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Objective To establish a CLCN3/MMTV-PyMT double transgenic mouse model of spontaneous breast tumor with simultaneously overexpressing ClC-3.Method CLCN3 transgenic mice were crossed with MMTV-PyMT spon-taneous mammary tumor model mice.The genotype was determined by PCR.The expression of ClC-3 in tissues was detec-ted by immunofluorescence and Western blot.Results CLCN3 and MMTV-PyMT transgenic mice were bred and CLCN3/MMTV-PyMT hybrid mouse model was successfully established.The ClC-3 expression in CLCN3/MMTV-PyMT hybrid mice was higher than that in the MMTV-PyMT mice, assessed by immunofluorescence and Western blot analysis.Conclu-sions Transgenic mouse models of spontaneous breast cancer with simultaneously overexpressing ClC-3 are successfully es-tablished.The double transgenic mice provide a good animal model for further research of ClC-3 in tumor growth and metas-tasis.
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AIM:To investigate the type of chloride channel activated by cisplatin in poorly differentiated na -sopharyngeal carcinoma cells (CNE-2Z cells).METHODS:The technique of whole-cell patch-clamp was used to investi-gate the role of Ca 2+in the activation of cisplatin-activated chloride currents and to analyze the effect of hypertonic stress on these currents in CNE-2Z cells.RESULTS:Chloride currents were induced when the cells were exposed to the calcium -free cisplatin solution , showing the similar density to the currents induced by cisplatin with the presence of extracellular cal -cium.However , the latency and the peak time of cisplatin-activated currents in the absence of extracellular calcium were prolonged.The activation of cisplatin-activated chloride currents was insensitive to the depletion of intra-and extracellular calcium.Calcium channel antagonist nifedipine had no effect on the cisplatin -activated chloride currents , while hypertonic solution completely inhibited those currents .CONCLUSION:The cisplatin-activated chloride currents are independent on intra/extracellular calcium .The chloride channels activated by cisplatin are not calcium-activated chloride channels , but are probably volume-sensitive chloride channels .
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This paper summarized the assessment model reform practiced in the postgraduate cell biology course for several years and successfully obtain a new testing method based on research papers re-porting and questioning. Before the assessment, each student searched and selected one required research paper to read and make a PowerPoint reporting document. During assessment , some students were ex-tracted to speak and comprehensive score, which was regarded as student's final examination results was made by teaching group. The rest of the students listened to speeches , questioned and filled the questions on the record sheet.Teaching group reported a score , which was regarded as part of student's final exami-nation result based on questioning record sheets and printed PowerPoint documents.This method can well evaluate students' scientific thinking and strengthen the training of scientific thinking of postgraduates.
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Background and purpose: Checkpoint kinase 1 and 2 have been proposed to be potential therapeutic targets to sensitize cancers to radio- or chemo-therapeutics. However, little is known about whether Chk1/2 is also a suitable target for sensitizing cancers to curcumin. In the present study, we investigated effects of Chk1/2 siRNA on curcumin-induced apopotosis in hepatoma cell line Huh7 and evaluated the effectiveness of Chkl/2as a therapeutic target to potentiate human hepatoma to curcumin. Methods: Effect of curcumin on the cell cycle checkpoint-associated proteins was detected by Westem blot. The knockdown efficacy of Chk1/2 siRNA was measured by RT-PCR and Westem blot. Effect of Chk1/2 siRNA on curcumin-induced apoptosis in Huh7 cells was evaluated by DAPI staining. Effect of Chk1/2 siRNA on cell cycle distribution in curcumin-treated Huh7 cells was analyzed by flow cytometry. Results: Curcumin significantly inhibited phosphorylation of cell cycle checkpoint-associtaed proteins Chk1(S317), Cdc25C(S216) and Cdk1(Y15). Chk1 siRNA decreased Chk1 mRNA and protein by 95% and 92% and Chk2 siRNA decreased Chk2 mRNA and protein by 60% and 55% respectively as compared with negative control siRNA (P<0.01). Inhibition of Chk1, but not Chk2, increased apoptotic rate from (21.3±1.8)% to (29.5±2.6)% (P<0.05). Neither Chk1 nor Cbk2 siRNA had any impact on cell cycle distribution in Huh7 cells induced by curcumin. Conclusion: Chk1 siRNA sensitized Huh7 cells to curcumin-induced apoptosis, suggesting that Chk1 is a potential therapeutic target to sensitize human hepatoma to curcumin.
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Objective: To investigate the effect of amiloride on in vitro invasive activity and uPA (urokinase-type plasminogen activator)system of human highly metastatic lung carcinoma cell line PGCL3. Methods: At 6 hours after treatment with amiloride at the concentrations of 25μmol/L，50μmol/L and 100μmol/L for PGCL3 cells，Transwell Chamber assay was performed to detect the effect of amiloride on the invasive and migratory capacity of PGCL3 cells.Effect of amiloride on the activity of uPA and PAI-1(plasminogen activator inhibitor-1)secreted by PGCL3 cells were measured by chromogenic substrate assay after PGCL3 cells were incubated with amiloride for 24 hours.RT-PCR was used to analyze the effect of amilorede on mRNA levels of uPA，uPAR(urokinase-type plasminogen activator receptor)and PAI-1.The expression levels of uPA，ERK2(extracellular regulated protein kinases 2)and ras protein were assessed by Western blot. Results: The number of cells through membrane was significantly decreased in invasion and migration test in vitro.The inhibitory rates of invasion and migration after treatment with amiloride of 100μmol/L were 37.7%±4.1%and 64.9%±4.9%.respectively,with a significant difference from those in the control group(P<0.01).At 24 hours after amiloride treatment，the chromogenic substrate assay showed direct inhibition of the activity of uPA and PAI-1 secreted by PGCL3 cells.No effect on the expression of uPAR in mRNA level was observed,but the expression of PAI-1 in mRNA level was significantly inhibited.Amiloride of 100μmol/L dramatically inhibited the expression of uPA mRNA.The expression level of uPA protein was decreased with the increase of the concentration of amiloride,but no effect was observed on the expression of ERK2 and ras in protein level.Conclusion: Amiloride can inhibit the invasion and migration of PGCL3 cells,through inhibiting the expression and activity of uPA and PAI-1.Amiloride is a potential agent to inhibit cancer invasion and metastasis.
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Objective:To investigate the effect of dimethyl amiloride (DMA) on invasive activity of PGCL3 cells from a human highly-metastatic lung carcinoma cell line in vitro and elucidate its possible mechanism. Methods:The invasion and migration capacities of PGCL3 cells were measured by using Transwell chamber assay after pretreatment with DMA. The effects of DMA on the activity of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) secreted by PGCL3 cells were measured by chromogenic substrate assay. The effects of DMA on uPA, urokinase-type plasminogen activator receptor (uPAR), and PAI-1 mRNAs transcription were determined by RT-PCR. The expression levels of extracellular regulated protein kinases 2 (ERK2) and ras protein were assessed by Western blot. Results:DMA inhibited invasion and migration capabilities of PGCL3 cells in vitro, down-regulated the mRNA transcription of uPA, uPAR and PAI-1, as well as up-regulated the expression of ras protein. After 24-hour treatment, DMA reduced the activity of uPA at higher concentration, but DMA had no effects on the activity of secreted PAI-1 protein and expression of ERK2 protein. Conclusion:DMA inhibits the invasion and migration of highly-metastatic lung cancer PGCL3 cells. The mechanism might be associated with down-regulation of the expression of uPA system.
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Background and purpose:Checkpoint kinase 1 and 2 have been proposed to be potential therapeutic targets to sensitize cancers to radioor chemo-therapeutics. However, little is known about whether Chk1/2 is also a suitable target for sensitizing cancers to curcumin. In the present study, we investigated effects of Chk1/2 siRNA on curcumin-induced apopotosis in hepatoma cell line Huh7 and evaluated the effectiveness of Chk1/2 as a therapeutic target to potentiate human hepatoma to curcumin. Methods:Effect of curcumin on the cell cycle checkpoint-associated proteins was detected by Western blot. The knockdown efficacy of Chk1/2 siRNA was measured by RT-PCR and Western blot. Effect of Chk1/2 siRNA on curcumin-induced apoptosis in Huh7 cells was evaluated by DAPI staining. Effect of Chk1/2 siRNA on cell cycle distribution in curcumin-treated Huh7 cells was analyzed by flow cytometry. Results:Curcumin significantly inhibited phosphorylation of cell cycle checkpoint-associtaed proteins Chk1(S317), Cdc25C(S216) and Cdk1(Y15). Chk1 siRNA decreased Chk1 mRNA and protein by 95% and 92% and Chk2 siRNA decreased Chk2 mRNA and protein by 60% and 55% respectively as compared with negative control siRNA (P
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S. The migration of CNE-2Z cells was inhibited by chloride channel blockers (ATP, NPPB and tamoxifen), but the inhibitory effect of the blockers varied with cells at different stages. CONCLUSIONS: The migratory ability is associated with the cell cycle in CNE-2Z cells. Chloride channels play an important role in cell migration of CNE-2Z cells.
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AIM: The roles of Cl-channels in regulatory volume decrease (RVD), cell proliferation and cell cycle progression in nasopharyngeal carcinoma cells (CNE-2Z) were investigated. METHODS: Image analysis of living cells was used to detect the volume changes following exposure to hypotonic solutions. Cell viability was determined by the trypan blue assay. MTT method was applied to detected cell proliferation. The effect of the blocker on the cell cycle distribution was monitored by the flow cytometry. RESULTS: 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) inhibited RVD and cell proliferation in a dose-dependent manner. NPPB at the concentration of 100 ?mol/L arrested cells in G 1 phase (G 1 population increased from 54% to 71% at 48 h after treatments), but did not significantly alter cell viability. CONCLUSION: Block of chloride channels suppressed cell proliferation by arresting cells in G 1 phase. The results suggest that activation of Cl-channels and RVD is necessary for facilitating cells to proceed to the S phase from G 1 phase and maintaining cell proliferation.
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gluconate in both G1 phase and S phase cells.The permeability of G1 phase cells to I-was higher than that in S phase cells,but to gluconate was lower than that in S phase cells.CONCLUSIONS: The density of the volume-activated Cl-current,the anion permeability of the channel and the sensitivity of the current to tamoxifen were different between the CNE-2Z cells in G1 phase and those in S phase.The results suggest that the expression of tamoxifen-sensitive,volume-activated chloride channels is differentiated at different stages of the cell cycle.