to understand biochemical features that are important to the selective antitumor activity of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine [Cl-F( upward arrow)-dAdo], we evaluated the biochemical pharmacology of three structurally similar compounds that have quite different antitumor activities. Cl-F( upward arrow)-dAdo was 50-fold more potent as an inhibitor of CEM cell growth than were either 2-chloro-9-(2-deoxy-2-fluoro-beta-D-ribofuranosyl)adenine [Cl-F( downward arrow)-dAdo] or 2-chloro-9-(2-deoxy-2, 2-difluoro-beta-D-ribofuranosyl)adenine [Cl-diF( upward arrow downward arrow)-dAdo]. The compounds were similar as substrates of deoxycytidine kinase. Similar amounts of their respective triphosphates accumulated in CEM cells, and the rate of disappearance of these metabolites was also similar. Cl-F( upward arrow)-dAdo was 10- to 30-fold more potent in its ability to inhibit the incorporation of cytidine into deoxycytidine nucleotides than either Cl-F( downward arrow)-dAdo or Cl-diF( upward arrow downward arrow)-dAdo, respectively, which indicated that ribonucleotide reductase was differentially inhibited by these three compounds. Thus, the differences in the cytotoxicity of these agents toward CEM cells were not related to quantitative differences in the phosphorylation of these agents to active forms but can mostly be accounted for by differences in the inhibition of ribonucleotide reductase activity. Furthermore, the inhibition of RNA and protein synthesis by Cl-F( downward arrow)-dAdo and Cl-diF( upward arrow downward arrow)-dAdo at concentrations similar to those required for the inhibition of DNA synthesis can help explain the poor antitumor selectivity of these two agents because all cells require RNA and protein synthesis.