Mechanisms of cell death induced by 5-fluoro-2′-deoxyuridine (FUdR)—Necrosis or apoptosis after treated with FUdR—

5-Fluoro-2'-deoxyuridine (FUdR) inhibits thymidylate synthase (TS). The inhibition of TS causes an imblance of intracellular deoxyribonucleoside triphosphate (dNTP) pools which subsequently induced cell death. We investigated the molecular mechanisms of cell death after treated with FUdR in F28-7 strain (which induced necrosis) and F287-A strain (mutant of F28-7 strain which induced apoptosis). After treated with FUdR, we observed different size of DNA fragmentations. F28-7 strain induced DNA cleavaged into 100-200 kbp fragments and F28-7-A strain induced DNA cleavaged into oligonucleosomal sized fragments. In F28-7 strain, FUdR induced the increased mRNA level of c-jun, c-fos and c-myc genes, caspase-3 like protease activity and the changes of mitochondrial membrane potential which were greater and earlier than those of F28-7-A strain. On the other hand, F28-7-A strain induced release of cytochrome c from mitochondria, but not F28-7 strain. Furthermore, caspase-5 inhibitor was strongly inhibited the cell death of F28-7 strain. We suggest that it is concerned with intensity of intracellular signals in the cell death of F28-7 strain and F28-7-A strain. INTRODUCTION Cell death can take place as either necrosis or apoptosis by determined cellular morphology. Necrosis is usually considered as result from physical injury and is accompanied by swelling of cells. It is not genetically controlled. On the contrary, apoptosis is genetically controlled and significant morphological change of nuclear including chromatin condensation, nuclear fragmentation and formation of apoptotic body. It also called " programed cell death ". Recently, some early events in the death program may be shared by the two types of cell death and that downstream events may contribute the guiding of cells toward apoptosis or necrosis. Thus, it is considerable investigation of the events in cell death is important to elucidate necrosis or apoptosis. We investigated the molecular mechanisms of cell death in F28-7 strain and F28-7-A strain after treated with FUdR. Cytotoxic effect is induced by inhibition of DNA synthesis resulted from which FUdR inhibits TS. The inhibition of TS causes an imblance of intracellular dNTP pool which subsequently induced cell death. In this report, it is suggested that the intensity of intracellular signals determined whether necrosis or apoptosis in F28-7 strain and F28-7-A strain treated with FUdR. RESULTS AND DISCUSSION Morphological changes were observed by confocal microscopy with propidium iodide-staining. In F28-7 strain, cell death was accompanied by necrosis-like cell swelling, whereas upon the cell death of F28-7-A strain, apoptotic bodies were observed. DNA fragmentation in F28-7 strain or F28-7-A strain treated with FUdR was examined by gel electrophoresis and orthogonal field alternation gel electrophoresis (OFAGE). In F28-7 strain, cell death induced by FUdR was accompanied by 100-200 kbp DNA fragments. On the contrary, upon the cell death of F28-7-A strain, oligonucleosomal DNA fragmentation was observed. This result indicated F28-7 strain induced apoptosis with biochemical characterization. Based on these findings, it is conceivable that the mechanisms of these DNA cleavaged patterns in 246 Nucleic Acids Research Supplement No. 2 Overactivati on of Caspase-5 (F28-7 Strain) Necrosis (F28-7 Strain) FUdR Common mechanisms of cell death in F28-7 strain and F28-7-A strain Expression of i . c-/os and c-myc The changes of mitochondrial membrane potential Activation of Caspase-3 like protease Strong