Tomáš Ruml

Differences in antitumor effects of various statins on human pancreatic cancer

Statins are widely used for the treatment of hypercholesterolemia. However, their inhibitory action on HMG‐CoA reductase also results in the depletion of intermediate biosynthetic products, which importantly contribute to cell proliferation. The aim of the present study was to compare the effects of the individual commercially available statins on experimental pancreatic cancer. The in vitro effects of individual statins (pravastatin, atorvastatin, simvastatin, lovastatin, cerivastatin, rosuvastatin and fluvastatin) on the viability of human pancreatic cancer were evaluated in CAPAN‐2, BxPc‐3 and MiaPaCa‐2 cell lines. The in vivo experiments were performed on nude mice xenotransplanted with CAPAN‐2 cells. The mice received oral treatments either with a placebo, or with the statins mentioned earlier in a daily dose corresponding to a hypocholesterolemic dose in humans. The effect of these statins on the intracellular Ras protein, trafficking in MiaPaCa‐2 transfected cells, was also investigated. Substantial differences in the tumor‐suppressive effects of all statins were detected in both in vitro and in vivo experiments. While simvastatin exerted the highest tumor‐suppressive effects in vitro, rosuvastatin (p = 0.002), cerivastatin (p = 0.002) and fluvastatin (p = 0.009) were the most potent compounds in an animal model. All statins (except pravastatin) inhibited intracellular Ras protein translocation. In summary, substantial tumor‐suppressive effects of various statins on the progression of experimental pancreatic adenocarcinoma were demonstrated, with marked differences among individual statins. These results support greatly the potential of statins for the chemoadjuvant treatment of pancreatic cancer.

Integrase of Mason-Pfizer monkey virus

The gene encoding an integrase of Mason–Pfizer monkey virus (M‐PMV) is located at the 3′‐end of the pol open reading frame. The M‐PMV integrase has not been previously isolated and characterized. We have now cloned, expressed, isolated, and characterized M‐PMV integrase and compared its activities and primary structure with those of HIV‐1 and other retroviral integrases. M‐PMV integrase prefers untranslated 3′‐region‐derived long‐terminal repeat sequences in both the 3′‐processing and the strand transfer activity assays. While the 3′‐processing reaction catalyzed by M‐PMV integrase was significantly increased in the presence of Mn2+ and Co2+ and was readily detectable in the presence of Mg2+ and Ni2+ cations, the strand transfer activity was strictly dependent only on Mn2+. M‐PMV integrase displays more relaxed substrate specificity than HIV‐1 integrase, catalyzing the cleavage and the strand transfer of M‐PMV and HIV‐1 long‐terminal repeat‐derived substrates with similar efficiency. The structure‐based sequence alignment of M‐PMV, HIV‐1, SIV, and ASV integrases predicted critical amino acids and motifs of M‐PMV integrase for metal binding, interaction with nucleic acids, dimerization, protein structure maintenance and function, as well as for binding of human immunodeficiency virus typeu20031 and Rous avian sarcoma virus integrase inhibitors 5‐CI‐TEP, DHPTPB and Y‐3.

The effect of point mutations within the N-terminal domain of Mason-Pfizer monkey virus capsid protein on virus core assembly and infectivity

Retroviral capsid protein (CA) mediates protein interactions driving the assembly of both immature viral particles and the core of the mature virions. Structurally conserved N-terminal domains of several retroviruses refold after proteolytic cleavage into a beta-hairpin, stabilized by a salt bridge between conserved N-terminal Pro and Asp residues. Based on comparison with other retroviral CA, we identified Asp50 and Asp57 as putative interacting partners for Pro1 in Mason-Pfizer monkey virus (M-PMV) CA. To investigate the importance of CA Pro1 and its interacting Asp in M-PMV core assembly and infectivity, P1A, P1Y, D50A, T54A and D57A mutations were introduced into M-PMV. The P1A and D57A mutations partially blocked Gag processing and the released viral particles exhibited aberrant cores and were non-infectious. These data indicate that the region spanning residues Asp50-Asp57 plays an important role in stabilization of the beta-hairpin and that Asp57 likely forms a salt-bridge with P1 in M-PMV CA.

The impact of altered polyprotein ratios on the assembly and infectivity of Mason-Pfizer monkey virus.

Most retroviruses employ a frameshift mechanism during polyprotein synthesis to balance appropriate ratios of structural proteins and enzymes. To investigate the requirements for individual precursors in retrovirus assembly, we modified the polyprotein repertoire of Mason-Pfizer monkey virus (M-PMV) by mutating the frameshift sites to imitate the polyprotein organization of Rous sarcoma virus (Gag-Pro and Gag-Pro-Pol) or Human immunodeficiency virus (Gag and Gag-Pro-Pol). For the Rous-like virus, assembly was impaired with no incorporation of Gag-Pro-Pol into particles and for the HIV-like virus an altered morphogenesis was observed. A mutant expressing Gag and Gag-Pro polyproteins and lacking Gag-Pro-Pol assembled intracellular particles at a level similar to the wild-type. Gag-Pro-Pol polyprotein alone neither formed immature particles nor processed the precursor. All the mutants were non-infectious except the HIV-like, which retained fractional infectivity.