Reduced RNA synthesis levels in isolated mouse liver nuclei following reaction with [(H2O)(NH3)5Ru(II)]2+

Abstract

The goal of this study is to establish the effect of [(H2O)(NH3)5Ru(II)]2+ reaction of nuclei on their RNA transcription levels. This question is important because ammineruthenium compounds share chemical and biological properties with the chemotherapeutic agent cis-dichlorodiammineplatinum(II) or cisplatin. First we demonstrate that mouse liver nuclei are active in RNA transcription in vitro and characterize the optimum conditions for in vitro transcription. Synthetic rates in the presence of inhibitors actinomycin D and alpha-Amanitin and measurements of oligo(dT)-cellulose RNA binding levels suggest that all three RNA Polymerases are active in synthesis at about the following percentages-RNA Polymerase I(30%), II(50%) and III(20%). Mouse liver nuclei reacted with [(H2O)(NH3)5Ru(II)]2+ and then oxidized had (NH3)5 Ru(III)3+n-DNA adduct levels inversely related to total RNA synthetic rates. Oligo(dT) cellulose RNA binding levels did not vary with DNA adduct density. These data suggest that direct DNA lesions rather than [(NH3)5Ru(III)]3+ effects on other aspects of the transcription system are responsible for the diminished RNA synthesis levels. Ammineruthenium complexes remain desirable candidates for chemotherapeutic agents that may be safely administered in the unreactive ruthenium(III) state and be activated toward DNA binding by reduction in the hypoxic environment of many tumour cells.

Non-Platinum Chemotherapeutic Metallopharmaceuticals

“…Modifying the ruthenium center to trans -[(H 2 O)py(NH 3 ) 4 Ru II ] causes the metal ion to bind specifically at G κ7 . Also consistent with DNA binding in vivo, a number of ammine, amine, and heterocyclic complexes of ruthenium exhibit inhibition of DNA replication, mutagenic activity and induction of the SOS repair mechanism, binding to nuclear DNA, , and reduction of RNA synthesis . More recently EDTA-type complexes of Ru III and even Ru IV have shown anticancer activity, apparently through DNA binding. ,

1 Structures of (a) trans -[(Im) 2 Cl 4 Ru] - 83 and (b) trans -[(Me 2 S O)(Im)Cl 4 Ru] - (NAMI)

…”

“…74 Also consistent with DNA binding in vivo, a number of ammine, amine, and heterocyclic complexes of ruthenium exhibit inhibition of DNA replication, 75 mutagenic activity and induction of the SOS repair mechanism, 76 binding to nuclear DNA, 71,77 and reduction of RNA synthesis. 78 shown anticancer activity, apparently through DNA binding. 79,80 Table 1 summarizes the anticancer activity of a representative selection of ruthenium complexes against animal tumor models.…”

The Effects of DNA Covalent Adducts on in Vitro Transcription

“…Purine nucleosides, particularly G (see Figure a,b for the formulas of the nucleosides), represent the preferential target of alkylating agents. The N7 position is attacked by nitrogen mustards, , cisplatin ( 3 ), − pentaammineruthenium(III) 15 ions as well as other ruthenium complexes, , vinyl chloride and its metabolites, and aflatoxin B 1 . , Methyl- ( 16 ) and ethylmethanesulfonate, dimethyl sulfate, N -nitroso- N -methylurea (17) , and other small alkylating agents also react mainly with N7 of G ( 18 ). In addition the exocyclic O6 of G ( 19 ), O2 ( 20 ) and O4 ( 21 ) of T as well as the N3 of A and C can also be alkylated. − The exocyclic 2-amino group of G can be alkylated by mitomycins ( 22 ), , the benzo[ a ]pyrene metabolite ( 23 ), and possibly by adriamycin as well as its cyanomorpholino derivative …”

“…The decrease in the amount of RNA synthesized on a modified template is assessed by measuring the incorporation of radioactive ribonucleotides into the transcript. Transcription, as well as replication assays, were used to characterize the covalent interactions of furocoumarins, ,− benzo[ a ]pyrene, , 2-aminofluorene, , cisplatin and trans -DDP, ammineruthenium complex, and nitracrine 57 with DNA.…”

“…Transcription assays can reveal new ligand−DNA interactions. Covalent binding of aflatoxin B 1 to C was first deduced by the inhibition of RNA synthesis on poly [d(I-C)]. , The formation of ammineruthenium complexes ( 15 ) with DNA has been first inferred by their inhibitory effect on RNA synthesis in isolated mouse liver nuclei. , Sometimes, however, structurally different covalent complexes, such as N -acetyl-2-aminofluorene ( 26 ) and 2-aminofluorene−DNA ( 25 ) adducts, inhibit transcription to a comparable extent . No dramatic differences are observed between 8-methoxypsoralen, a cross-linking drug, and monofunctionally binding furocoumarins, such as 4,6-dimethylangelicin 98 and benzopsoralen (Table ).…”