CLONING AND EXPRESSION OF A GENE MEDIATINGγ-RADIATION-INDUCED APOPTOSIS IN HL-60 CELLS / 药物分析学报

Objective To identify the member of the caspase family proteases involved in γ-radiation-induced apoptosis in HL-60 cells and to study the expression of the caspase gene in normal, apoptotic cells and in immortal tu mor cells. Methods By using degenerate oligonucleotide primers encoding the highly conserved peptides that were pre sent in all known caspases, we performed RT-PCR on poly(A)RNA from γ-radiation-induced apoptotic HL-60 cells. Caspase-3 mRNA in apoptotic HL-60 cells and in human tumor cell lines was analyzed by Northern blot. Results The amplified DNA fragment was identified with caspase-3 cDNA by cloning and sequencing. The Northern blot analysis of caspase-3 mRNA of different human tumor cell lines showed that the caspase-3 gene transcript was more highly ex pressed in leukemia cell lines and the SH-SY5Y cell line than in HeLa and MCF-7 cells. It was more highly expressed in the radiation-induced apoptotic HL-60 cells than in control HL-60 cells. Conclusion These results indicated that caspase-3 was involved in γ-radiation-induced apoptosis in HL-60 cells. The high level of expression of caspase-3 may aid efforts to understand the insensitivity of some tumor cells to radiation, their inherent ability to survive, and apop tosis.

The Electron Microscopic Study on the Development of Knee Joint in Rat / 대한해부학회지

These study was designed to observe the appearance and the characteristics of apoptotic cells during the development of knee joint in rat. The fetus were collected on the 16th, 17th, 18th, 19th, and 20th day of pregnancy. In this study, TUNEL staining, electron microscopic investigation and immunocytochemical gold labeling techniques were used. In the immuno-cytochemical gold labeling techniques, primary antibodies were used, which were to be polyclonal rabbit anti-mouse/ rat Bax, polyclonal rabbit anti-tissue transglutaminase C, and polyclonal goat anti-cpp32p20. The samples were observed under JEOL 1200 EX-II transmission electron microscope. The results were as follows. 1. In a 16-day-old fetus, between femur and tibia cartilages, mesenchymal cells were observed. Mesenchymal cells had marginated heterochromatin and dilated rough endoplasmic reticulum. 2. In a 17-day-old fetus, the knee joint clefts were first formed. In the primordial cruciate ligaments between the cartilages, capillaries were scattered. The apoptotic cells, which had fragmented and condensed nucleus, showed in the synovium. And necrotic cells, which had nuclear chromatin margination, perinuclear cisternae, and dilated rough endoplasmic reticulum, also were observed in the joint cleft surface. 3. From the 18-day-old fetus, phagocytic synovial cells and secretory synovial cells could be confirmed. The apoptotic cells were not seen. 4. In a 17-day-old fetus, a few cells were positive for TUNEL reaction in the joint cleft region. 5. In a 17-day-old fetus, Bax were marked on the mitochondria, endoplasmic reticulum of apoptotic cells. Also, it was marked at the phagocytosed apoptotic bodies in the neighboring cells. 6. In a 17-day-old fetus, the tissue Transglutaminase C were marked in the perinuclear region, vacuoles, cell membrane and extracellular matrix of the apoptotic cells. Also, it was marked at the phagocytosed apoptotic bodies in the neighboring cells. 7. In a 17-days-old fetus, CPP32 labeling were marked in the cytoplasm of the apoptotic cells. Practically, it was distributed between the phagocytosed apoptotic bodies and the neighboring cells. On the basis of above findings, it is obvious that the joint cleft are first formed in a 17-day-old fetus, a few cells are to be TUNEL positive signals, and the apoptotic cells contain Bax, tissue Transglutaminase C, and CPP32. Therefore the apoptotic cells and the necrotic cells are appeared in the 17-day-old fetus, and these cells are concerned with joint cleft formation.

Cell Beath Induced by Ethanol : Prevention of Cell Death by the bcl-2 Proto-Oncogene

The Bcl-2 protein has been shown to block apoptosis induced by a variety of stimuli. We have performed the experiments which cell death can be blocked by the bcl-2 proto-oncogene under moderate(50-100mM) or high ethanol treatment(400-600mM). As a result of morphological changes, and MTT assay, cell death was blocked by Bcl-2 under 100mM ethanol. However, the results of DNA fragmentation and RT-PCR(ICE, and CPP32), immunoblotting(CPP32, and PARP) for SK-pcDNA3 cells(vector only) and SK-Bcl-2 cells(stably expressed bcl-2 gene) were showen to be no significant differences between two cell lines. These result suggested that cell death induced by ethanol was not followed by apoptosis mechanism, and was blocked by the bcl-2 proto-oncogene with moderate ethanol.

The dynamic changes of CPP32 mRNA in hippocampus following temporary whole cerebral ischemia in rats / 临床神经病学杂志

Objective To observe the dynamic changes of CPP32 mRNA in hippocampus following temporary whole cerebral ischemia in rats and to explore the mechanism of neuron apoptosis induced by cerebral ischemia.Methods The model of temporary whole cerebral ischemia in rats were induced by carotid artery negative pressure shunt and the expression of CPP32 mRNA were detected with fluorescence quantify RT PCR.Results Compared with the naive group, the expression of CPP32 mRNA did not increase insignificantly in sham operation group. Compared with sham operation group, the expression of CPP32 mRNA in ischemia group trended to increase at 6 h after ischemia and increased by 53% at 12 h and 223% at 24 h after ischemia. It kept in high level and decreased at 72 h after ischemia.Conclusion The expression of CPP32 mRNA in hippocampus could be induced by temporary whole cerebral ischemia. It began to increase at 6 h, reached its fastigium at 24 h and tended to decrease at 72 h after ischemia. The enhanced gene transcription of CPP32 plays an important role in the neuron apoptosis induced by cerebral ischemia.