Apoptosis plays an important role in embryonic development, tissue remodeling, immune regulation and tumor regression. Two groups of molecules (Bcl-2 family and "Death factor" family) are involved in regulat...Apoptosis plays an important role in embryonic development, tissue remodeling, immune regulation and tumor regression. Two groups of molecules (Bcl-2 family and "Death factor" family) are involved in regulating apoptosis. In order to know about the effect of Bcl-2 on apoptosis induced by Fas, a typical member of "Death factor" family, the transfection experiments with expression vectors pcDNA3-fl and pcDNA3-bcl-2 were performed in BEL-7404 cells, a human hepatocellular carcinoma cell line which expresses endogenous Fas, but not FasL and Bcl-2. The data showed that the expression of FasL in pcDNA3-fl transfected hepatoma cells obviously induced the apoptosis of the cells. However, the overexpression of Bcl-2 in pcDNA3-bcl-2 transfected 7404/b-16 cells counteracted pcDNA3-fl transient transfection mediated apoptosis. Further study by cotransfection experiments indicated that Bid but not Bax (both were pro-apoptotic proteins of Bcl-2 family) blocked the inhibitory effect of Bcl-2 on Fas-mediated apoptosis. These results suggested that Fas-mediated apoptosis in human hepatoma cells is possibly regulated by Bcl-2 family proteins via mitochondria pathway.展开更多
Mad protein has been shown as an antagonist of cMyc protein in some cell lines. The effect of Mad protein to the malignant phenotype of human hepatoma BEL7404 cell line was investigated experimentally. An eukarryotic ...Mad protein has been shown as an antagonist of cMyc protein in some cell lines. The effect of Mad protein to the malignant phenotype of human hepatoma BEL7404 cell line was investigated experimentally. An eukarryotic vector pCDNA Ⅲ containing full ORF fragmentof mad cDNA was transfected into targeted cells. Under G418 selection, stable Mad-overexpressed cells were cloned.Studies on the effect of Mad over-expression in cell proliferation and cell cycle revealed that cell morphology of the Mad-overexpressed BEL-7404-M1 cells was significantly different from the parent and control vector transfected cells. DNA synthesis, cell proliferation and anchorage-independent growth in soft-agar of the madtransfected cel1s were partially inhibited in comparison to control cells.Flow Cytometry analysis indicated that mad over-expression might block more transfectant cells at G0/G1 phase, resulting in the retardation of cell proliferation. RT-PCR detected a marked inhibition of the expression of cdc25A, an important regulator gene of G0/G1to S phase in cell cycle. It was also found that Mad protein overexpression could greatly suppress p53-mediated apoptosis in BEL-7404-M1 cells in the absence of serume.Thus, Mad proteins may function as a negative regulator antagonizing c-Myc activity in the control of cell growth and apoptosis in human hepatocellular carcinoma BEL7404 cells.madoverexpression and regulation of cell growth and展开更多
Epidermal growth factor （EGF） induced intracellular free calcium ([Ca2+]i) response was studied in fura-2- or fluo-3-loaded human hepatoma cells of BEL-7404 cell line. Single cell [Ca2+]i analysis and [Ca2+], measur...Epidermal growth factor （EGF） induced intracellular free calcium ([Ca2+]i) response was studied in fura-2- or fluo-3-loaded human hepatoma cells of BEL-7404 cell line. Single cell [Ca2+]i analysis and [Ca2+], measurement in cell populations revealed that EGF triggered a rapid [Ca2+]iincrease in the dose-dependent and time- dependent manner. Pretreatment of cells with an endoplasmic reticulum （ER） Ca2+-ATPase inhibitor, thapsigargin （TG） at 100 nM concentration for 20 min, completely abolished EGF-induced [Ca2+]i increase, and chelating extracellular calcium by excess EGTA partially inhibited the increase.Furthermore, the expression of antisense EGF receptor sequence in BEL-7404 cells suppressed the [Ca2+]i response to EGF. The results suggest that EGF receptor-mediated [Ca2+]i increase in the human hepatoma cells is essentially dependent on the Ca2+ storage in ER.展开更多
Effects of thapsigargin, an inhibitor of Ca2+-ATPase in surface of endoplasmic reticulum, on apoptotic cell death were studied in human hepatoma cells of BEL-7404 cell line by using both flow cytometry and electron mi...Effects of thapsigargin, an inhibitor of Ca2+-ATPase in surface of endoplasmic reticulum, on apoptotic cell death were studied in human hepatoma cells of BEL-7404 cell line by using both flow cytometry and electron mi-croscopy Propidium iodide staining and flow cytome- try revealed that in the serum-free condition, thapsigar-gin increased the rate of apoptosis of BEL- 7404 cells in a dose-dependent manner. Prolongation of the period of serum-free condition enhanced the apoptosis induced by thapsigargin treatment. Morphological observation with electron microscope further demonstrated that chromatin condensation and fragmentation, apoptotic bodies existed in TG-treated cells, supporting that thapsigargin is a po-tent activator of apoptosis in the cells.展开更多
Telomerase activity was inhibited in a dose and time-dependent manner with the treatment of cisplatin for 24, 48, or 72 h in a concentration ranged from 0.8 to 50 1uM in BEL-7404 human hepatoma cells. There were no ch...Telomerase activity was inhibited in a dose and time-dependent manner with the treatment of cisplatin for 24, 48, or 72 h in a concentration ranged from 0.8 to 50 1uM in BEL-7404 human hepatoma cells. There were no changes in expression pattern of three telomerase subunits, its catalytic reverse transcriptase subunit (hTERT), its RNA component (hTR) or the associated protein subunit (TP1), after cisplatin treated for 72 h with indicated concentrations. Mean telomere lengths were decreased by the cisplatin treatment. Cell growth inhibition and cell cycle accumulation in G2/M phase were found to be correlated with telomerase inhibition in the present study, but percentages of cell apoptosis did not change markedly during the process.展开更多
Effects of antisense epidermal growth factor receptor (EGFR) sequence on apoptotic cell death were examined in a human hepatoma cell line BEL-7404 cells. In the cells of JX-1, a sub clone of BEL-7404 stably transfecte...Effects of antisense epidermal growth factor receptor (EGFR) sequence on apoptotic cell death were examined in a human hepatoma cell line BEL-7404 cells. In the cells of JX-1, a sub clone of BEL-7404 stably transfected with antisense EGFR vector (Cell Research, 3:75, 1993), an enhanced rate (9.5%) of spontaneous apoptosis was detected by flow cytometry, whereas the rates of spontaneous apoptosis in JX-0 cells, a sub-clone of BEL-7404 transfected by control vector, and the parellt BEL-7404 cells were almost equal and about 1.7%. Serum-starvation for 72 h increased the rate of apoptosis of JX-1cells up to 33.7%, while JX-0 and BEL-7404 cells, under the same condition, produced less than 5% of apoptotic cells. Observation with electron microscope demonstrated that condensation and fragmentation of chromatin and formation of apoptotic bodies of ten occurred in JX-1 cells, especially during serumstarvation. These results, combined with the data of DNA fragmentation Elisa test, suggested that antisense EGFR sequence enhances apoptosis in the human hepatoma cells.Comparison of intracellular Ca2+ level and the responsiveness of JX-1 cells to the induced action of EGF and tharpsigargin (TG) treatment with that of control JX-0cells indicated that antisense EGFR might interrupt the EGF/EGFR signaling pathway resulting in the decreass of intracellular Ca2+ pool content as well as the responsiveness of these cells to the extracellular signals. These findings suggest that antisense EGFR either directly or indirectly reglllates Ca2+ storage in endoplasmic reticulum,thereby enhances apoptosis in the hnman hepatoma cells.展开更多
基金Major State Basic Reaearch (973) Program of China.
文摘Apoptosis plays an important role in embryonic development, tissue remodeling, immune regulation and tumor regression. Two groups of molecules (Bcl-2 family and "Death factor" family) are involved in regulating apoptosis. In order to know about the effect of Bcl-2 on apoptosis induced by Fas, a typical member of "Death factor" family, the transfection experiments with expression vectors pcDNA3-fl and pcDNA3-bcl-2 were performed in BEL-7404 cells, a human hepatocellular carcinoma cell line which expresses endogenous Fas, but not FasL and Bcl-2. The data showed that the expression of FasL in pcDNA3-fl transfected hepatoma cells obviously induced the apoptosis of the cells. However, the overexpression of Bcl-2 in pcDNA3-bcl-2 transfected 7404/b-16 cells counteracted pcDNA3-fl transient transfection mediated apoptosis. Further study by cotransfection experiments indicated that Bid but not Bax (both were pro-apoptotic proteins of Bcl-2 family) blocked the inhibitory effect of Bcl-2 on Fas-mediated apoptosis. These results suggested that Fas-mediated apoptosis in human hepatoma cells is possibly regulated by Bcl-2 family proteins via mitochondria pathway.
文摘Mad protein has been shown as an antagonist of cMyc protein in some cell lines. The effect of Mad protein to the malignant phenotype of human hepatoma BEL7404 cell line was investigated experimentally. An eukarryotic vector pCDNA Ⅲ containing full ORF fragmentof mad cDNA was transfected into targeted cells. Under G418 selection, stable Mad-overexpressed cells were cloned.Studies on the effect of Mad over-expression in cell proliferation and cell cycle revealed that cell morphology of the Mad-overexpressed BEL-7404-M1 cells was significantly different from the parent and control vector transfected cells. DNA synthesis, cell proliferation and anchorage-independent growth in soft-agar of the madtransfected cel1s were partially inhibited in comparison to control cells.Flow Cytometry analysis indicated that mad over-expression might block more transfectant cells at G0/G1 phase, resulting in the retardation of cell proliferation. RT-PCR detected a marked inhibition of the expression of cdc25A, an important regulator gene of G0/G1to S phase in cell cycle. It was also found that Mad protein overexpression could greatly suppress p53-mediated apoptosis in BEL-7404-M1 cells in the absence of serume.Thus, Mad proteins may function as a negative regulator antagonizing c-Myc activity in the control of cell growth and apoptosis in human hepatocellular carcinoma BEL7404 cells.madoverexpression and regulation of cell growth and
文摘Epidermal growth factor （EGF） induced intracellular free calcium ([Ca2+]i) response was studied in fura-2- or fluo-3-loaded human hepatoma cells of BEL-7404 cell line. Single cell [Ca2+]i analysis and [Ca2+], measurement in cell populations revealed that EGF triggered a rapid [Ca2+]iincrease in the dose-dependent and time- dependent manner. Pretreatment of cells with an endoplasmic reticulum （ER） Ca2+-ATPase inhibitor, thapsigargin （TG） at 100 nM concentration for 20 min, completely abolished EGF-induced [Ca2+]i increase, and chelating extracellular calcium by excess EGTA partially inhibited the increase.Furthermore, the expression of antisense EGF receptor sequence in BEL-7404 cells suppressed the [Ca2+]i response to EGF. The results suggest that EGF receptor-mediated [Ca2+]i increase in the human hepatoma cells is essentially dependent on the Ca2+ storage in ER.
文摘Effects of thapsigargin, an inhibitor of Ca2+-ATPase in surface of endoplasmic reticulum, on apoptotic cell death were studied in human hepatoma cells of BEL-7404 cell line by using both flow cytometry and electron mi-croscopy Propidium iodide staining and flow cytome- try revealed that in the serum-free condition, thapsigar-gin increased the rate of apoptosis of BEL- 7404 cells in a dose-dependent manner. Prolongation of the period of serum-free condition enhanced the apoptosis induced by thapsigargin treatment. Morphological observation with electron microscope further demonstrated that chromatin condensation and fragmentation, apoptotic bodies existed in TG-treated cells, supporting that thapsigargin is a po-tent activator of apoptosis in the cells.
文摘Telomerase activity was inhibited in a dose and time-dependent manner with the treatment of cisplatin for 24, 48, or 72 h in a concentration ranged from 0.8 to 50 1uM in BEL-7404 human hepatoma cells. There were no changes in expression pattern of three telomerase subunits, its catalytic reverse transcriptase subunit (hTERT), its RNA component (hTR) or the associated protein subunit (TP1), after cisplatin treated for 72 h with indicated concentrations. Mean telomere lengths were decreased by the cisplatin treatment. Cell growth inhibition and cell cycle accumulation in G2/M phase were found to be correlated with telomerase inhibition in the present study, but percentages of cell apoptosis did not change markedly during the process.
文摘Effects of antisense epidermal growth factor receptor (EGFR) sequence on apoptotic cell death were examined in a human hepatoma cell line BEL-7404 cells. In the cells of JX-1, a sub clone of BEL-7404 stably transfected with antisense EGFR vector (Cell Research, 3:75, 1993), an enhanced rate (9.5%) of spontaneous apoptosis was detected by flow cytometry, whereas the rates of spontaneous apoptosis in JX-0 cells, a sub-clone of BEL-7404 transfected by control vector, and the parellt BEL-7404 cells were almost equal and about 1.7%. Serum-starvation for 72 h increased the rate of apoptosis of JX-1cells up to 33.7%, while JX-0 and BEL-7404 cells, under the same condition, produced less than 5% of apoptotic cells. Observation with electron microscope demonstrated that condensation and fragmentation of chromatin and formation of apoptotic bodies of ten occurred in JX-1 cells, especially during serumstarvation. These results, combined with the data of DNA fragmentation Elisa test, suggested that antisense EGFR sequence enhances apoptosis in the human hepatoma cells.Comparison of intracellular Ca2+ level and the responsiveness of JX-1 cells to the induced action of EGF and tharpsigargin (TG) treatment with that of control JX-0cells indicated that antisense EGFR might interrupt the EGF/EGFR signaling pathway resulting in the decreass of intracellular Ca2+ pool content as well as the responsiveness of these cells to the extracellular signals. These findings suggest that antisense EGFR either directly or indirectly reglllates Ca2+ storage in endoplasmic reticulum,thereby enhances apoptosis in the hnman hepatoma cells.
