Pyridoxal 5 ¶-phosphate (PLP) -dependent ornithine decarboxylase (ODC) is the key enzyme in polyamine synthesis. ODC is overexpressed in many tumor cells and thus a potential drug target. Here we show the design and synthesis of a coenzyme-substrate analogue as a novel precursor inhibitor of ODC. Structural analysis of the crystal structure of human ODC disclosed an additional hydrophobic pocket surrounding the E-amino group of its substrate ornithine. Molecular modeling methods showed favorable interactions of the BOC-protected pyridoxylornithine conjugate, termed POB, in the active site of human ODC. The synthesized and purified POB completely inhibited the activity of newly induced ODC activity at 100 Mmol/L in glioma LN229 and COS7 cells. In correlation with the inhibition of ODC activity, a timedependent inhibition of cell growth was observed in myeloma, glioma LN18 and LN229, Jurkat, COS7, and SW2 small-cell lung cancer cells if DNA synthesis and cell number were measured, but not in the nontumorigenic human aortic smooth muscle cells. POB strongly inhibited cell proliferation not only of low-grade glioma LN229 cells in a dose-dependent manner (IC 50 f50 Mmol/L) but also of high-grade glioblastoma multiforme cells. POB is much more efficient in inhibiting proliferation of several types of tumor cells than A-DL-difluoromethylornithine, the best known irreversible inhibitor of ODC. [Mol Cancer Ther 2007;6(6):1831 -9]

No abstract

Ewing sarcoma (EWS) proto-oncoprotein, an RNA-binding protein, is involved in DNA recombination and repair, gene expression, RNA processing and transport, as well as cell signalling. Chimeric EWS oncoproteins generated by chromosomal translocations between EWSR1 and the genes of transcription factors cause malignant tumors. To understand the loss of function by these translocations, the role of the intact EWS protein has to be investigated. The predominantly nuclear localization of the EWS protein via a transportin-1-mediated mechanism is dependent on the recently identified C-NLS (also known as PY-NLS). Among other residues in the C-NLS, Y656 interacts with transportin-1 and is essential for its nuclear localization. Here, we show that Y656 is phosphorylated, which seems to be a critical factor for transportin-1-mediated nuclear import. If Y656 was mutated cytosolic aggregates of the EWS protein, colocalized with transportin-1, were observed, similar to those described with mutants of the closely related FUS/TLS protein that had amino acid substitutions in the PY-NLS causing familial amyothrophic lateral sclerosis.

No abstract

In the course of the apoptotic cell death, cells fragment into apoptotic bodies, the elimination of which by phagocytosis is thought to avoid the release of cytosolic constituents whose occurrence is indicative for necrotic cell death. Confluent cultures of chicken embryo fibroblasts, however, show a different behaviour. After serum deprivation, they transiently released with the same time course mitogenic activity, lactate dehydrogenase and cytosolic peptidyl prolyl cis-trans isomerases into the serum-free culture medium. The release correlated in time with a decrease of the cell number which started approximately 3 h after serum removal and ceased within approximately 10 h at about half of the initial cell density. Morphological features like cell shrinkage, membrane blebbing and cell fragmentation as well as internucleosomal DNA fragmentation indicated apoptotic cell death whereas necrotic cell death could be excluded. Conditioned medium (M(r) > or = 30 kDa) from serum-deprived cultures of chicken embryo fibroblasts completely prevented chicken embryo fibroblasts to undergo apoptosis as did phorbol 12-myristate, 13-acetate and, to -60%, L-cysteine. Cycloheximide had no effect on serum deprivation-induced apoptosis. From the present results it can be concluded that chicken embryo fibroblasts and possibly other cells undergoing apoptosis release cytosolic components and endogenous survival factor(s) which prevent apoptosis.