Novel RNA polymerase I inhibitor CX-5461 suppresses imiquimod-induced experimental psoriasis.

Clinical treatment of psoriasis remains challenging because of possible long-term drug toxicities and loss of therapeutic effects over time. CX-5461 is a novel selective inhibitor of RNA polymerase I. Our previous studies have shown that CX-5461 has potent anti-inflammatory effects. Here we investigated whether CX-5461 could inhibit the development of imiquimod-induced experimental psoriasis in mice. Adult male C57BL/6 mice were used, and psoriasis-like lesions were induced by topical imiquimod treatment. In vivo, we demonstrated that topical application of CX-5461 prevented the development of imiquimod-induced psoriasis, with decreases in keratinocyte proliferation, T-cell infiltration and pathological angiogenesis. CX-5461 also reversed existing skin inflammation induced imiquimod and retarded the development of 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperplasia and inflammation. In vitro, CX-5461 induced cell cycle arrest in keratinocytes, inhibited expressions of interleukin-17, interleukin-23 receptor and retinoic acid receptor-related orphan receptor-γt in activated T cells, and reduced angiogenic functions of endothelial cells. In conclusion, CX-5461 exhibits therapeutic effects on experimental psoriasis in mice, likely via multiple mechanisms including anti-proliferative, anti-inflammatory and anti-angiogenic activities.

Action of alpha-amanitin during pyrophosphorolysis and elongation by RNA polymerase II.

Using defined elongation complexes formed on dC-tailed templates with Drosophila RNA polymerase II, we have examined elongation, pyrophosphorolysis, and DmS-II-mediated transcript cleavage and the inhibitory effect of alpha-amanitin on these processes. Analysis of pyrophosphorolysis on soluble or immobilized and templates confirmed that NTPs are liberated instead of dinucleotides that are released during DmS-II-mediated transcript cleavage. 10 microgram/ml alpha-amanitin completely inhibited DmS-II-mediated transcript cleavage but allowed extended pyrophosphorolysis and nucleotide addition to occur. alpha-Amanitin dramatically decreased the Vmax for nucleotide addition but only slightly affected the Km for nucleotides. Although the processes ae mechanistically distinct, both pyrophosphorolysis and DmS-II-mediated transcript cleavage frequently resulted in similar patterns of shortened transcript. Since polymerase molecules encounter similar kinetic barriers during both processes, it is possible that there is a common step in the reverse movement of the polymerase.

Presence of a limited number of essential nucleotides in the promoter region of mouse ribosomal RNA gene.

Point mutations are introduced into a mouse rDNA fragment containing the promoter region by a sodium bisulfite method and the mutants are tested for the ability of accurate transcription initiation in vitro. The results indicate that the change, G to A, at -7 completely eliminates the promoter activity, and those at -16 and at -25 decrease it to about 10% and 50%, respectively. On the other hand, the substitutions at +9, +4, -2, -9 and -39 do not alter the template activity significantly. It is concluded that there are limited but distinct nucleotides that are essential for the transcription initiation of this gene. This sort of absolute requirement for single specific bases is not reported in protein coding genes transcribed by RNA polymerase II. We propose that these rigid recognition signals which we have found are the molecular basis for the strong species-dependency of the transcription machinery of RNA polymerase I system. A model is presented in which a transcription factor interacts with the rDNA promoter from one side of the DNA double-helix with essential contacts at these bases.

Transcription of adenovirus 2 DNA by human RNA polymerase II in vitro.

The interaction of Adenovirus 2 DNA and human placental RNA polymerase II in vitro satisfies criteria that suggest that at least some fraction of our purified polymerase preparations corresponds to prokaryotic holoenzyme and is able to initiate transcription at "true" promoters: (1) The purified enzyme forms highly stable complexes at specific sites on Ad 2 DNA; Kass = 1--2 X 10(12) M-1. (2) Transcription of Ad 2 DNA from pre-formed complexes with human RNA polymerase II is resistant to poly I. (3) Many of the stable-binding sites correspond to Ad 2 promoters known to be active in vivo. We also present evidence consistent with a two-state (I and RS) model (Chamberlin et al. 1976; Travers 1974) for the interaction of human RNA polymerase II with Ad 2 DNA. These experiments, which are similar to those described previously in studies of wheat germ RNA polymerase II (Seidman et al. 1979), indicate that the mechanisms of transcription inhibition and promoter site selection in eukaryotic and prokaryotic systems may be very similar.