hygromycin-a and Prostatic-Neoplasms

The differential sensitivity of tumor cells to TRAIL-induced apoptosis may be mediated by different intracellular inhibitors of apoptosis, and only a few reports have described the pathway(s) that are activated in response to TRAIL in prostate cells.. LNCaP was transfected with a dominant-negative form of FADD (FADD-DN) and cells were selected in the presence of hygromycin. Cell viability was estimated by calcein assay. Apoptosis was estimated by caspase activation using both fluorogenic substrates and Western blot analysis of activated caspases. To detect cytochrome c release, mitochondria-free cytosol was prepared and Western blot analysis was performed.. LNCaP is resistant to TRAIL but TRAIL transiently induces DEVDase activity and activation of caspase-8; caspase-2, -3, -7, and -9 were not activated. Wortmannin, an inhibitor of the PI3K/Akt pathway, converted the phenotype of LNCaP from TRAIL-resistant to -sensitive. In the presence of wortmannin TRAIL induced activation of caspase-2, -3, -7, -8, and -9, as well as dissipation of mitochondrial transmembrane potential and release of cyto-chrome c from mitochondria into the cytosol. In addition, combined TRAIL and wortmannin treatment resulted in cleavage of several proteins: PARP, Akt, p21/WAF1, and MDM2 as well as dephosphorylation of Akt. The proteolysis of p21/WAFI and Akt, which are known survival factors, presumably amplify the apoptotic cascade in LNCaP. Transfection of FADD-DN in LNCaP resulted in inhibition of caspase activation as well as in resistance to combined treatment with TRAIL and wortmannin.. These results suggest that caspase-8 activation is necessary but not sufficient for TRAIL-mediated apoptosis and is presumably blocked downstream of caspase-8 by the PI3K/Akt pathway.

Topics: Adaptor Proteins, Signal Transducing; Androstadienes; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Carrier Proteins; Caspase 8; Caspase 9; Caspases; Cinnamates; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Drug Resistance; Enzyme Activation; Enzyme Inhibitors; Fas-Associated Death Domain Protein; Humans; Hygromycin B; Male; Membrane Glycoproteins; Nuclear Proteins; Peptide Hydrolases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-mdm2; TNF-Related Apoptosis-Inducing Ligand; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Wortmannin

Changes in the outer membrane of apoptotic cells can induce neighboring cells to become phagocytic. Using genetically marked prostate cancer cell lines, we explored the possibility that genetic information might be transferred from an apoptotic cell to a phagocytic neighbor. Neomycin-resistant LNCaP cells that overexpress bcl-2 (LNCaP(bcl-2/neo-r)) were cocultured with hygromycin-resistant LNCaP cells (LNCaP(hygr-r)). The cocultures were then transiently exposed to serum starvation to induce apoptosis of LNCaP(hygr-r) cells. Surviving cells were then coselected in medium containing both antibiotics. Whereas monocultures of LNCaP(bcl-2/neo-r) or LNCaP(hygr-r) treated this way yielded no colonies, cocultures yielded dual-antibiotic-resistant clones at a frequency of approximately 1 in 10(5). Pre-exposure to an apoptotic agent was required; cocultures not exposed to serum starvation yielded no dual-selectable colonies. Analysis of DNA extracted from a dual-resistant clone demonstrated that the restriction endonuclease pattern of the neo-r gene was unaltered when compared with the parental LNCaP(bcl-2/neo-r). However the hygr-r gene demonstrated an altered restriction endonuclease pattern in the dual-resistant derivative compared with the parental LNCaP(hygr-r) cell line. This is evidence that genetic information can be transferred from one prostate cancer cell to another through the process of apoptosis, and we term this form of genetic transfer "apoptotic conversion."

Topics: Anti-Bacterial Agents; Apoptosis; Blotting, Southern; Cinnamates; Clone Cells; Coculture Techniques; DNA, Complementary; Drug Resistance, Microbial; Humans; Hygromycin B; Male; Neomycin; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured