Remarkably reduced expression of FoxO3a in metaplastic colorectum, primary colorectal cancer and liver metastasis.

The forkhead family members of transcription factors (FoxOs) are expected to be potential cancer-related drug targets and thus are being extremely studied recently. In the present study, FoxO3a, one major member of this family, was identified to be down-regulated in colorectal cancer through micro-array analysis, which was confirmed by RT-PCR and Western blot in 28 patients. Moreover, immunohistochemistry (IHC) showed that the expression levels of FoxO3a were remarkably reduced in 99 cases of primary colorectal cancer, liver metastasis, and even in metaplastic colorectal tissue. IHC also revealed an exclusion of FoxO3a from the nucleus of most cells of tumor-associated tissues. Silencing FoxO3a by siRNA led to elevation of G2-M phase cells. We conclude that the downregulation of FoxO3a may greatly contribute to tumor development, and thus FoxO3a may represent a novel therapeutic target in colorectal cancer.

[Overexpression of hSav1 promotes Mst1-induced apoptosis in HeLa cells].

OBJECTIVE: To elucidate the effect of hSav1 expression on Mst1-mediated apoptosis in HeLa cells. METHODS: Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 micromol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. RESULTS: Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSav1 could be detect from the anti-Mst1 immunoprecipitation complex. The immunofluorescent labeling showed that hSav1 and Mst1 had the same localization in cells. Overexpressed protein hSav1 did not induce a significant cell apoptosis. However, co-expression of hSav1 with Mst1 resulted in a significant increase of apoptosis above the level seen with Mst1 alone (24.5% +/- 2.4% vs. 39.3% +/- 4.0%, P < 0.05). CONCLUSION: Our findings indicate that hSav1 is a newly identified protein that interacts with Mst1 and augments Mst1-mediated apoptosis.

[Apoptosis of sperm induced by murine cytomegalovirus infection and its mechanism regulated by sperm mitochondria: experiment with mice].

OBJECTIVE: To study the effects of murine cytomegalovirus (MCMV) infection on mature sperm apoptosis in mice, and to explore the mechanism of MCMV-induced apoptosis regulated by mitochondria. METHODS: MCMV was inoculated into the testes of 15 BALB/c mice to establish acute MCMV infection models and 15 mice were used as controls. 1, 2, 4, 6, and 9 days after the infection 3 mice from each group were killed. Flow cytometry was used to observe the apoptosis of sperms. Laser scanning confocal microscopy was performed to examine the mitochondrial membrane potential (delta psi psi m) of sperm. The mitochondria ultrastructure of sperm was observed under electron microscope. RESULTS: The sperm apoptotic ratebeganto increase from day 2 after inoculation (D2 PI), peaked to the level of (39.3 +/- 1.0)% compared with that of the control group on D4 PI, and then fell-off (F = 362.822, P < 0.05). The delta psi m of sperm began to increase on D1 PI at the level of (74.0 +/- 1.4), began to decrease on D2 PI [( 63.0 +/- 2.2)], dropped to the minimum on D4 PI [(40.2 +/- 2.3)], then ascended slowly again (F = 32.257, P < 0.05). The mitochondria ultrastructure of sperm showed damage after MCMV infection that was especially severe from D2 PI to D6 PI. CONCLUSION: MCMV acute infection in reproductive organ induces apoptosis of mature sperm in the cauda epididymidis. Sperm mitochondria participate in and regulate initiatively the apoptosis of sperm.

[Inhibitory effect of N-terminal 24 amino acids of the p55 gamma, a regulatory subunit of phosphoinositide 3-kinase, on proliferation of colon carcinoma cell line HT29].

BACKGROUND & OBJECTIVE: p55 gamma is one of the regulatory subunits of phosphoinositide 3-kinase (PI3K), which plays an important role in the regulation of PI3K activity. This study was to explore the inhibitory effect of the N-terminal 24 amino acids of the p55 gamma on proliferation of colon carcinoma cell line HT29. METHODS: Ad-N24p55-GFP, containing N-terminal 24 amino acids of the p55 gamma, and control vector Ad-GFP were constructed, and used to infect HT29 cells. The cell cycle progression was detected using flow cytometry. DNA synthesis was analyzed using the BrdU/PI method. The nude mice model bearing HT29 xenograft tumors was used to study the effect of Ad-N24p55-GFP against the tumor. RESULTS: Compared with cells infected with Ad-GFP, cells infected with Ad-N24p55-GFP at the G0/G1 phase were increased from 65.11% to 73.39% P < 0.05, and cells at S and G2/M phases were decreased from 17.37% to 15.08% and from 17.51% to 11.13%, respectively; BrdU-positive cells were decreased from 24.82% to 9.27%. In the nude mice model, the tumor growth was inhibited after the administration of Ad-N24p55-GFP. The tumor weight was (0.32+/-0.08)g vs. (10.67+/-0.3)g and (0.72+/-0.28)g in the Ad-N24P55-GFP group,the blank control group and the Ad-GFP group, respectively. CONCLUSION: In HT29 cells, overexpression of the N-terminal 24 amino acids of the p55 gamma can induce cell cycle arrest, inhibit DNA synthesis and inhibit tumor growth in the nude mice model of HT29 xenograft tumors.

[Effect of starvation-induced autophagy on cell cycle of tumor cells].

BACKGROUND & OBJECTIVE: No serum starvation could induce autophagy and cell cycle arrest. Although autophagy and cell cycle have been widely explored, little is known about their relationship. This study was to observe the change of Cyclin expression during starvation-induced autophagy to discuss the effect of autophagy on cell cycle. METHODS: In control group, HeLa cells were treated with d-Hanks solution (a medium with no serum). In experiment group, HeLa cells were treated with d-Hanks solution containing 3-methyladenine (3-MA, a specific inhibitor of autophagy). Cells were harvested after being starved for 0, 3, 6 and 12 h. Flow cytometry (FCM) and Weston blot were used to detect Cyclin and microtubule-associated protein 1 light chain 3(LC-3) which marked autophagy specifically. RESULTS: In control group, the expression of LC-3 protein was detected early after being starved for 3 h, and gradually increased along with starvation; the expression of Cyclin D3 and Cyclin E was decreased evidently after a short-time starvation (3 h) and descended to the minimum when cells were being starved for 6 h; the expression of Cyclin A and Cyclin B1 were apparently decreased after being starved for 6 h. In experiment group, LC-3 protein could not be detected during starvation when cells were exposed to 3-MA and the down-regulation of Cyclins was suppressed. CONCLUSIONS: Autophagy is involved in starvation-induced hydrolysis of Cyclins. The hydrolysis of Cyclin D3 and Cyclin E is quicker than that of Cyclin A and Cyclin B1.

[Preparation of suspension of gastric mucous membrane single cell and expression of cyclins in cells].

OBJECTIVE: To explore a method which can remove the gastric mucus in order to prepare mucous membrane single cell suspension for the research of cytomics. METHODS: Enzymology was used to remove the mucus gel and to separate mucous layer from the normal fresh gastric tissue. The mucous layer was broken to prepare single cell suspension with machine method. Expression of major cyclins in mucous layer cells was examined by cytoimmunochemistry, flow cytometry(FCM) and confocal microscopy. RESULTS: The 0.1% pepsin could dissolve the mucus gel and 1.2-2.4 U/L dispase could separate the mucous layer completely. The single mucous cell suspension was prepared successfully. FCM results from mucous single cell suspension revealed that expression of cyclin D(3), B(1) was obvious, that of cyclin D(2) was weak and that of cyclin D(1), A, E was the least. Similar results were found with confocal microscopy. CONCLUSIONS: Single cell suspension from mucous layer can be easily prepared by pepsin and dispase. Cyclins schedule expression in vivo is different from cyclins schedule expression in vitro.

[Effects of mitofusin-2 gene on proliferation and chemosensitivity of human breast carcinoma cell line MCF-7].

BACKGROUND & OBJECTIVE: Mitofusin-2(mfn2), a proliferation-inhibiting gene, targets to the outer membrane of mitochondria. Its overexpression suppresses the proliferation of vascular smooth muscle cells. This study was to explore the effects of mfn2 gene on the proliferation and chemosensitivity of human breast carcinoma cell line MCF-7. METHODS: Plasmid pEGFP-mfn2 containing mfn2 cDNA was constructed and transfected into MCF-7 cells by sofast. The expression of green fluorescent protein (GFP) in MCF-7 cells was detected by Western blot. Cell proliferation was measured by MTT assay and cell counting. Cell cycle and chemosensitivity of MCF-7 cells to camptothecin (CAM) was observed by flow cytometry (FCM). RESULTS: After transfection of pEGFP-mfn2, the stable expression of GFP protein was detected in MCF-7 cells, and cell cycle was arrested: the S phase proportion was significantly higher in pEGFP-mfn2-transfected cells than in pEGFP-transfected and untransfected cells [(42.7+/-1.3)% vs. (17.2+/-2.0)% and (19.6+/-1.7)%, P<0.05]. The apoptosis rate were significantly higher in pEGFP-mfn2-transfected cells than in pEGFP-transfected and untransfected cells [(16.0+/-0.3)% vs. (4.5+/-0.9)% and (3.6+/-0.6)% before treatment of CAM, P<0.05; (69.6+/-4.3)% vs. (31.0+/-1.8)% and (23.4+/-2.8)% after 4-hour treatment of CAM, P<0.05]. CONCLUSION: mfn2 gene can inhibit the proliferation of MCF-7 cells and increase their chemosensitivity to CAM.

[Correlations of GRIM-19 and its target gene product STAT3 to malignancy of human colorectal carcinoma].

BACKGROUND & OBJECTIVE: GRIM-19 (gene associated with retinoid-interferon-induced mortality-19) gene is a specific protein to inhibit signal transducers and activators of transcription 3 (STAT3). STAT3 and its pathway are involved in modulating cell proliferation, apoptosis, differentiation, and mediating malignant transformation of cells. This study was to investigate the expression of GRIM-19 and its target gene STAT3 in human colorectal carcinoma tissues, and explore their roles in the tumorigenesis of colorectal carcinoma. METHODS: The expression of GRIM-19, STAT3 and its activated form p-STAT3 in 40 specimens of colorectal carcinoma, adjacent tissue, and normal tissue was determined by immunohistochemistry and Western blot. The correlations of the expression of GRIM-19, STAT3, and p-STAT3 to various clinicopathologic characteristics of colorectal carcinoma were analyzed statistically. The mRNA expression and gene mutation of GRIM-19 in colon cancer cell line SW480 and 23 specimens of colorectal carcinoma, adjacent tissue, and normal tissue were detected by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. RESULTS: The expression of both STAT3 and p-STAT3 were up-regulated in colorectal carcinoma. The mRNA and protein expression of GRIM-19 was obviously lower in colorectal carcinoma than in normal tissues. The expression of GRIM-19 was correlated to clinical stage and cell differentiation of colorectal cancer (P< 0.05). GRIM-19 expression in colorectal cancer was negatively correlated to STAT3 and p-STAT3 expression (Chi2 = 9.95, P = 0.00; Chi2 = 5.10, P = 0.02). No mutation of GRIM-19 gene was detected in colorectal carcinoma tissues. CONCLUSIONS: The low expression or absence of GRIM-19 may play an important role in the tumorigenesis of colorectal carcinoma. The high expression of STAT3 and the low expression of GRIM-19 co-exist in colorectal carcinoma, and may be related to malignant transformation and abnormal proliferation of cells.

Cytochrome P-450 epoxygenases protect endothelial cells from apoptosis induced by tumor necrosis factor-alpha via MAPK and PI3K/Akt signaling pathways.

Endothelial cells play a vital role in the maintenance of cardiovascular homeostasis. Epoxyeicosatrienoic acids (EETs), cytochrome P-450 (CYP) epoxygenase metabolites of arachidonic acid in endothelial cells, possess potent and diverse biological effects within the vasculature. We evaluated the effects of overexpression of CYP epoxygenases on tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in bovine aortic endothelial cells. CYP epoxygenase overexpression significantly increased endothelial cell viability and inhibited TNF-alpha induction of endothelial cell apoptosis as evaluated by morphological analysis of nuclear condensation, DNA laddering, and fluorescent-activated cell sorting (FACS) analysis. CYP epoxygenase overexpression also significantly inhibited caspase-3 activity and downregulation of Bcl-2 expression induced by TNF-alpha. The antiapoptotic effects of CYP epoxygenase overexpression were significantly attenuated by inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK signaling pathways; however, inhibition of endothelial nitric oxide synthase activity had no effect. Furthermore, CYP epoxygenase overexpression significantly attenuated the extent of TNF-alpha-induced ERK1/2 dephosphorylation in a time-dependent manner and significantly increased PI3K expression and Akt phosphorylation in both the presence and absence of TNF-alpha. Collectively, these results suggest that CYP epoxygenase overexpression, which is known to increase EET biosynthesis, significantly protects endothelial cells from apoptosis induced by TNF-alpha. This effect is mediated, at least in part, through inhibition of ERK dephosphorylation and activation of PI3K/Akt signaling.

[DNA damage and apoptosis of human airway epithelial cell lines caused by cigarette smoke extract].

BACKGROUND & OBJECTIVE: Cigarette smoke exposure has been reported to induce DNA damage in a variety of cell types. This study was to investigate DNA damage and apoptosis in normal human bronchial epithelial cell line NHBE and lung carcinoma cell line SPC-A1 caused by cigarette smoke extract. METHODS: NHBE and SPC-A1 cells were incubated with different concentrations of cigarette smoke extract. Cell viability was evaluated by MTT assay. Fluorescence-labeled anti-histone gamma-H2AX polyclonal antibody was used to detect DNA double-strand breaks (DSBS) in chromatin. DNA damage was analyzed by flow cytometry. The expression of gamma-H2AX was detected by Western blot. Cigarette smoke extract-induced cell apoptosis was detected by sub G1 peak method and annexin V-FITC/propidium iodide (PI) staining assay. Cell morphology of DNA damage and apoptosis was observed by confocal laser microscopy. RESULTS: MTT assay results showed that cigarette smoke extract decreased the viability of NHBE and SPC-A1 cells in time-and concentration-dependent manners. Cigarette smoke extract induced DSBS in NHBE cells and SPC-A1 cells, and led to H2AX phosphorylation (denoted gamma-H2AX) in time-and concentration-dependent manners. The maximal value of gamma-H2AX was seen about 4 h after the treatment, and the value decreased subsequently, but did not reduce to a normal level. Cell apoptosis appeared about 12 h after DNA damage. Cigarette smoke extract also initiated the accumulation of gamma-H2AX in these cells, and cell apoptosis morphology was observed 4 h later. CONCLUSION: Cigarette smoke extract can induce DSBS and apoptosis in NHBE and SPC-A1 cells in time-and concentration-dependent manners.

[Paclitaxel induces apoptosis of acute leukemia cells in S phase].

BACKGROUND & OBJECTIVE: Cell cycle specificity, an important feature of anticancer drugs, is commonly used in the design of combined chemotherapy. Paclitaxel is widely accepted as an G2/M phase-specific agent. However, cumulative evidences revealed that the cell cycle specificity of anticancer drugs in vitro is not always consistent as in vivo. This study was to observe the effect of paclitaxel on the cell cycle specificity in different cell models. METHODS: Effects of paclitaxel on cell apoptosis of human lymphocyte leukemia cell line Molt-4 and 17 clinical specimens of acute leukemia were investigated using flow cytometry. RESULTS: Paclitaxel induced G2/M phase-specific apoptosis in exponentially growing Molt-4 cells, G0/G1 phase-specific apoptosis in high-density cultured Molt-4 cells, and S phase-specific apoptosis in acute leukemia specimens. CONCLUSIONS: Paclitaxel exhibits different cell-cycle specificity in different models. Different from other studies, paclitaxel induces S phase-specific apoptosis in clinical leukemia specimens. This difference is most likely related to the growing status of the target cells.

[Inducement of tumor cell autophagy and cell cycle analysis].

BACKGROUND & OBJECTIVE: Autophagy is the main phenomenon of type II programmed cell death which is also named as autophagic cell death, and autophagy has a close relationship with autophagic cell death. The relationship of apoptosis and cell cycle has been explored deeply, but little is known about the relationship of autophagic cell death and cell cycle. This study was to observe the correlation of autophagy induced by different methods to cell cycle. METHODS: Exponentially growing HeLa and SW480 cells, and peripheral blood lymphocytes (PBLs) from healthy donors, with or without 48 h stimulation of phytohemagglutinin (PHA), were treated with Hanks' solution (to produce starvation) or vincristine. Confocal laser microscope and transmission electron microscope (TEM) were used to detect autophagy; flow cytometry (FCM) was innovatively used to detect the cell cycle of autophagic cells with dipl-parameters of microtubule-associated protein 1 light chain 3 (MAP1-LC3-II)/PI. RESULTS: Autophagy of HeLa and SW480 cells induced by starvation or vincristine was observed in G1, S, and G2/M phases and increased along with the inducement time; no autophagy was observed in unstimulated PBLs. The positive rate of LC3-II, indicating the occurrence of autophagy, was lower than 2.62% when induced by starvation in Hanks' solution for 48 h, or 6.16% when induced by vincristine for 48 h. After PBLs were stimulated into cell cycle by PHA, autophagy was markedly detected 2 h after the indicated inducements. CONCLUSIONS: MAP1-LC3-II/DNA dipl-parameter analysis by FCM is a convenient and reliable method for simultaneously analyzing autophagy and cell cycle. Autophagy could be induced when cells are in cell cycle, while the cells in G0 phase are insensitive to the inducers.

[Effect of caffeine on camptothecin-induced apoptosis of Molt-4 cells].

BACKGROUND & OBJECTIVE: Caffeine could act on cell cycle checkpoints and affect the progression of cell cycle, but its impact on apoptosis of tumor cells is in debate. This study was carried out to investigate the effects of caffeine on camptothecin-induced apoptosis and cell cycle checkpoints of leukemia cell line Molt-4. METHODS: The cell apoptosis was induced by camptothecin, and caffeine was used to interfere with cell cycle checkpoints. The apoptosis rate and cell cycle during apoptosis were analyzed using sub-G1 method and Annexin V-propidium iodide (Annexin V/PI) staining. RESULTS: Caffeine (2.0-20.0 mmon/L) had no effect on proliferation of Molt-4 cells in exponentially growth phase. Camptothecin selectively induced apoptosis of Molt-4 cells in S phase; when induced with camptothecin (0.15 micromol/L) for 4 or 6 h, the apoptosis rates were (23.69+/-2.26)% and (36.99+/-1.42)%. This cell cycle-specific apoptosis were inhibited obviously by caffeine with the apoptosis rates of (4.79+/-0.64)% and (2.69+/-0.56)%. When caffeine was removed, the apoptosis rates increased obviously to (46.23+/-0.21)% and (55.81+/-0.41)%, and still mainly happened in S phase. CONCLUSIONS: Caffeine could inhibit camptothecin-induced apoptosis of Molt-4 cells. As a drug acting on cell cycle checkpoints, caffeine could transiently shield the surveillance of checkpoints to damaged cells and inhibit cell apoptosis. The effect may be reversed when caffeine is removed away.

[Analysis of cell cycle checkpoints by cyclins/DNA flow cytometry].

BACKGROUND & OBJECTIVE: Eukaryotic cell cycle events progress strictly in order which is controlled by the mechanism of checkpoint. At present, most analyses of checkpoint use the flow cytometry (FCM) based on DNA histogram to detect cell cycle distribution. This study was designed to set up and evaluate a new method, Cyclins/DNA multiparameter FCM based on the model of late G1 phase (G1L) checkpoint, for analyzing cell cycle checkpoint. METHODS: After irradiation by ultraviolet (UV), human acute lymphatic leukemia MOLT-4 cells were gathered and fixed at different time points, and divided into 2 groups. In one group, the total G0/G1 phase cells were calculated by Modifit software using DNA histogram method; in the other group, fluorescence intensity and threshold of Cyclin E in G1L cells and G0/G1 phase cells were quantitatively analyzed by Cyclins/DNA multiparameter method. RESULTS: When analyzed by DNA histogram method, the percentage of G0/G1 phase cells was unchanged after irradiated for 0-4 h, but increased to 12.6% after irradiated for 6 h. When analyzed by Cyclins/DNA multiparameter method, the Cyclin E fluorescence intensity of G1L cells was increased from 295.1 (control) to 341.2 (15.6%) after irradiated for 1 h, and increased to 577.6 (95.7%) with the threshold increased from 2.0 (control) to 5.4 after irradiated for 6 h; G1L cells was slightly decreased after irradiated for 6 h when the apoptosis rate was 5.61%, and early G1 phase (G1E) cells was increased slowly. CONCLUSION: Cyclin E/DNA multiparameter FCM could quantitatively detect fluorescence intensity and threshold of Cyclin E, and is more sensitive and precise than DNA histogram FCM in detecting G1L checkpoint.

[Expressions of cyclins A, B1, D3, and E in asynchronous and synchronous MOLT-4 cells of G1 phase].

BACKGROUND & OBJECTIVE: We have testified expression imbalance of Cyclins A, B1, D3, and E in MOLT-4 cells, which were synchronized by "double thymidine blocks", with flow cytometry (FCM). However, comparisons of Cyclins expressions between synchronized cells and asynchronized cells haven't been performed at that time because of technique limitations. This study was to compare expressions of Cyclins A, B1, D3, and E in G(1) phase of asynchronized and synchronized cells by newly established "postsorting Western blot", and to conform the unreasonableness of using double thymidine blocks to synchronize cells to analyze normal cell cycles. METHODS: MOLT-4 cells were synchronized at G(1) phase by double thymidine blocks, asynchronous cells of G(1) phase were sorted by FCM. Western blot and double parameters analysis of DNA/Cyclins were performed to detect Cyclins A, B1, D3, and E expressions in asynchronous and synchronous MOLT-4 cells of G(1) phase. RESULTS: There were almost no expressions of Cyclins A, B1 in asynchronous MOLT-4 cells of G(1) phase, and obvious expressions in synchronized cells of G(1) phase. Expressions of Cyclins D3, E in synchronous MOLT-4 cells of G(1) phase were higher than those in asynchronous cells of G(1) phase. The FCM results were accordant with Western blot results. CONCLUSIONS: The expressions of Cyclins in synchronized cells obtained through "double thymidine blocks" can't represent their expressions in normal cells. Thus, synchronous cells produced by "double thymidine blocks" are not ideal experimental models for analyzing normal cell cycles.

[Promotive effect of decreased cyclin E threshold on cell proliferation].

BACKGROUND & OBJECTIVE: Many studies showed that high expression of Cyclin E promotes cell proliferation, but contrary data was also reported that cell proliferation didn't decrease with low expression of Cyclin E. In addition, we observed that many tumor cells have strong capability of proliferation with low expression of Cyclins, including Cyclin E. This study was to analyze effect of reduced Cyclin E threshold on proliferation of acute lymphocyte leukemia cell line MOLT-4 to explain the above phenomena. METHODS: We have established the model of decreased Cyclin E threshold in MOLT-4 cells by treating cells with low concentration (5 mmol/L) of caffeine for 2, and 4 h. The positive rates of proliferation cell nuclear antigen (PCNA), Ki67, and DNA strand break induction by photolysis (SBIP) were analyzed by flow cytometry. RESULTS: MOLT-4 cells presented sharply decrease of Cyclin E threshold, and increase of positive rates of PCNA, Ki67, and SBIP after treated with caffeine, especially at 2-h point. CONCLUSIONS: Decrease of Cyclin E threshold was accompanied by increase of cell proliferation. MOLT-4 cells may remain high proliferation capability with low level of Cyclin expression.

[Expression of estrogen receptor in different breast cancer cell lines transfected with estrogen receptor plasmid HEGO5 mediated by FuGENE6].

BACKGROUND & OBJECTIVE: Gene transfection is a major approach in studies of estrogen receptor (ER)expression in breast cancer cells,and ER's functional mechanism. This study was designed to detect the transfection efficiency of different human breast cancer cell lines mediated by FuGENE6 reagent, and ER expression in these cells after transfected with ER plasmid HEGO5. METHODS: Breast cancer cell lines were transfected with plasmid pEGFP-N1,and the transfection efficiency was measured by flow cytometry (FCM). The cell lines with high transfection efficiency were transfected with HEGO5,and then the expression of ER was determined by FCM and Western blot. RESULTS: There were obvious differences in transfection efficiencies of breast cancer cell lines MM-231, MM330, MM134 VI, MM175VII, MM157, MM361, MM436, MM453, UaCC812, UaCC893, BT-549, BT-20, HBL-100, Hs578t, MCF-7, T-47d, and ZR-75-1. Repetitious transfection test in HBL-100 cells showed that co-efficient of variation was 5.1. HEGO5 was transfected into the cells with high plasmid transfection efficiency (MCF-7, HBL-100, Hs578t, MM436, MM453, and BT-20), positive rates of ER in these cells ranged from 12.9% to 54.8%, corresponding to the pEGFP-N1 transfection efficiency, and the expression of ER detected by FCM were in accordance with those tested by Western blot. To some extent, the expression of ER in transfected cells was cell cycle specific. CONCLUSIONS: Transfection of breast cancer cell lines mediated by FuGENE6 reagent has a good repetition,and the results were stable and reliable. In the cells transfected with HEGO5,the expression of ER can be qualitatively detected by Western blot, and quantitatively detected by FCM.

[Correlation of intra-cellular localization and variations of active caspase-3 to morphology changes in apoptotic MOLT-4 cells induced by X-ray].

BACKGROUND & OBJECTIVE: How pro-caspase-3 activation lead to serial morphology changes during progress of cell apoptosis is unclear. This study was to investigate the variations and intra-localization of active Caspase-3, determine cell morphology changes in apoptotic MOLT-4 cells induced by X-ray, and evaluate their relationship. METHODS: MOLT-4 cells were irradiated by 10 Gy X-ray. Sub G(1)peak method, and DNA fragmentation assay were used to detect variations of DNA in apoptotic cells. Annexin V/PI method was used to determine the cell membrane reversion, and fluorescence labeled inhibitor of Caspases (FLICA) was used to detect the active Caspase-3 in apoptotic cells. Cell morphology and Caspase-3 intra-localization were determined by confocal microscopy. RESULTS: MOLT-4 cells irradiated by 10 Gy X-ray presented classical apoptotic morphology changes such as membrane reversion, and apoptotic body. Caspase-3 was activated after irradiation, and increased remarkably after irradiated for 4 hours. Activated Caspase-3 moved from sub-membrane toward cytoplasm and nucleus. Caspase-3 activity was detected 2 hours earlier than membrane reversion. CONCLUSIONS: Caspase-3 was activated in MOLT-4 cells induced by X-ray, and its intra-localization correlated with the apoptotic morphology changes. The spatial shift of active Caspase-3 in MOLT-4 cells induced by X-ray is one of the mechanisms of apoptosis.

[Western blot analysis and flow cytometric analysis of estrogen and progesterone receptors in fixed breast cancer cells].

BACKGROUND & OBJECTIVE: Quantitative and qualitative detection of estrogen receptor (ER) and progesterone receptor (PR) levels are very important for predicting prognosis and evaluating the outcome of endocrine therapy of breast cancer patients. Western blot analysis and Flow cytometry (FCM) are important means for quantitative and qualitative analysis of proteins, but conventional Western blot analysis need to extract proteins from fresh cells. The present study was designed to establish Western blot analysis of human estrogen receptor (ER) and progesterone receptor (PR) in fixed breast cancer cells, and to explore the possibility of ER and PR analysis in fixed cells by flow cytometry and Western blot analysis simultaneously. METHODS: Proteins extracted from fresh and fixed cells of different exponentially growing breast cancer cell lines were labelled using 1D5 and PgR636, which were monoantibodies to ERalpha and PR, respectively. The expression of ER and PR were determined using Western blot analysis, and the results were compared with that of fixed cells measured with flow cytometry. RESULTS: Clear and correct ERalpha bands were observed with Western blot analysis in cell lines T-47d, MCF-7, and ZR-75-1, and the band density of fixed T-47d and ZR-75-1 was higher than that of fresh cells. The expression of ERalpha in MM231 cells was negative. Clear and correct PR bands were visible in T-47d and ZR-75-1 cells with Western blot analysis, and the band density of fixed cells was higher than that of fresh cells. And the expression of PR in MM231 and MCF-7 cells were negative. In addition, positive expression of ER and PR in different cell lines measured by flow cytometry were the same with that analyzed by Western blot analysis. CONCLUSION: After fixed with 0.25% paraformaldehyde and 75% ethanol, breast cancer cells can be used for not only quantitative measurement of ER and PR, but also Western blot analysis of ER and PR.