Effects of alpha-difluoromethylornithine on thrombospondin-1 production by human breast cancer cells.

We have previously observed that inhibition of polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) upregulates production of thrombospondin-1 (TSP-1), an extracellular matrix protein with potent anti-angiogenic and antimetastatic properties, by MDA-MB-435 human breast cancer cells in culture. The present experiments were designed to investigate the mechanisms by which DFMO regulates TSP-1 production in this system. 35S-methionine pulse chase experiments indicated that DFMO administration increased TSP-1 synthesis by approximately 6-fold, while it slightly but significantly decreased protein half-life from 35 to 28 min. DFMO treatment increased steady state TSP-1 mRNA levels by 2-fold in MDA-MB-435 cells. TSP-1 promoter reporter studies indicated that this increase was largely due to activation of transcription. Analysis of distribution of TSP-1 mRNA levels between non-polysomal, subpolysomal and polysomal fractions in control and DFMO-treated cells suggested a major stimulatory effect of the drug on TSP-1 translation. A similar increase in TSP-1 transcription and translation in response to DFMO treatment was also observed in vivo in MDA-MB-435 breast cancer xenografts. Surprisingly however, we failed to detect an increase in TSP-1 protein as assessed by Western blot analysis. The reason for this unexpected finding is unknown but may be due to DFMO-induced stimulation of TSP-1 secretion into the systemic circulation, thus preventing its accumulation within the tumor.

Inability of L22 ribosomal protein alteration to increase macrolide MICs in the absence of efflux mechanism in Haemophilus influenzae HMC-S.

BACKGROUND: Haemophilus influenzae HMC-C with high-level macrolide resistance after multi-step selection by clarithromycin reverted spontaneously and became hypersusceptible to macrolides. OBJECTIVE: Determination of macrolide resistance mechanism(s) in hypersusceptible and hyperresistant strains. METHODS: The presence of macrolide efflux in the strains was studied by radioactive erythromycin accumulation. Ribosomal mutations were investigated by sequencing. The possible role of acrAB clusters in macrolide resistance was studied by sequencing and expression analysis. RESULTS: The parent strain had no ribosomal alteration, but both high-level resistant and hypersusceptible strains had R88P mutations in ribosomal protein L22. Radioactive macrolide accumulation studies pointed to the presence of macrolide efflux in the high-level resistant and parent strains, but not in the hypersusceptible derivative. Transformation of hypersusceptible strains using total DNA from the parent strain restored the macrolide efflux system in the hypersusceptible strain, which was confirmed by MIC levels and radioactive erythromycin accumulation similar to that of the mutant resistant strain. Analysis of sequence and transcription of acrAB gene clusters showed no significant differences between resistant and hypersusceptible derivatives. CONCLUSION: Mutation in ribosomal protein L22 alone does not confer high-level macrolide resistance unless efflux is present.