ABSTRACT
Prion protein (PrP(C)) via its isoform PrP(SC) is involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). We observed that murine erythroleukemia (MEL) cells arrested in phase G(1) undergo transcriptional activation of Prn-p gene. Here, we explored the potential role of activation of Prn-p gene and cytosolic accumulation of PrP(C) in growth arrest, differentiation, and apoptotic DNA fragmentation by stably transfecting MEL and N2a cells with Prn-p cDNA. Stably transfected MEL cells (clones # 6, 12, 20, 38, and 42) were assessed for growth and differentiation, while clones N2a13 and N2a8 of N2a cells for growth and apoptosis by flow cytometry using Annexin V and propidium iodide (PI). Our results indicate that (a) Induction of terminal differentiation of stably transfected MEL cells led to growth arrest, activation of Prn-p gene, concomitant expression of transfected Prn-p cDNA, suppression of bax gene, cytosolic accumulation of PrP(C), and DNA fragmentation. The latter was also induced in non-differentiated MEL cells growing under serum-free conditions; (b) similarly, serum deprivation promoted growth arrest, apoptosis/necrosis associated with DNA fragmentation in parental N2a and N2a13 cells that produced relative high level of PrP(C) and not PrP(SC). These data indicate that activation of Prn-p gene and expression of transfected Prn-p cDNA in cells of both hematopoietic and neuronal origin occurred concomitantly, and led to cytosolic accumulation of PrP(C) and DNA damage induced by serum deprivation. PrP(C) production failed to protect DNA fragmentation induced by serum deprivation. The question how does PrP(C) contribute to growth arrest and DNA fragmentation is discussed.
Subject(s)
Apoptosis , DNA Fragmentation , Leukemia, Erythroblastic, Acute/metabolism , Neuroblastoma/metabolism , PrPC Proteins/biosynthesis , Prions/biosynthesis , Transcriptional Activation , Animals , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Proliferation , Culture Media, Serum-Free/metabolism , Cytosol/metabolism , Dimethyl Sulfoxide/pharmacology , Flow Cytometry , Gene Expression Regulation, Neoplastic , Leukemia, Erythroblastic, Acute/genetics , Leukemia, Erythroblastic, Acute/pathology , Leukemia, Erythroblastic, Acute/physiopathology , Mice , Neuroblastoma/genetics , Neuroblastoma/pathology , Neuroblastoma/physiopathology , Prion Proteins , Prions/genetics , RNA, Messenger/biosynthesis , Time Factors , Transfection , Up-Regulation , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
PURPOSE: The aim of this study was to assess the cytotoxicity of 4 resin composites' eluates on WEHI 13 Var fibroblasts as they aged in a biologic medium. MATERIALS AND METHODS: Cytotoxicity was determined by counting the number of viable cells by trypan blue exclusion. Morphologic changes attributable to cytotoxicity were observed by May-Grunwald-Giemsa cytologic staining and microscopic examination. DNA gel electrophoresis was performed to detect possible genotoxicity and DNA damage. RESULTS: All resin composite eluates, except that for Targis, caused a pronounced cytotoxicity during the first 72 hours that gradually decreased after 2 weeks of aging. Severe morphologic alterations and pronounced DNA damage were also observed. CONCLUSION: These findings indicate that resin-based dental restorative materials release agents cytotoxic and genotoxic to fibroblasts. Cytotoxicity is gradually decreased as the composite resins age in a biologic-relevant medium.