Differentiated inhibition of DNA, RNA and protein synthesis in L1210 cells by 8-methoxypsoralen

The synthesis of DNA, RNA and protein was measured in L1210 cells following treatment with 8-methoxypsoralen in combination with long wavelength ultraviolet irradiation. The results show that the DNA synthesis is strongly inhibited (approximately 95%) at 200 ng/ml reaching a minimum within 2 hours while RNA synthesis is only weakly affected at this concentration (approximately 40% inhibition). At 2 micrograms/ml the RNA synthesis is inhibited approximately 90%. Even at this concentration only a moderate effect is seen on the protein synthesis. These results strongly indicate that the phototoxic action of 8-methoxypsoralen is primarily due to inhibition of DNA synthesis.     

Inhibition of DNA-dependent RNA polymerase from Escherichia coli by pyran copolymer

Pyran copolymer, a potent inhibitor of DNA-dependent RNA polymerase from Escherichia coli, prevented polyribonucleotide synthesis by blocking both the initiation and elongation steps. The inhibition was noncompetitive with respect to template and nucleotide triphosphate substrates. Template binding and the stability of the nascent RNA chain were not affected by the inhibitor.     

DNA-dependent RNA polymerase from T8 Guerin tumor

DNA-dependent RNA polymerases from nuclei of T8 Guerin tumor were studied. Two enzymes were purified several hundred times by the use of ammonium sulfate precipitation, DEAE-cellulose and phosphocellulose chromatography. One of them belongs to A(I) RNA polymerases and the second to B(II) as was established from their metal and ionic strength requirements. activity in the presence of native and denatured DNA and the resistance to a-amanitin inhibition. The quantity of class A enzyme was increased compared to B, a fact observed with most neoplastic tissues so far studied. This increase of the polymerase responsible for ribosomal RNA synthesis could probably be related to malignant transformation in animals.     

Photobinding of 8-methoxypsoralen to transfer RNA and 5-fluorouracil-enriched transfer RNA.

The photobinding of [3H]8MOP to tRNA upon irradiation at 365 nm in the absence of O2 was determined by gel filtration. The maximum photobinding was found to be ca. 4 mol of 8MOP er mol of tRNA and 5FU-tRNA, with an overall quantum yield of 2.3 X 10(-3). The photobinding kinetics for 8MOP-tRNA showed an apparent induction period or sigmoidal kinetic curve, indicating a specific initial photobinding site on tRNA which was identified as 4-thiouridine at position 8 from the 5'-end of Escherichia coli tRNA. Photobinding of 8MOP to 5FU-tRNA proceeded without an apparent induction period. 8MOP-tRNA and 8MOP-5FU-tRNA adducts were characterized by absorption, fluorescence, and CD spectroscopy. A modified procedure was also developed to analyze the nucleoside composition in modified 8MOP-tRNA and 8MOP-5FU-tRNA. The results showed that 8MOP photochemically added mainly to pyrimidine bases. The photobinding of 8MOP changed the conformation (secondary in particular) of tRNA and inhibited aminoacyl-tRNA synthetase activity.     

Bacterial radiosensitization by 8-methoxypsoralen.

8-Methoxypsoralen has been shown to act as a radiosensitizer of hypoxic bacterial cells with uvrA, recA and uvrB and/or lexA mutations. No effect of the drug on the radiosensitivity of oxic bacteria with these mutations was observed. This drug differs from O2 and electron-affinic radiosensitizers in that its effect is not purely dose-modifying and can exceed the oxygen effect in certain mutants.     

Evidence for uptake of 8-methoxypsoralen and 5-methoxypsoralen by cellular nuclei

The fluorescent appearance of oral mucosa cells treated with 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen (5-MOP) was observed by means of fluorescence microscopy. Fluorescence at the nuclei was weakened in 8-MOP-treated cells, while it was intensified in 5-MOP-treated cells. These findings were consistent with changes in the fluorescence intensities on association of the psoralen derivatives with DNA in aqueous solution. This intensity change of fluorescence and also the blue shift of the fluorescence maximum of the derivatives on association suggested that the environment around the psoralen molecules is as little polar as methanol. From the results of these fluorescence microscopic observations and spectroscopic analysis of fluorescence of derivatives interacting with DNA during equilibrium dialysis, we concluded that 8-MOP, as well as 5-MOP, is incorporated by nuclei of human cells.     

Mechanistic analysis of RNA synthesis by RNA-dependent RNA polymerase from two promoters reveals similarities to DNA-dependent RNA polymerase.

The brome mosaic virus (BMV) RNA-dependent RNA polymerase (RdRp) directs template-specific synthesis of (-)-strand genomic and (+)-strand subgenomic RNAs in vitro. Although the requirements for (-)-strand RNA synthesis have been characterized previously, the mechanism of subgenomic RNA synthesis has not. Mutational analysis of the subgenomic promoter revealed that the +1 cytidylate and the +2 adenylate are important for RNA synthesis. Unlike (-)-strand RNA synthesis, which required only a high GTP concentration, subgenomic RNA synthesis required high concentrations of both GTP and UTP. Phylogenetic analysis of the sequences surrounding the initiation sites for subgenomic and genomic (+)-strand RNA synthesis in representative members of the alphavirus-like superfamily revealed that the +1 and +2 positions are highly conserved as a pyrimidine-adenylate. GDP and dinucleotide primers were able to more efficiently stimulate (-)-strand synthesis than subgenomic synthesis under conditions of limiting GTP. Oligonucleotide products of 6-, 7-, and 9-nt were synthesized and released by RdRp in 3-20-fold molar excess to full-length subgenomic RNA. Termination of RNA synthesis by RdRp was not induced by template sequence alone. Our characterization of the stepwise mechanism of subgenomic and (-)-strand RNA synthesis by RdRp permits comparisons to the mechanism of DNA-dependent RNA synthesis.     

Inhibition of poliovirus RNA synthesis by brefeldin A.

Brefeldin A (BFA), a fungal metabolite that blocks transport of newly synthesized proteins from the endoplasmic reticulum, was found to inhibit poliovirus replication 10(5)- to 10(6)-fold. BFA does not inhibit entry of poliovirus into the cell or translation of viral RNA. Poliovirus RNA synthesis, however, is completely inhibited by BFA. A specific class of membranous vesicles, with which the poliovirus replication complex is physically associated, is known to proliferate in poliovirus-infected cells. BFA may inhibit poliovirus replication by preventing the formation of these vesicles.