Mike Thorne | PubMed Health

PubMed2005

Key role for p27Kip1, retinoblastoma protein Rb, and MYCN in polyamine inhibitor-induced G1 cell cycle arrest in MYCN-amplified human neuroblastoma cells.

Alpha-difluoromethylornithine (DFMO) inhibits the proto-oncogene ornithine decarboxylase (ODC) and is known to induce cell cycle arrest. However, the effect of DFMO on human neuroblastoma (NB) cells and the exact mechanism of DFMO-induced cell death are largely unknown. Treatment with DFMO in combination with SAM486A, an S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor, has been shown to enhance polyamine pool depletion. Therefore, we analysed the mechanism of action of DFMO and/or SAM486A in two established MYCN-amplified human NB cell lines. DFMO and SAM486A caused rapid cell growth inhibition, polyamine depletion, and G1 cell cycle arrest without apoptosis in cell lines LAN-1 and NMB-7. These effects were enhanced with combined inhibitors and largely prevented by cotreatment with exogenous polyamines. The G1 cell cycle arrest was concomitant with an increase in cyclin-dependent kinase inhibitor p27Kip1. In a similar fashion, DFMO and DFMO/SAM486A inhibited the phosphorylation of the G1/S transition-regulating retinoblastoma protein Rb at residues Ser795 and Ser807/811. Moreover, we observed a dramatic decrease in MYCN protein levels. Overexpression of MYCN induces an aggressive NB phenotype with malignant behavior. We show for the first time that DFMO and SAM486A induce G1 cell cycle arrest in NB cells through p27Kip1 and Rb hypophosphorylation.

Adenosylmethionine Decarboxylase↗

PubMed2004

Recombinant cobra venom factor.

Cobra venom factor (CVF) is the complement-activating protein from cobra venom. CVF is a three-chain protein that functionally resembles C3b, the activated form of complement component C3. Like C3b, CVF forms a C3/C5 convertase with factor B in the presence of factor D and Mg(2+). Although CVF exhibits functional activity of C3b, it structurally resembles the C3b degradation product C3c, which is not able to form a C3/C5 convertase. CVF has become an important research tool to decomplement laboratory animals in order to study the role of complement in host defense, immune response, and pathogenesis of disease. As the Asian cobras of the Naja species are on the list of endangered species, cobra venom as the source for CVF has become increasingly difficult to obtain. Methods have been developed to recombinantly produce active forms of CVF. This manuscript reviews the production of recombinant pro-CVF using both prokaryotic and eukaryotic expression systems. The recombinant production of pro-CVF in two insect cell expression systems (baculovirus-infected Sf9 Spodoptera frugiperda cells, stably transfected S2 Drosophila melanogaster cells) generates three forms of pro-CVF: single-chain pro-CVF resembling pro-C3, a two-chain form of pro-CVF resembling C3, and another two-chain form of pro-CVF resembling C3b. All three forms of pro-CVF exhibit functional activity of mature, natural CVF. Recombinant pro-CVF supports the activation of factor B in the presence of factor D and Mg(2+), forms a bimolecular convertase pro-CVF,Bb that exhibits cleaving activity for both C3 and C5, and depletes the serum complement activity. The activity of pro-CVF and the resulting C3/C5 convertase is indistinguishable from CVF and the CVF,Bb convertase. Recombinant production of functionally active forms of pro-CVF ensures the availability of an important research reagent for future research involving complement depletion. The experimental systems to recombinantly produce active forms of CVF will also be invaluable for studies to delineate the structure/function relationship of CVF and its differences from C3, and to generate human C3 derivatives with CVF-like function ("humanized CVF") for therapeutic complement depletion.

Animals↗