Objective To investigate the effect of the 8-bromum-cyclic adenosine monophosphate (8-Br-cAMP) on the telomerase activity and changes of cell cycle in retinoblastoma （RB） cells. Methods The cultured RB cells were divided into the experimental group (8-Br-cAMP) and control group. After cultured for 24, 48 and 72 hours in vitro, the telomerase activity of RB cells was detected by polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) and the changes of cell cycle were detected by flow-cytometry. Results The difference of telomerase activity was significant between the experimental groups and control group (Plt;0.01). There was a negative correlation between the A value of absorbance and the time in the experimental groups (r=-0.778 9, F=33.936, Plt;0.01). The changes of the cell cycle were that the percentages increased in G1 phase and decreased in S phases. Conclusion 8-Br-cAMP may weaken telomerase activity, affect the cell cycle, and inhibit the proliferation of RB cells. (Chin J Ocul Fundus Dis，2004，20：358-360)
Objective To investigate inhibited effects of melatonin (MLT) on proliferative activity of retinoblastoma cell line HXORB44 and its related mechanism. Methods HXO-RB44 cells were treated by MLT of different concentration (10-10, 10-9, 10-8, 10-7 mmol/L. Cell counting and tetrazolium dyereduction assay (MTT) were used to determine the effect of MLT on the survival and proliferation of HXO-RB44 cells. Apoptotic nuclei were further analyzed by HoechstPI fluorescence staining. Flow cytometry was used to measure the fluorescent intensity of ROS, cell cycle distribution and apoptosis. Results 10 -6 mmol/L (or exceed) of MLT could inhibit the proliferation of HXO-RB44cells in vitro while 10-7 mmol/L (or below) of MLT couldn't. With the increase of MLT concentration from 10-10 mmol/L to 10-7 mmol/L, HXO-RB44 cells gradually increased the expression of ROS. Hoechst staining showed that 4, 8, 12 and 24 hours after the incubation with MLT, the nuclear pyknosis and nuclear fragmentation increased in HXORB44 cells. The extent of apoptosis was proportional to the concentrations of MLT. Flow cytometry revealed that with the increasing of MLT concentration, G0/G1 and G2/M phase cells increased, S phase cells decreased. The apoptotic rate was also increased. Conclusion 10 -6 M of MLT could inhibit the proliferation of HXO-RB44 cells. This effect may relate to the increased ROS expression, cell cycle arrest at G0/G1 phase and apoptosis of HXO-RB44 cells.
ObjectiveTo investigate the effects of dipyridamole (DP), one of the human equilibrative nucleoside transporters (hENTs) blockers, on 5-fluorouracil (5-FU) induced cell apoptosis and cell cycle changes of the pancreatic cancer Panc-1 cell line. MethodsPancreatic cancer cell line Panc-1 was divided into hENTs blocked group and hENTs unblocked group. The hENTs blocked group was further divided into two subgroups according to the DP concentration, 5 μmol/L DP group and 10 μmol/L DP group. Each group was incubated in culture medium with or without 1.5×106 ng/L 5-FU for 24 h. Cell apoptosis and cell cycle were detected by flow cytometry. Results①The apoptosis results of each group： Incubated in culture medium with 1.5×106 ng/L 5-FU for 24 h, the apoptosis rates of 5 μmol/L DP group and 10 μmol/L DP group were higer than those of hENTs unblocked group （Plt;0.05）, and which of 10 μmol/L DP group was higer than that of 5 μmol/L DP group (Plt;0.05). Incubated in culture medium without 5-FU for 24 h, there were no significant differences of the apoptosis rates among three groups (Pgt;0.05). ②The cell cycle results of each group: Incubated in culture medium with 1.5×106 ng/L 5-FU for 24 h, the percentages of S phase cells in the 5 μmol/L DP group and 10 μmol/L DP group were less than those of hENTs unblocked group (Plt;0.05). The percentage of S phase cell of 5 μmol/L DP group reduced to 87.09% and that of 10 μmol/L DP group reduced to 74.06% as compared with hENTs unblocked group. 5-FU had little influence on G2 phase (Pgt;0.05) except for the percentage of G2 phase cells in the 5 μmol/L DP group increased significantly (Plt;0.05) as compared with the hENTs unblocked group. Incubated in culture medium without 5-FU for 24 h, there were no significant differences of the cell cycles among three groups (Pgt;0.05). ConclusionsIn pancreatic cancer Panc-1 cell, DP could enhance the cytotoxicity of 5-FU by blocking hENTs. The enhanced cytotoxicity is related to elevation of 5-FU concentration in cells, and unrelated to DP itself.
ObjectiveTo investigate the effect and mechanism of caveolion-1 on the growth and proliferation of human pancreatic carcinoma cell Panc1, in vitro. MethodsThe plasmid pCI-neo-cav-1 and its corresponding empty vector (pCI-neo) were transfected into Panc1 cell line (study group and control group, respectively). Expressions of caveolin-1, Akt, and Aktphosphate (p-Akt) were determined in transfectants by Western blot analysis. The cell growth curve was drawn and the double time was calculated in each group, and the cell cycle was analyzed by flow cytometry. The colony formation ability of tumor cells was detected by anchorageindependent growth assay. ResultsCaveolin-1 expression was up-regulated (Plt;0.01) and the growth of Panc1 cell was inhibited significantly (Plt;0.01) in the study group comparing with the control group. Caveolin-1 overexpression inhibited proliferation of Panc1 cell by arresting the cell cycle in the G0/G1 phase (Plt;0.05), the rate of S phase in the study group was lower than that of the control group (Plt;0.01). Proliferation index of the study group was also lower than that of the control group (Plt;0.01). Caveolin-1 overexpression reduced the capacity of the cells to form colonies in soft agar (Plt;0.01). p-Akt protein was reduced in the study group as compared with the control group (Plt;0.05). ConclusionCaveolin-1 can function as a cancer suppressor through inhibiting the activation of PI3K/Akt signaling pathway in Panc1 cell.
Objective To know the abnormal expression of the cell cycle-regulated proteins in pancreatic adenocarcinoma and their effect on tumor cell growth. Methods The expression of p16, p21, Rb and p53 protein in 47 cases were investigated by immunohistochemistry with wet autoclave pretreatment for antigen retriaval. Furthermore, tumor growth index were assessed by a novel anti-ki-67 antibody (ki-s5). Results All the expression of p53, p16, p21 and Rb protein were the nuclear stainning. The positive rates of p53, p16, p21 and Rb protein were 55%, 53%, 74% and 98% respectively. There was negative correlation between of p16, p21 or Rb protein expression and ki-67 growth index. No relation of p53 protein stainning and the expression of p21 protein was found. Conclusion In pancreatic adenocarcinoma, the negative expression of p16 protein and p21 protein may play an important role in tumor cell growth, but tumor proliferation caused by abnormality of Rb protein is rare. The expression of p21 protein was not associated with the expression of p53 protein.
Objective To evaluate the role of the cell cycle related genes cyclinD1 and Bcl-2 protein expression in the pathogenesis and infilt ration of the uveal melanoma． Methods Using immunohis tochemistry to detect the cyclinD1 and bcl-2 protein expression in 96 cases of uveal melanoma． Results The expression content of bcl-2 was high in uveal melanoma, and there wasn't any relationship between bcl-2 cell positivity and tumor cell type and extrascleral extension． In contrast, cyclinD1 expression was higher in epithelial cell uveal melanoma than mix cell and spindle cell varieties． There was a positive correlation between cyclinD1 cell positivity and extrascleral extension． Conclusion The expression of bcl-2 protein is important for the survival of the uveal melanoma． CyclinD1 may serve as a sensitive index of its malignancy． (Chin J Ocul Fundus Dis, 2001,17:44-46)
Objective To investigate the effect on proliferation and apoptosis of vascular endothelial cells exposed to high glucose. Methods The cultured human umbilical vein endothelial cells (HUVEC) were treated with high glucose at concentrations of 10,20,30,40,50 mmol/L for 2,4,6,8,10,12,14 days,cpm value was studied by using tritium-labelled thymidine deoxyribose(3H-TdR)incorporation assay.Flow cytometry was used to detect apoptosis index,proliferation index and cell cycle. Results Treated with high glucose,the proliferation index and cpm were significantly reduced in a dose and time dependent manner than the control groups(Plt;0.05).The apoptosis index of HUVEC were higher compared with the control groups(Plt;0.05).The percent of the cultured cells in G1 phase in all high glucose groups were increased,the percent in S phase were largely decreased(Plt;0.05). Conclusion Exposed to high glucose,the apoptosis of HUVEC was accelerated and the suppressive effect of high glucose on the proliferation of HUVEC was observed.Endothelial cells were possibly arrested in G1/S checkpoint. (Chin J Ocul Fundus Dis,2000,16:177-180)
Objective To study the effects of L-arginine （L-Arg） on cell proliferation, inducible nitric oxide synthase (iNOS) expression and cell cycle in human colon carcinoma cell line LS174 through nitric oxide (NO) pathway. Methods LS174 cells were cultured in medium with L-Arg at different concentrations for different times. MTT method was employed to evaluate the level of the cell proliferation. The production of NO in culture supernatants of LS174 cell was detected with enzyme reduction of nitrate. The distribution of the cell cycle was detected with the flow cytometry (FCM). The expression level of iNOS in the cells was determined by Western blot and SP immunocytochemical staining method. Results The growth of LS174 was promoted by the L-Arg at low concentration (0.125 mmol/L) and inhibited at high concentrations (0.5， 2， 8 and 32 mmol/L). The level of NO was increased with the increasing concentration of L-Arg in culture medium. To compare with the control group, the ratio of cells at S phase was increased after 48 hours’ treatments with high concentrations (0.5, 2, 8 and 32 mmol/L) of L-Arg (P＜0.05, P＜0.01); while there was no obvious difference after treatments with low concentration (0.125 mmol/L) of L-Arg (Pgt;0.05). With the increase of the concentration of L-Arg, the expression of iNOS was increased as compared with control group. The higher the concentration of L-Arg was, the better the effect. Conclusion L-Arg can induce the expression of iNOS resulting in increase the production of nitric oxide (NO). Low concentration of L-Arg can promote the growth of LS174 cells, while high concentration ones can inhibit growth and proliferation. The high concentration of L-Arg could induce S phase arrestion in the cell cycle.
【Abstract】ObjectiveTo investigate the proliferation rate of HepG2 cell after multiple thermotherapy and the possible reasons related to it. MethodsAfter HepG2 cell were treaded by ten repeated cycles of heat exposure at 43 ℃ for 80 minutes twice a day, the doubling time of cell was analyzed， and the cell cycle, bcl2 mRNA and bax mRNA were detected. ResultsThe proliferation rate of HepG2 cell which treated with heat speeded up, the percentage of G2 and S in cell cycle increased, the expression of bcl2 mRNA strengthened and the rate of bcl2/bax increased. ConclusionThe speeded proliferation of HepG2 cell after multiple thermotherapy is related to its high percentage of DNA duplicated and dividing cell, strengthened expression of bcl2 mRNA and increased rate of bcl2/bax.
Objective To investigate the effect of exogenous Rb gene on the cell cycle of vitreous retinoblastoma (RB) transplantation tumor in nude mouse. Methods Based on establishing vitreous RB transplantation tumor in nude mouse,constructing retrovirus vector of Rb gene PBabe-Rb and transfecing it into the RB transplantation model by liposome Dosper,the change of cell cycle of the RB transplantation tumor by flow cytometry(FCM)was analysed． Results FCM showed that the cells of G1phase of the treated eyes were obviously more than the control eyes with the value of DNA index(DI)and S phase fraction(SPF) decreased by the Rb gene expression. Conclusion The exogenous PBabe-Rb gene can partially suppress the progress of the cell cycle of RB transplantation tumor in vivo. (Chin J Ocul Fundus Dis，2000，16：1-70)