Inhibition of methylation of nuclear ribonucleic acid in L1210 cells by tubercidin, 8-azaadenosine and formycin

The adenosine analogs tubercidin (7-deazaadenosine), formycin (7-amino-3-[β-d-ribofuranosyl] pyrazolo[4,3-d]pyrimidine) and 8-azaadenosine were examined for their effects on the synthesis and methylation of nuclear RNA in L1210 cells in vitro. Total RNA and DNA synthesis was affected to the greatest extent by tubercidin (IC₅₀ = 7 × 10^?6M) and to an insignificant degree by 8-azaadenosine and formycin; however, the effects of the latter two drugs, but not of tubercidin, were potentiated by 2'-deoxycoformycin, an inhibitor of adenosine deaminase. In the presence of 2'-deoxycoformycin, RNA synthesis was inhibited by 40 per cent at 1 × 10^?4 M 8-azaadenosine and by 50 per cent at 2 × 10^?4 M formycin, while DNA synthesis was inhibited less extensively. Alkaline hydrolysis of nuclear RNA labeled with [¹⁴C]uridine and l-[methyl-³H]methionine showed preferential inhibition of base methylation in mononucleotides, but not of 2′-O-methylation in dinucleotides, for all three drugs. This differential effect persisted to varying degrees in ?18S and 4S nuclear RNA separated by electrophoresis. The reduction in base methylation in 4S RNA was associated with seven of the eight methylated nucleosides in 4S RNA separated by two-dimensional thin-layer chromatography. These results indicate that tubercidin, 8-azaadenosine and formycin can preferentially inhibit the base methylation of nuclear RNA relative to its synthesis.     

Effects of 8-azaadenosine and formycin on cell lethality and the synthesis and methylation of nucleic acids in human colon carcinoma cells in culture

The cytocidal and biochemical effects of formycin and 8-azaadenosine in the presence and absence of the adenosine deaminase inhibitor, 2′-deoxycoformycin, were studied in human colon carcinoma (HT-29) cells in culture. Logarithmically growing cells were unaffected by 24-hr exposure to either 10^?6M formycin or 8-azaadenosine, but 1 to 1.4 log reductions in colony formation were produced by 10^?5M of each analog. In the presence of 10^?6M 2′-deoxycoformycin, a 3- and 30-fold potentiation of the cytocidal activity of 8-azaadenosine and formycin, respectively, was produced. Inhibition of DNA synthesis but not RNA synthesis by 8-azaadenosine paralleled its cytocidal activity; however, neither variable correlated closely with the cytotoxic effects of formycin. In addition, the methylation of nuclear RNA was unaffected by both drugs while the methylation of 5-methyl-deoxycytidine in DNA was inhibited to a lesser extent than DNA synthesis. Measurements of the incorporation of [³H]formycin and [³H]8-azaadenosine into nuclear RNA and DNA in the presence and absence of 2′-deoxycorformycin indicated that formycin substitution in RNA and DNA was enhanced 10- and 20-fold, respectively, while [³H]8-azaadenosine incorporation into both nucleic acids was increased 6- to 7-fold. These results suggest that the incorporation of formycin into nucleic acids, particularly DNA, correlates closely with its lethal effect on cell viability. On the other hand, the cytocidal activity of 8-azaadenosine more clearly parallels its inhibitory effect on DNA synthesis rather than its substitution into nucleic acids.     

Potentiation by 2'-deoxycoformycin of the inhibitory effect of xylosyladenine on nuclear RNA synthesis in L1210 cells in vitro

The effect of the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), on the inhibitory effect of 9-β-d-xylofuranosyladenine (XA) on nuclear RNA synthesis was examined in L1210 cells in vitro. Pretreatment of cells for 15 min with a 100 per cent inhibitory dose (1 × 10^?6 M) of dCF resulted in approximately a 3- to 8-fold reduction in the 50 per cent inhibitory dose (id₅₀) of XA for [³H]uridine and [³H]thymidine incorporation into RNA and DNA respectively. The id₅₀ for XA for RNA synthesis vs DNA synthesis was 5-fold lower in the absence of dCF and 20-fold lower in the presence of dCF, indicating the greater sensitivity of RNA synthesis to this inhibitor. Fractionation of nuclear RNA into rRNA, non-poly(A) heterogeneous RNA and poly(A)heterogeneous RNA revealed the latter species of RNA to be less sensitive to XA in the absence of dCF; however, in the presence of dCF, all three species of nuclear RNA showed similar sensitivities. Nuclear polyadenylic acid synthesis was among the most sensitive RNA fractions to XA, and was also inhibited to a greater degree by pretreatment of cells with dCF. These results indicate that XA is potentiated markedly by inhibition of adenosine deaminase, and that deamination serves as a major catabolic route for this drug.