aphidicolin and Necrosis

Apoptosis is a physiological mode of death where the dying cell plays an active part in its own demise, which contrasts sharply with what is seen in necrosis. In the present paper I have shown that when cells of the immune system are exposed to a range of cytotoxic agents, such as the RNA synthesis inhibitor actinomycin D, the DNA polymerase inhibitor aphidicolin or the topoisomerase I inhibitor campthothecin, they rapidly undergo cell death via apoptosis. This is characterized by DNA fragmentation to yield the now hallmark ladder pattern of death by this mechanism. All of these agents are capable of inducing apoptosis irrespective of what phase of the cell cycle a cell is in. These studies also indicate that apoptosis can occur in immune cells without recourse to macromolecular synthesis.

Topics: Animals; Aphidicolin; Apoptosis; Camptothecin; Cell Cycle; Dactinomycin; Humans; Immune System; Necrosis

Recently, the three-dimensional structure of the active site of human DNA polymerase alpha (pol alpha) was proposed based on the application of molecular modeling methods and molecular dynamic simulations. The modeled structure of the enzyme was used for docking selective inhibitors (nucleotide analogs and the non-nucleoside inhibitor aphidicolin) in its active site in order to design new drugs for actinic keratosis and squamous cell carcinoma (SCC). The resulting complexes explained the geometrical and physicochemical interactions of the inhibitors with the amino acid residues involved in binding to the catalytic site, and offered insight into the experimentally derived binding data. The proposed structures were synthesized and tested in vitro for their influence on human keratinocytes and relevant tumor cell lines. Effects were compared to aphidicolin which inhibits pol alpha in a non-competitive manner, as well as to diclofenac and 5-fluorouracil, both approved for therapy of actinic keratosis. Here we describe three new nucleoside analogs inhibiting keratinocyte proliferation by inhibiting DNA synthesis and inducing apoptosis and necrosis. Thus, the combination of modeling studies and in vitro tests should allow the derivation of new drug candidates for the therapy of skin tumors, given that the agents are not relevant substrates of nucleotide transporters expressed by skin cancer cells. Kinases for nucleoside activation were detected, too, corresponding with the observed effects of nucleoside analogs.

Topics: Aphidicolin; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; DNA Polymerase I; Humans; Keratinocytes; Keratosis, Actinic; Models, Chemical; Models, Molecular; Necrosis; Nucleic Acid Synthesis Inhibitors; Nucleotide Transport Proteins; Protein Binding; Purines; Skin Neoplasms; Thymidine

Smooth muscle cells and endothelial cells play an important role in cardiovascular diseases and may therefore be a potential target for gene therapy. Most in vitro experiments are performed using proliferating cell cultures. Nevertheless, non-dividing cells would represent more realistic in vivo conditions for gene therapy. Therefore, a simple method to achieve physiologically quiescence in cell cultures is needed for experiments. Growth to confluence is sufficient for endothelial cells to reach quiescence, in contrast to smooth muscle cells. Alternative techniques were investigated to achieve quiescence for smooth muscle cells. N-acetyl-cysteine, heparin, aphidicolin and serum-free medium are known inhibitors of smooth muscle cell proliferation and were tested for cell viability, necrosis and apoptosis. The inhibition status was evaluated counting cells in a cell counter. Toxicity, necrosis and apoptosis were determined using FACS analysis. Then, smooth muscle cells and endothelial cells were transfected with plasmid containing the beta-galactosidase gene using liposomes. Analysis of gene expression in transfected cells included a quantitative beta-galactosidase assay and X-gal staining. Growth inhibition was achieved with all agents tested. Using N-acetyl-cysteine, only slightly reduced growth rates were observed. Aphidicolin stopped cell growth almost immediately, but demonstrated enhanced toxicity. The amount of apoptotic and necrotic cells was lowest using heparin in the presence of foetal calf serum. Transfection experiments using stationary cultures of smooth muscle cells using heparin or aphidicolin demonstrated 5-10-fold lower transfection rates compared to transfected proliferating cell cultures serving as controls. Transfection experiments using stationary cultures of endothelial cells using growth inhibition through confluence demonstrated 40-fold lower transfection rates than transfected proliferating cell cultures. Transfer efficiency was much lower in endothelial cells compared to smooth muscle cells. In conclusion, quiescent cells simulate more realistically the in vivo situation and may therefore represent a better model for future in vivo experiments based on in vitro findings.

Topics: Animals; Aorta; Aphidicolin; Apoptosis; beta-Galactosidase; Cell Division; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Endothelium, Vascular; Flow Cytometry; Growth Inhibitors; Heparin; Kinetics; Muscle, Smooth, Vascular; Necrosis; Recombinant Proteins; Swine; Transfection