Objective To investigate the roles of the cyclin D1/CDK4 and E2F-1/4 pathways and compare their work patterns in cell cycle changes induced by different doses of B[a]E Methods Human embryo lung fibroblasts （HELFs） w...Objective To investigate the roles of the cyclin D1/CDK4 and E2F-1/4 pathways and compare their work patterns in cell cycle changes induced by different doses of B[a]E Methods Human embryo lung fibroblasts （HELFs） were treated with 2 μmol/L or 100 μmol/L B[a]P which were provided with some characteristics of transformed cells （T-HELFs）. Cyclin D l, CDK4 and E2F-1/4 expressions were determined by Western blotting. Flow cytometry was used to detect the distribution of cell cycle. Results After B[a]P treatment, the proportion of the first gap （G 1） phase cells decreased. CDK4 and E2F-4 expression did not change significantly. In 2 μmol/L treated cells, a marked overexpression of cyclin D1 and E2F-1 was observed. However, in T-HELFs overexpression was limited to cyclin D1 only, and no overexpression of E2F-1 was observed. The decreases of G1 phase in response to B[a]P treatment were blocked in antisense cyclin D1 and antisense CDK4 transfected HELFs （A-D1 and A-K4） and T-HELFs （T-A-D1 and T-A-K4）. After 2 μmol/L B[a]P treatment, overexpression of E2F-1 was attenuated in A-D1, and E2F-4 expression was decreased significantly in A-K4. In T-A-D1 and T-A-K4, E2F-4 expression was increased significantly, compared with T-HELFs. The E2F-1 expression remained unchanged in T-A-D1 and T-A-K4. Condusions Cyclin DI/CDK4-E2F-1/4 pathways work in different patterns in response to low dose and high dose B[a]P treatment. In HELFs treated with 2 μmol/L B[a]P, cyclin D1 positively regulates the E2F-1 expression while CDK4 negatively regulates the E2F-4 expression; however, in HELFs treated with 100 μmol/L B[a]P, both cyclin D1 and CDK4 negatively regulate the E2F-4 expression.展开更多
Objective To study the molecular mechanism of the inhibitory effects of vitamin C on benzo[a]pyrene （B[a]P）-induced changes of cell cycle in human embryo lung fibroblast （HELF） cells. Methods The stable transfecta...Objective To study the molecular mechanism of the inhibitory effects of vitamin C on benzo[a]pyrene （B[a]P）-induced changes of cell cycle in human embryo lung fibroblast （HELF） cells. Methods The stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established. Cells were cultured and pretreated with vitamin C before stimulation with B[a]P for 24 h. The expression levels of cyclin DI, CDK4, E2FI, and E2F4 were determined by Western blot. Flow cytometric analysis was employed to detect the distributions of cell cycle. Results B[a]P significantly elevated the expression levels of cyclin D 1, E2F1, and E2F4 in HELF cells. Vitamin C decreased the expression levels of cyclin D 1, E2F1, and E2F4 in B [a]P-stimulated HELF cells. Dose-dependent relationships were not found between the different concentrations of vitamin C （10, 100, 500, 1000, and 5000 lamol/L） and the expression levels of cyclin D 1, E2F1, and E2F4 in HELF cells. The expression levels of cyclin D1, E2FI, and E2F4 in B[a]P-treated transfectants were lower than those in B[a]P-treated HELF cells. The expression levels of cyclin DI and E2F4 treated with vitamin C and antisense cyclin D1 were decreased compared with those treated with antisense cyclin DI alone. The effects of vitamin C combined with antisense CDK4 on the expression levels of cyclin DI and E2FI/E2F4 were similar to those of antisense CDK4 alone. B[a]P progressed HELF cells from GI to S phase. Both vitamin C and antisense cyclin DI suppressed the changes of cell cycle progressed by B[a]P. However, antisense CDK4 did not attenuate the above changes. Vitamin C combined with antisense CDK4 markedly suppressed B[a]P-induced changes of cell cycle as compared with antisense CDK4. But the inhibitory effects of vitamin C combined with antisense cyclin DI on B[a]P-induced changes of cell cycle were similar to those of vitamin C alone or antisense cyclin DI alone. Conclusions B[a]P progressed HELF cells from G1 to S phase via intracellular signaling pathway of cyclin D I/E2F. Vitamin C may modulate this signaling pathway to protect cells from injury caused by B[a]P.展开更多
AIM:To evaluate the effects of the proteasome inhibitor bortezomib(BZB)on E2Fs and related genes in hepatocellular carcinoma(HCC)cells.METHODS:The mRNA levels of the E2F family members(pro-proliferative:E2F1-3 and ant...AIM:To evaluate the effects of the proteasome inhibitor bortezomib(BZB)on E2Fs and related genes in hepatocellular carcinoma(HCC)cells.METHODS:The mRNA levels of the E2F family members(pro-proliferative:E2F1-3 and anti-proliferative:E2F4-8)and of their related genes cyclins and cyclindependent kinases(cdks)were evaluated in two HCC cell lines following a single BZB administration.mRNA levels of the epithelial-mesenchymal transition(EMT)genes were also measured in both cell lines after BZB treatment.The BZB concentration(40 nmol/L)used was chosen to stay well below the maximal amount/cm2recommended for in vivo application,and 2 d incubation was chosen as this time point has been found optimal to detect BZB effects in our previous studies.The HCC cell lines,HepG2 and JHH6,were chosen as they display different phenotypes,hepatocyte-like for HepG2and undifferentiated for JHH6,thus representing an in vitro model of low and high aggressive forms of HCC,respectively.The mRNA levels of the target genes were measured by two-color microarray-based gene expression analysis,performed according to Agilent Technologies protocol and using an Agilent Scan B.For the E2F family members,mRNA levels were quantified by realtime reverse transcription polymerase chain reaction(RT-PCR).Using small interfering RNA’s,the effects of E2F8 depletion on cell number was also evaluated.RESULTS:After BZB treatment,microarray analysis of the undifferentiated JHH6 revealed a significant decrease in the expression of the pro-proliferative E2F member E2F2.Quantitative RT-PCR data were in keeping with the microarray analysis,and showed a significant increase and decrease in E2F8 and E2F2 mRNA levels,respectively.In contrast,BZB treatment of the hepatocyte-like HCC cell line HepG2 had a significant impact on mRNA levels of 5 of the 8 E2F members.In particular,mRNA levels of the pro-proliferative E2F members E2F1,E2F2,and of the anti-proliferative member E2F8,decreased over 80%.Notably,a reduction in E2F8 expression in HepG2 and JHH6 cells following siRNA treatment had no impact on cell proliferation.As observed with JHH6,BZB treatment of HepG2cells induced a significant increase in mRNA levels of an anti-proliferative E2F member,E2F6 in this case.As was observed with E2F’s,more dramatic changes in mRNA levels of the E2F related genes cyclins and Cdks and EMT genes were observed after BZB treatment of HepG2 compared to JHH6.CONCLUSION:The differential expression of E2Fs and related genes induced by BZB in diverse HCC cell phenotypes contribute to bortezomib’s mechanism of action in hepatocellular carcinoma.展开更多
基金Grants of National Natural Science Foundation of China (30371206, 30028019)973 National Key Basic Research and Development Program (2002 CB 512905)
文摘Objective To investigate the roles of the cyclin D1/CDK4 and E2F-1/4 pathways and compare their work patterns in cell cycle changes induced by different doses of B[a]E Methods Human embryo lung fibroblasts （HELFs） were treated with 2 μmol/L or 100 μmol/L B[a]P which were provided with some characteristics of transformed cells （T-HELFs）. Cyclin D l, CDK4 and E2F-1/4 expressions were determined by Western blotting. Flow cytometry was used to detect the distribution of cell cycle. Results After B[a]P treatment, the proportion of the first gap （G 1） phase cells decreased. CDK4 and E2F-4 expression did not change significantly. In 2 μmol/L treated cells, a marked overexpression of cyclin D1 and E2F-1 was observed. However, in T-HELFs overexpression was limited to cyclin D1 only, and no overexpression of E2F-1 was observed. The decreases of G1 phase in response to B[a]P treatment were blocked in antisense cyclin D1 and antisense CDK4 transfected HELFs （A-D1 and A-K4） and T-HELFs （T-A-D1 and T-A-K4）. After 2 μmol/L B[a]P treatment, overexpression of E2F-1 was attenuated in A-D1, and E2F-4 expression was decreased significantly in A-K4. In T-A-D1 and T-A-K4, E2F-4 expression was increased significantly, compared with T-HELFs. The E2F-1 expression remained unchanged in T-A-D1 and T-A-K4. Condusions Cyclin DI/CDK4-E2F-1/4 pathways work in different patterns in response to low dose and high dose B[a]P treatment. In HELFs treated with 2 μmol/L B[a]P, cyclin D1 positively regulates the E2F-1 expression while CDK4 negatively regulates the E2F-4 expression; however, in HELFs treated with 100 μmol/L B[a]P, both cyclin D1 and CDK4 negatively regulate the E2F-4 expression.
基金This work was supported by grants of National Natural Science Foundation of China (30371206, 30440420593), 973 National Key Basic Research and Development Program (2002 CB 512905) and Taishan Charitable Association LTD. HK.
文摘Objective To study the molecular mechanism of the inhibitory effects of vitamin C on benzo[a]pyrene （B[a]P）-induced changes of cell cycle in human embryo lung fibroblast （HELF） cells. Methods The stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established. Cells were cultured and pretreated with vitamin C before stimulation with B[a]P for 24 h. The expression levels of cyclin DI, CDK4, E2FI, and E2F4 were determined by Western blot. Flow cytometric analysis was employed to detect the distributions of cell cycle. Results B[a]P significantly elevated the expression levels of cyclin D 1, E2F1, and E2F4 in HELF cells. Vitamin C decreased the expression levels of cyclin D 1, E2F1, and E2F4 in B [a]P-stimulated HELF cells. Dose-dependent relationships were not found between the different concentrations of vitamin C （10, 100, 500, 1000, and 5000 lamol/L） and the expression levels of cyclin D 1, E2F1, and E2F4 in HELF cells. The expression levels of cyclin D1, E2FI, and E2F4 in B[a]P-treated transfectants were lower than those in B[a]P-treated HELF cells. The expression levels of cyclin DI and E2F4 treated with vitamin C and antisense cyclin D1 were decreased compared with those treated with antisense cyclin DI alone. The effects of vitamin C combined with antisense CDK4 on the expression levels of cyclin DI and E2FI/E2F4 were similar to those of antisense CDK4 alone. B[a]P progressed HELF cells from GI to S phase. Both vitamin C and antisense cyclin DI suppressed the changes of cell cycle progressed by B[a]P. However, antisense CDK4 did not attenuate the above changes. Vitamin C combined with antisense CDK4 markedly suppressed B[a]P-induced changes of cell cycle as compared with antisense CDK4. But the inhibitory effects of vitamin C combined with antisense cyclin DI on B[a]P-induced changes of cell cycle were similar to those of vitamin C alone or antisense cyclin DI alone. Conclusions B[a]P progressed HELF cells from G1 to S phase via intracellular signaling pathway of cyclin D I/E2F. Vitamin C may modulate this signaling pathway to protect cells from injury caused by B[a]P.
基金Supported by The "Fondazione Cassa di Risparmio of Triestethe "Fondazione Benefica Kathleen Foreman Casali of Trieste"the Italian Minister of Instruction,University and Research(MIUR),PRIN 2010-11,No.20109PLMH2(in part)
文摘AIM:To evaluate the effects of the proteasome inhibitor bortezomib(BZB)on E2Fs and related genes in hepatocellular carcinoma(HCC)cells.METHODS:The mRNA levels of the E2F family members(pro-proliferative:E2F1-3 and anti-proliferative:E2F4-8)and of their related genes cyclins and cyclindependent kinases(cdks)were evaluated in two HCC cell lines following a single BZB administration.mRNA levels of the epithelial-mesenchymal transition(EMT)genes were also measured in both cell lines after BZB treatment.The BZB concentration(40 nmol/L)used was chosen to stay well below the maximal amount/cm2recommended for in vivo application,and 2 d incubation was chosen as this time point has been found optimal to detect BZB effects in our previous studies.The HCC cell lines,HepG2 and JHH6,were chosen as they display different phenotypes,hepatocyte-like for HepG2and undifferentiated for JHH6,thus representing an in vitro model of low and high aggressive forms of HCC,respectively.The mRNA levels of the target genes were measured by two-color microarray-based gene expression analysis,performed according to Agilent Technologies protocol and using an Agilent Scan B.For the E2F family members,mRNA levels were quantified by realtime reverse transcription polymerase chain reaction(RT-PCR).Using small interfering RNA’s,the effects of E2F8 depletion on cell number was also evaluated.RESULTS:After BZB treatment,microarray analysis of the undifferentiated JHH6 revealed a significant decrease in the expression of the pro-proliferative E2F member E2F2.Quantitative RT-PCR data were in keeping with the microarray analysis,and showed a significant increase and decrease in E2F8 and E2F2 mRNA levels,respectively.In contrast,BZB treatment of the hepatocyte-like HCC cell line HepG2 had a significant impact on mRNA levels of 5 of the 8 E2F members.In particular,mRNA levels of the pro-proliferative E2F members E2F1,E2F2,and of the anti-proliferative member E2F8,decreased over 80%.Notably,a reduction in E2F8 expression in HepG2 and JHH6 cells following siRNA treatment had no impact on cell proliferation.As observed with JHH6,BZB treatment of HepG2cells induced a significant increase in mRNA levels of an anti-proliferative E2F member,E2F6 in this case.As was observed with E2F’s,more dramatic changes in mRNA levels of the E2F related genes cyclins and Cdks and EMT genes were observed after BZB treatment of HepG2 compared to JHH6.CONCLUSION:The differential expression of E2Fs and related genes induced by BZB in diverse HCC cell phenotypes contribute to bortezomib’s mechanism of action in hepatocellular carcinoma.
