Analysis of DNA Ploidy, Cell Cycle and Ki67 Antigen in Nasopharyngeal Carcinoma by Flow Cytometry

Summary: The expression of DNA ploidy, the cell cycle and Ki67 antigen in nasopharyngeal carcinoma (NPC) were studied and their relationship with the clinical biological behaviors and prognosis of NPC was evaluated. Biopsied specimens of NPC were made into cell suspension. By using cytometric double labeling Ki67 and DNA method, the expression of DNA ploidy, the cell cycle and Ki67 antigen were analyzed. The patients were followed-up for about 3 years and the relationship between the above-mentioned parameters and the clinical biological behavior and prognosis of NPC were evaluated. Of the 62 cases of NPC, the DNA aneuploid accounted for 29.03 %. The S phase cells accounted for 0 to 54 % in the cell cycle and the positive expression of Ki67 ranged from 0 to 52 %. There were 40 cases of LPI (64.5 %) including 15 negative cases and 22 cases of HPI (35 5 %) respectively. The DNA anueploid content was positively related to the S phase cells. The patients having a low expression of Ki67 or DNA aneuploid in tumor cells were not sensitive to chemotherapy, liable to metastasis to distant organs and had a poor prognosis, while Ki67 showed no correlation with DNA ploidy and the cell cycle. It was suggested that DNA ploidy and Ki67 could be used as an independent and objective marker to evaluate the radiosensitivity and prognosis of NPC.

Probing the Cell Cycle with Flow Cytometry

Flow cytometry is a versatile technique to study different aspects of the cell cycle from subpopulations of cells to detailed cell kinetic information. In this paper a basic review of cell kinetic parameters is presented followed by detailed descriptions of the different flow cytometric methodologies that can be used to extract pertinent information for a particular study. The methodologies range from simple DNA profile analysis, the use of bromodeoxyuridine to cell cycle-associated proteins such as the cyclins.

A Novel Three-parameter Flow Cytometric Analysis for Cell Cycle

To set up a three-parameter method for cell cycle analysis by two-laser flowcy-tometer, which can detect two types of cyclin plus DNA content in one measurement, and thatanalyze unscheduled expression of cyclins. Methods: Three-color fluorescence was used for analysisof two types of cyclins and DNA content simultaneously in individual cells by two-laser flowcytometry. MOLT-4 cells were used to study the expression of major cyclins in mammalian cells. ATriton-X100 permeabilization procedure was optimized for detection of two types of cyclins. Onecyclin was stained directly with a FITC-conjugated monoclonal antibody (mAb), and the other,indirectly with RPE-Cy5-conjugated secondary antibody, while DNA was stained with the fluorochromeDAPI. mAMSA and mimosine treated MOLT-4 cells were used to test this three-parameter method.Results: Permeabilization with 0.5% Triton-XlOO in PBS containing 1% BSA for 5 min on ice providedoptimal conditions for the simultaneous labelling of two cyclins plus DNA in single cells. It wasfound that the emission spectrum of the three dyes (DAPI, FITC and RPE-Cy5) could be measured withno compensation. Based on cyclinA/cyclinE/DNA flow cytometric analysis, asynchronously growingMOLT-4 cells could be divided into 6 compartments (G1o, G1e, G1l, S, G2, and M) simultaneously,allowing for analysis of cell cycle phase specific perturbations without the necessity of cellsynchronization. Unscheduled cyclin B1 expression was observed in G1 cells treated with mimosine andcyclin E in G2 cells treated with mAMSA. We found that unscheduled cyclin expression paralleledexpected cyclin expression. Conclusion: Thus, three-color FCM analysis of cells may not only beapplied to measure unscheduled vs. expected cyclin expression but may also be used to estimate thefraction of cycling cells in up to 6 cell populations.

Suppression of renal cell proliferation, induction of apoptosis and cell cycle arrest: Cytotoxicity of vanadium in broilers

The aims of this study were to clarify the effects of high vanadium on the renal cell cycle and apoptosis in broilers. 420 one-day-old avian broilers were divided into six groups and fed on a control diet (vanadium 0.073 mg/kg), and five high vanadium diets (vanadium 5 mg/kg, high vanadium group I;15 mg/kg, high vanadium group II;30 mg/kg, high vanadium group III;45 mg/kg, high vanadium group IV;60 mg/kg, high vanadium group V) throughout the experimental period of 42 days. As tested by flow cytometry, the percentage of apoptotic renal cells was increased in high vanadium group II, III, IV and V when compared with that of control group. The Proliferating index (PI) of renal cell and the ratio of S, G2 + M phase cells were markedly decreased and population of G0/G1 cells was increased in high vanadium group II, III, IV and V. The results showed that dietary vanadium in excess of 15 mg/kg was toxic to kidney by the renal cells cycle arrest and increased apoptosis, which caused the growth depression of the kidney in broilers.

The Apoptosis and CellCycle Changes of SUD4 and DOHH2 Induced by Topotecan

Topotecan (TPT), a semisynthetic analogue of the natural product camptothecin is a cell cycle-specific drug with antitumor activity. To clarify the effect of TPT on SUD4 and DOHH2 cell line in this study, we examined the apoptosis and cell cycle changes of the two human cancer cell lines by exposing to TPT for 18 hours at various concentrations. The linear relationship between apoptosis cell number and the concentration of TPT was observed by means of Flow Cytometry and Annexin V assay. Then, DOHH2 cell is much more sensitive to TPT than SUD4 cell. In addition, Cell Question Software Assay showed positive relationship between the frequency of cells accumulated in S-phase and the concentration of TPT. The least concentration of TPT to change cell cycle is 5 nmol·L?1 in both cell lines. These results suggest that the inducing apoptosis of cancer cells is one of mechanism of TPT antitumor activity.

The approaches in detecting cell cycle specificity of Fas-mediated apoptosis in leukemia cell lines and activated PBLs in vitro

Objective： To establish a system in detecting the cell cycle specificity induced by recombinant human Fas ligand in vitro, so as to provide a reliable platform for further exploring the mechanism of cell cycle control and regulation in Fas-mediated apoptosis. Methods： The target cells-leukaemia cell lines and activated peripheral blood lymphocytes stimulated by phytohemagglutinin were incubated with recombinant human Fas ligand for 6 to 36 h, apoptosis was detected by sub-G1, common annexin-Ⅴ/PI and modified annexin Ⅴ and propidium iodide （API） methods and analysed by flow cytometry. Results： The modified API method demonstrated that Fas-mediated apoptosis was cell cycle specific and initiated at G1 phase. The common annexinⅤ/PI method showed the most appropriate condition for the detection of typical cell cycle-specific apoptosis. The sub-G1 method could only illuminate late apoptosis and DNA histogram. Conclusion： Fas-mediated apoptosis was cell cycle-specific and initiated at G 1 phase. Based on the modified API and common AnnexinⅤ/PI methods, the establishment of stable and typical cell cycle-specific model in Fas-mediated apoptosis in vitro was feasible.

Flow Cytometric Evidence for Hydroxyl Radical-induced Apoptosis in Tobacco Protoplasts

Protoplasts prepared from tobacco (Nicotiana tabacum L., cultivar BY-2) suspension cells have similar morphological characteristics to those in animal cells. The hallmarks of apoptosis such as condensation and peripheral distribution of nuclei, TUNEL positive reaction, and DNA ladders were observed when tobacco protoplasts were treated with the hydroxyl radical generating system (1.0 mmol/L FeSO4/0.5 mmol/L H2O2). In animals, the loss of transmembrane potential (DeltaPsi(m)) and the exposure of phospholipid phosphatidylserine (PS) are believed to be the main apoptosis events. To test whether these significant processes take place in plants, flow cytometry was used to detect annexin V binding and changes in DeltaPsi(m). Results showed that the PS turned out from inner membrane and DeltaPsi(m) gradually decreased during the apoptosis. All these apoptotic characteristics proved that hydroxyl radicals can cause typical programmed cell death (PCD) in tobacco protoplasts and this design can be served as an effective experiment system to explore the mechanism of plant apoptosis.

Gender difference in the neuroprotective effect of rat bone marrow mesenchymal cells against hypoxiainduced apoptosis of retinal ganglion cells

Bone marrow mesenchymal stem cells can reduce retinal ganglion cell death and effectively prevent vision loss. Previously, we found that during differentiation, female rhesus monkey bone marrow mesenchymal stem cells acquire a higher neurogenic potential compared with male rhesus monkey bone marrow mesenchymal stem cells. This suggests that female bone marrow mesenchymal stem cells have a stronger neuroprotective effect than male bone marrow mesenchymal stem cells. Here, we first isolated and cultured bone marrow mesenchymal stem cells from female and male rats by density gradient centrifugation. Retinal tissue from newborn rats was prepared by enzymatic digestion to obtain primary retinal ganglion cells. Using the transwell system, retinal ganglion cells were co-cultured with bone marrow mesenchymal stem cells under hypoxia. Cell apoptosis was detected by flow cytometry and caspase-3 activity assay. We found a marked increase in apoptotic rate and caspase-3 activity of retinal ganglion cells after 24 hours of hypoxia compared with normoxia. Moreover, apoptotic rate and caspase-3 activity of retinal ganglion cells significantly decreased with both female and male bone marrow mesenchymal stem cell co-culture under hypoxia compared with culture alone, with more significant effects from female bone marrow mesenchymal stem cells. Our results indicate that bone marrow mesenchymal stem cells exert a neuroprotective effect against hypoxia-induced apoptosis of retinal ganglion cells, and also that female cells have greater neuroprotective ability compared with male cells.

Aquaporin-1 down regulation associated with inhibiting cell viability and inducing apoptosis of human lens epithelial cells

AIM：To investigate the role of Aquaporin-1（AQP-1）in lens epithelial cells（LECs）and its potential target genes.AQP-1 is specifically expressed in LECs of eyes and is significant for lens homeostasis and transparency maintenance.Herein,AQP-1 expression in LECs was investigated to evaluate its influence on cell survival in association with its potential role in cataract formation.·M ETHODS：LECs were transfected with lentivirus carrying AQP-1 small interfering RNA（si RNA）.Real-time polymerase chain reaction（PCR）and Western blotting were conducted to detect AQP-1 expression in LECs from different groups.Meanwhile,cell counting kit-8（CCK-8）assay and flow cytometry were performed to measure LEC proliferation and apoptosis,respectively.·RESULTS：AQP-1 expression was significantly reduced in LECs,both at m RNA and protein levels（〈0.05）,after si RNA treatment.Decreased cell viability was detected by CCK-8 assay in LECs with si RNA interference,compared to control cells（〈0.05）.The apoptosis rate significantly increased in cells after si RNA interference（〈0.05）.·CONCLUSION：The decreased cell viability following AQP-1 down regulation is largely due to its induction of apoptosis of LECs.AQP-1 reduction might lead to changes of physiological functions in LECs,which might be associated with the occurrence and development of cataracts.

Autophagy occurs within an hour of adenosine triphosphate treatment after nerve cell damage:the neuroprotective effects of adenosine triphosphate against apoptosis

After hypoxia, ischemia, or inflammatory injuries to the central nervous system, the damaged cells release a large amount of adenosine triphosphate, which may cause secondary neuronal death. Autophagy is a form of cell death that also has neuroprotective effects. Cell Counting Kit assay, monodansylcadaverine staining, flow cytometry, western blotting, and real-time PCR were used to determine the effects of exogenous adenosine triphosphate treatment at different concentrations （2, 4, 6, 8, 10 mmol/L） over time （1, 2, 3, and 6 hours） on the apoptosis and autophagy of SH-SY5Y cells. High concentrations of extracellular adenosine triphosphate induced autophagy and apoptosis of SH-SYSY cells. The enhanced autophagy first appeared, and peaked at 1 hour after treatment with adenosine triphosphate. Cell apoptosis peaked at 3 hours, and persisted through 6 hours. With prolonged exposure to the adenosine triphosphate treatment, the fraction of apoptotic cells increased. These data suggest that the SH-SY5Y neural cells initiated autophagy against apoptosis within an hour of adenosine triphosphate treatment to protect themselves against injury.

Relationship between Fas/ FasL expression and apoptosis of colon adenocarcinoma cell lines

INTRODUCTIONFas/ FasL system has been identified as a keymediator of apoptosis in tumor cells[1-4]. Theoccurrence and development of neoplasm are closelyrelated to apoptosis[5-7] Most chemotherapeuticdrugs kill cancer cells mainly by inducingapoptosis[8-14].'

Growth Inhibition and Apoptosis Induction in Human Hepatoma Cells by Tanshinone Ⅱ_A

In order to .study the effect of tanshinone ⅡA on growth and apoptosis in human hepatoma cell line BEL-7402 in vitro, the human hepatoma cell line BEL-7402 was treated with tanshinone ⅡA at various concentrations for 72 h. Growth suppression was evaluated by MTT assay; apoptosis-relat-ed alterations in morphology and biochemistry were ascertained under cytochemical staining (Hoechst 33258), transmission electron microscopy (TEM), and DNA agarose gel electrophoresis. Apoptotic rate was quantified by flow cytometry (FCM). The results showed that Tanshinone ⅡA could inhibit the growth of hepatoma cells in a dose-dependent manner, with IC50 value being 6. 28μg/ml. After treatment with 1-10μg/ml tanshinone ⅡA for 72 h, BEL-7402 cells apoptosis with nuclear chro-matin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodies were observed. DNA ladder could be demonstrated on DNA electrophoresis. FCM analysis showed hypodiploid peaks on histogram, and the apoptotic rates at μg/ml concentration for 12 h> 24 h, 36 h, 48 h and 72 h were (2. 32±0. 16)%, (3. 01±0. 35) %, (3. 87±0. 43)%, (6. 73±0. 58)% and (20. 85 ± 1. 74) % respectively, which were all significantly higher than those in the control group (1. 07±0. 13) %. It is concluded that Tanshinone ⅡA could induce human hepatoma cell line BEL-7402 apoptosis, which may be related to the mechanism of growth inhibition.

Approach for mechanism of BH3 domain counterpart BH3I-2' inducing colorectal cancer cell apoptosis

Objective To discuss mechanism of BH3 domain counterpart BH3I-2' inducing colorectal cancer cell apoptosis.Methods Detected inhibition ratio and apoptosis of colorectal cancer cells HCT-116,which were treated by BH3I-2',with microplate reader and flow cytometry.Results Inhibition ratio of colorectal cancer cells,which were treated by BH3I-2',could reach about 50%.Ratio of viable apoptotic cell decreased and that of non-viable apoptotic cell increased as time went.Conclusions BH3I-2' can induce colorectal cancer cell apoptosis.

Oleanolic acid-induced apoptosis and its relation with intracellular calcium in human lung adenocarcinoma A549 cells

Objective To investigate the effect of oleanolic acid (OA) on apoptosis,correlation between apoptosis and intracellular calcium,and its mechanism in human lung adenocarcinoma cell line A549. Methods Human lung adenocarcinoma A549 cells were incubated in vitro and assigned with OA concentrations of 0,10,20 and 40μg/mL. The apoptosis status of A549 cell line was detected with Annexin V-FITC/PI by flow cytometry (FCM); fluorescence intensity (FI) of A549 cells was assessed and the level of intracellular calcium was calculated at 24 hour of OA intervention. The relation between apoptosis and calcium FI was illustrated by curve fitting. Results FCM showed that 10,20 and 40μg/mL of OA could induce A549 cell apoptosis,which followed a concentration-effect pattern; 24-hour intervention with 20μg/mL and 40μg/mL OA showed increased A549 cell apoptosis,and was significantly different from that with 0μg/mL OA (P<0.01). The FI of intracellular calcium concentration in 10,20 and 40μg/mL OA groups was significantly higher than that in 0μg/mL group after 24 hours’ intervention,and the FI showed a trend of increase with increased OA concentration (P<0.01). Curve fitting showed a significant correlation between apoptosis rate and intracellular calcium concentration in A549 cells (r=0.981,P<0.01). Regression equation was Y=0.508X-1.627. Conclusion OA plays a role in inducing apoptosis of human lung adenocarcinoma cells in a concentration-dependent manner. The OA-induced apoptosis is responsible for intracellular calcium overload of the tumor.

Apoptosis of HeLa Cells Induced by Cisplatin and Its Mechanism

To study the apoptosis induced by cisplatin in cervical cancer cell line HeLa and its mechanism, cell growth inhibition of cisplatin on HeLa cells was analyzed by MTT assay. Cell apoptosis was examined by cytometry and Hoechst33258 staining after treatment with cisplatin. The effects of cisplatin on transcription of E6 were analyzed by RT-PCR. The protein expressions of E6, p53, p21, Bax and Bcl-2 were studied by Western blotting. Cisplatin inhibited proliferation in a time- and dose-dependant manner. Cytometically, sub-G1 peak showed higher apoptosis rates in the experimental group than those in the control. Hoechst33258staining exhibited apoptosis induced by cisplatin. RT-PCR revealed that cisplatin decreased transcription of E6. Western blotting showed that cisplatin decreased protein expression of E6 and increased protein expression of p53, p21and Bax. It had no effect on protein expression of Bcl-2. It is concluded that cisplatin can induce apoptosis in HeLa cells by suppressing HPV E6 and thereby restoring the function of p53.

Apoptosis-inducing effect of Jinke on Molt-4 cells and its mechanism

Objective： To investigate the apoptosis-inducing effect of Jinke on Molt-4 cells and its possible mechanism. Methods： The Molt-4 cells were treated with different concentrations of Jinke and then cultured for necessary time. The Annexin-V / PI method was used to detect the apoptosis rate. The cell cycle was analyzed by DNA content with flow cytometry. Double parameters analysis of cyclins / DNA was performed to detect the expression of cyclin E. API method was used to confirm the cell cycle-specific apoptosis. The expressions of Bcl-2 and Bax were detected by westem blot. Results： 24 h after the treatment of 0.5, 1.0, 1.5, 2.0 and 3.0 mg/mL Jinke, the apoptosis rate of Molt-4 cells was evaluated in a concentration-dependent manner, from 5.2% of the control group to 41.0% of the 3.0 mg/mL Jinke group. When the Molt-4 cells were cultured with 1.5 mg/mL Jinke, the apoptosis rate was evaluated in a time-dependent manner. DNA content analysis showed that G0/G1 phase of Molt-4 cells increased in a time-dependent manner. The expression of cyclin E increased gradually. API assay showed the apoptosis cells were almost in G0/G1 phase. Western blot showed the Bcl-2 was down-regulated and the Bax was up-regulated. Conclusion： Jinke could induce G1 phase-specific apoptosis in Molt-4 cells in time-and concentration-dependent manners involving G1 phase arrest. The mechanism of apoptosis inducing effect may be related to the upregulation of Bax and the down-regulation of Bcl-2.

Analysis of cell cycle's correlation of γ-H2AX

Objective： To analyze and discuss cell cycle＇s correlation of y-H2AX, so as to accumulate the data for the further studies of y-H2AX. Methods： MOLT-4 cells, and peripheral blood lymphocytes （PBLs）, with or without 48 h stimulation of phytohemagglutinin （PHA）, were irradiated by ultraviolet rays （UV rays）. Fluorescence-labeled y-H2AX antibody was used to detect γ-H2AX foci at the DNA double-strand breaks （DSBs） in chromatin, DNA damage was analyzed by flow cytometry, cell cycle and cell apoptosis were detected by sub-G1 peak method, the expression of γ-H2AX was detected by Western blot. Results： With the progression of time, sub-G1 peak emerged apparently in the DNA histograms, and the cells of apoptosis increased gradually; with the progression of time, the increase of γ-H2AX emerged and firstly raised, then decreased; PBLs with 48 h stimulation of PHA entered apparently cell cycle, cells of S and G2/M phase emerged, and PBLs without stimulation of PHA did not enter cell cycle; Western blot showed the increase of the expression of γ-H2AX, and the increase also firstly raised, then decreased. Conclusion： γ-H2AX expressed in the cells of stationary phase and proliferative phase, and with the progression of time, the increase of γ-H2AX firstly raised, and then decreased.

The Mechanism of Cisplatin-induced Apoptosis in HeLa Cells

OBJECTIVE To study the mechanism of apoptosis induced by cisplatin in vitro in HeLa cells cervical cancer cell line. METHODS The inhibitory effect of cisplatin on HeLa cell growth was analyzed by the MTT assay. Cell apoptosis was measured with flow cytometry and Hoechst 33258 staining following cisplatin treatment. The effect of cisplatin on transcription of HPV E6 was analyzed by RT-PCR and protein expression of E6, P53, p21, Bax and Bcl-2 was studied by Western blots. RESULTS Cisplatin inhibited cellular proliferation in a time and dose- dependant manner. The sub-G1 peak by flow cytometry showed a higher apoptotic rate in the experimental group compared to the controls and Hoechst 33258 staining indicated that apoptosis was induced by cisplatin. Results of RT-PCR demonstrated that cisplatin decreased tran- scription of E6. Western Blots showed that cisplatin decreased protein expression of E6 and increased protein expression of P53, p21 and Bax but had no effect on protein expression of Bcl-2. CONCLUSION Cisplatin induces apoptosis and death of HeLa cells through the suppression of HPV E6 and restoration of p53 function.

流式细胞术检测细胞周期影响因素及不同免疫细胞亚群周期分析

细胞周期检测对于了解细胞增殖状态、细胞功能研究、药物筛选与评估等领域具有重要意义。流式细胞仪分析细胞周期是目前较为经典且广泛应用的检测手段之一。碘化丙啶(propidium iodide,PI)单染是最常用的基于流式的检测细胞周期方法,然而,该方法在使用过程有诸多处理因素会对检测结果造成影响。此外,不同免疫细胞亚群在不同细胞周期阶段的分布差异也有助于研究免疫反应,了解疾病状态。本文以B16-F10细胞系为例,采用PI单染法,从固定条件、上机条件和软件分析3个角度,评价多种处理因素对细胞周期检测结果的影响。根据结果分析,在进行细胞周期检测时,建议取3×10^(6)个细胞,用300μL预冷PBS重悬细胞后逐滴滴入700μL预冷的无水乙醇,放置于4℃或-20℃过夜固定,低速收样,收样速率为每秒400~600颗粒数,去黏连后需收集至少3000个细胞。另外,通过EdU、PI双指标染色可以精确划分细胞周期,而细胞表面标志物染色联合Ki-67和PI染色法,可在不进行细胞分选的情况下进行不同免疫细胞亚群周期分析。本文较为系统的探讨了PI单染法检测细胞周期的影响因素,提供了标准的实验操作流程。建立了EdU联合PI检测细胞周期和针对不同免疫细胞亚群周期分析方案,拓宽了细胞周期检测方式。

Cell cycle analysis by cyclin E+A/DNA multiparameter flow cytometry in exponential growth MOLT-4 cells

Objective To establish a new method for analyzing the cell cycle in tumor which is a kind of cell cycle disease.Methods Mixtures of cyclin E and cyclin A antibodies were incubated with fixed MOLT-4 cells, and measured by flow cytometry.Results We developed a cyclin E+A/DNA flow cytometry analysis method, which may distinguish G0, early G1, late G1, S, G2 and M phase cells, rather than three phases in the DNA content histogram.Conclusion Cyclin E+A/DNA multiparameter flow cytometry can simultaneously differentiate in the same sample six cell groups: G0, early G1, late G1, S, G2 and M phase cells. It performed better than any other cell cycle analysis methods that we have used and has a definite cell biology foundation.