Cell cycle dependence of drug initiated apoptosis in HL-60 cells

The extent- and cell cycle specificity of apoptotic cell death were studied in human leukemia HL-60 cells treated with 1.25-20 mu M etoposide, 0.125-2.0 mu g/ml dactinomycin, 12.5-200 mu M 3-deazaadenosine (c(3)Ado) and 10-50 Gy gamma-irradiation. Flow cytometry was used to measure the fraction of apoptotic cells and determining their cell cycle position. With all agents the extent of apoptotic cell death showed a clear dose- and time-dependency. Specific apoptosis of S-phase cells was found in cultures treated with 1.25-2.5 mu M etoposide. At 5-20 mu M etoposide, 0.125-2.0 mu g/ml dactinomycin and 50-200 mu M c(3)Ado cells from all cell cycle phases responded by apoptosis. Exposure of the cells to 10 Gy gamma-irradiation resulted in apoptosis of G(2)+M-phase cells. At 50 Gy cells from the S- and G(0)/G(1)-phases also entered apoptosis. Our findings indicate that the cell cycle specificity of an anti-cancer agent initiated apoptotic response depends upon the drug/irradiation dose and exposure time used. This demonstrates that the resistance of subpopulations of cells to apoptosis is relative.     

X线照射剂量率对A549肺癌细胞周期的影响

目的：了解不同剂量率X线照射对非小细胞肺癌细胞周期的影响,以期为临床制定放疗计划提供一定的实验依据。方法选取A549肺癌细胞进行培养48 h,采用直线加速器行X线照射,照射剂量为6 Gy,照射剂量率分别为1、2、4、6 Gy/min,照射后24 h收集细胞,用70%乙醇固定,RNA酶处理,碘化丙啶染色,采用流式细胞学的方法检测细胞周期。结果 X线照射后细胞周期分布发生明显的改变,细胞在G2/M期发生明显阻滞,S期的细胞明显减少,其中以2 Gy/min照射的细胞G2/M期的阻滞最为明显。结论 X线照射影响A549细胞周期的进程,相同的剂量,不同的剂量率对细胞周期的影响存在明显差异。选取最佳剂量率进行治疗,可能有助于提高肿瘤放射治疗效果。     

The late effects of proton irradiation on cell growth, cell cycle arrest and apoptosis in a human melanoma cell line

The aim of this work is the in vitro study of the late effects of single proton irradiation on HTB63 human melanoma cell growth, cell cycle and cell death. The experimental conditions were focused on analyzing the effects of irradiation on the periphery of tumour that can be, in clinical practice, close to critical organs. Confluent cell monolayers were irradiated with single doses ranging from 1 - 20 Gy, using proton beams having an energy of 22.6 MeV at the target. Antiproliferative effect of protons, cell cycle analysis and initiation of cell death, were followed 48 hours after irradiation. The inhibition of melanoma cell growth was observed, especially after single application of 12 and 16 Gy. Cell cycle analysis and cell viability have shown the G2/M and G1/G0 arrest of irradiated cells correlating with the increase of the applied dose. The flow cytometric analysis has shown presence of apoptotic nuclei. These data demonstrate that irradiation with protons, under the chosen experimental conditions, have significant effects on melanoma cell growth inhibition being dose dependent, G2/M cell cycle arrest and appearance of apoptotic nuclei, even 48 hours after irradiation. The results obtained may help the understanding of the relationship between cell proliferation, death and cell cycle regulation of melanomas after proton irradiation.     

Deregulation of p53/p21Cip1/Waf1 pathway contributes to polyploidy and apoptosis of E1A+cHa-ras transformed cells after gamma-irradiation.

The p53/p21Cip1/Waf1-dependent checkpoint control of G1/S and G2/M phases of the cell cycle in response to DNA damage is an important mechanism of genome stability maintenance in normal cells. In many tumor cells, due to frequent point mutations and deletions of p53, the stringent control of the cell cycle and apoptosis is compromised. We have examined the cell cycle control and cell death of the rat embryo fibroblast cells (REF) transformed by E1A+cHa-ras oncogenes and expressing wild type p53. Gamma-irradiation at a dosage of 6 Gy has been used to analyse the p53-dependent trans-activation of the target p21cip1/waf1 gene and the levels of activity of cyclin-dependent kinases. Our results show that the cell cycle inhibitors p21Cip1/Waf1 and p27KIP accumulate in response to irradiation both in REF and E1A+cHa-ras cells. In contrast to normal REF cells, the accumulation of p21Cip1/Waf1 and p27KIP inhibitors, however, does not lead to inhibition of Cdk2 and cyclins E, A-associated kinase activities and to a G1/S block in E1A+cHa-ras cells. It is unlikely that the lack of inhibitory function of p21Cip1/Waf1 can be explained by its inability to bind Cdk2 and Cdk4 kinases or PCNA. Moreover, the p21Cip1/Waf1-associated kinase activity is increased upon gamma-irradiation of E1A+cHa-ras cells. We suggest that inactivation of p21Cip1/Waf1 may be accounted for by its interaction with E1A oncoproducts as the inhibitor is detected in immunoprecipitates using E1A-specific antibodies. During a temporary G2/M delay induced by gamma-irradiation, E1A+cHa-ras transformants continue DNA replication, which leads to accumulation of polyploid cells with lobulated nuclei and micronuclei. Thus, DNA damage of E1A+cHa-ras transformed cells, with a combination of functionally active wild type p53 and inactive p21Cip1/Waf1, contributes to formation of polyploid cells which then die due to apoptosis.     

电离辐射对食管癌细胞周期及MDC1、53BP1蛋白表达的影响

目的 观察60Co γ射线照射后食管癌细胞周期、细胞凋亡及其相关蛋白表达的变化,为食管癌放射治疗、靶向治疗提供理论依据。方法 食管癌细胞株TE 13进行不同剂量（0、1、2、5、10、15Gy）照射后,应用流式细胞术分别检测照射后1、2、12、24和48h细胞周期和凋亡指数的变化;同时采用Western blot方法检测MDC1和53BP1蛋白表达情况。结果 TE 13细胞照射后12、24、48h,TE 13细胞的G0/G1期、G2/M期和S期的变化呈现明显剂量依赖性,1Gy和2Gy照射后12h,细胞G2/M期阻滞开始出现;5、10、15Gy照射后24h,细胞G2/M期阻滞最为明显,与对照组（0Gy组）相比,差异具有统计学意义（P<0.05）;15Gy照射后12h、24、48h,TE 13细胞的凋亡增加非常显著（P<0.01）;不同剂量照射后1、2、24h,TE 13细胞MDC1和53BP1蛋白表达未见明显变化（P>0.05）。结论 TE 13细胞经不同剂量放射线照射后,细胞周期出现明显的G2/M期阻滞,细胞凋亡指数明显增加,但对MDC1和53BP1蛋白表达未见明显影响。     

食管癌细胞照射后细胞周期及相关蛋白表达的变化

目的：探讨食管癌细胞照射后细胞周期、细胞凋亡及其相关蛋白表达的变化。方法：食管癌细胞株TE-1和TE-13照射2、5、10、15Gy后，应用流武细胞仪分别检测照射后6、24和48h细胞周期和凋亡指数变化、Western blot检测细胞周期相关蛋白的表达。结果：2、5、10、15Gy照射0～48h，TE-1和TE-13细胞均出现明显剂量依赖性的G2／M期阻滞和解除变化；5～15Gy照射后48h，TE-13细胞在G2／M期阻滞逐渐解除时伴随着细胞凋亡的明显增加，而TE-1细胞凋亡不增加。2株细胞胞浆CHK2-p68磷酸化水平均呈随照射后时间延长而出现时相性变化，但与照射剂量无关；而CHK1、CHK2、CHK1-p345和CDK1蛋白表达均无明显变化。15Gy照射后24h，TE-13细胞胞浆中eyelin B1表达下降而TE1细胞中无明显变化。结论：病理不同分化的食管癌细胞照射后细胞周期、细胞凋亡以及细胞周期相关蛋白表达变化不尽相同。     

Accumulation of HL-60 leukemia cells in G2/M and inhibition of cytokinesis caused by two marine compounds,bistratene A and cycloxazoline

The effects on the cell cycle of two biologically active compounds, bistratene A and cycloxazoline, from the marine ascidianLissoclinum bistratum were studied in HL-60 human leukemia cells using flow cytometry. Both compounds were shown to cause an apparent accumulation of cells in the G2/M phase. This effect was shown to be both time- and dose-dependent. At the longer time points (30 and 48 h after addition of the compounds) polyploidy was apparent. The fate of cells labeled in the S phase with 5-bromo-2-deoxyuridine (BrdUrd) was analysed using a bivariate BrdUrd/PI (propidium iodide) technique. Bistratene A and cycloxazoline treatment prevented the majority of BrdUrd-labeled cells from progressing through to the G1 phase. Approximately 50% of the cells were delayed at G2/M, and a significant proportion of cells appeared to be polyploid. Light and electron microscopy revealed the presence of multinucleated cells accounting for the apparent polyploidy. The progression of cells out of the G1 phase was also examined by synchronising cells with mimosine and releasing them from mimosine block in the presence of bistratene A. There was no evidence of a block at the G1/S phase transition or through the S phase since DNA synthesis was not inhibited. The mechanism by which these compounds interfere with cytokinesis is presently unknown but, in the case of bistratene A, may be linked to altered phosphorylation of cellular proteins involved in cell-cycle control.This paper is dedicated to the memory of Karen Marshall, who died tragically in a car accident during the course of this study     

Radiosensitization of colon cancer cell lines by docetaxel: mechanisms of action

The radiation-modifying action of docetaxel in experimental systems is well established. Docetaxel is also an increasingly important drug for the treatment of cancer in concurrent radiotherapy protocols. However, the mechanisms of docetaxel radiosensitization are not fully understood. We have investigated the magnitude and mechanisms of docetaxel radiosensitization in vitro in four human colorectal cancer cell lines (SW480, SW707, SW48, and HT29) with widely differing radiosensitivities. Cell survival curves were generated for a range of docetaxel concentrations (5-20 nM) alone and for X-rays (1-5 Gy) +/- 10 or 20 nM docetaxel (for 24 h before irradiation). Cell cycle distributions and apoptotic frequencies were measured during the treatments. Sensitivity to docetaxel alone was similar in all cell lines and could be attributed to massive induction of apoptosis (60-80% by 24 h). Radiosensitivity varied widely; the surviving fractions at 2 Gy in the most resistant (HT29) and most sensitive (SW28) lines were 0.81 and 0.13, respectively. Exposure to 10 nM docetaxel induced a progressive accumulation of SW480, SW707, and SW48 cells in G2/M. After 24 h, 55-70% of the cells were in G2/M. It is likely, therefore, that accumulation in this radiosensitive phase of the cell cycle contributes significantly to radiosensitization by the drug.     

Late G1 accumulation after 2 Gy of gamma-irradiation is related to endogenous Raf-1 protein expression and intrinsic radiosensitivity in human cells.

We have previously reported a correlation between high endogenous expression of the protein product of the RAF-1 proto-oncogene, intrinsic cellular radiosensitivity and rapid exit from a G2/M delay induced by 2 Gy of gamma-irradiation. Raf1 is a positive serine/threonine kinase signal transduction factor that relays signals from the cell membrane to the MAP kinase system further downstream and is believed to be involved in an ionizing radiation signal transduction pathway modulating the G1/S checkpoint. We therefore extended our flow cytometric studies to investigate relationships between radiosensitivity, endogenous expression of the Raf1 protein and perturbation of cell cycle checkpoints, leading to alterations in the G1, S and G2/M populations after 2 Gy of gamma-irradiation. Differences in intrinsic radiosensitivity after modulation of the G1/S checkpoint have generally been understood to involve p53 function up to the present time. A role for dominant oncogenes in control of G1/S transit in radiation-treated cells has not been identified previously. Here, we show in 12 human in vitro cancer cell lines that late G1 accumulation after 2 Gy of radiation is related to both Raf1 expression (r = 0.91, P = 0.0001) and the radiosensitivity parameter SF2 (r = -0.71, P = 0.009).     

Gamma-irradiation deregulates cell cycle control and apoptosis in nevoid basal cell carcinoma syndrome-derived cells.

The nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by nevi, palmar and plantar pits, falx calcification, vertebrate anomalies and basal cell carcinomas. It is well known in NBCCS that gamma-irradiation to the skin induces basal cell carcinomas or causes an enlargement of the tumor size, although the details of the mechanism remain unknown. We have established lymphoblastoid cell lines from three NBCCS patients, and we present here the first evidence of abnormal cell cycle and apoptosis regulations. A novel mutation (single nucleotide deletion) in the coding region of the human patched gene, PTCH, was identified in two sibling patients, but no apparent abnormalities were detected in the gene of the remaining patient. Nevertheless, the three established cell lines showed similar features in the following analyses. Flow cytometric analyses revealed that the NBCCS-derived cells were accumulated in the G2M phase after gamma-irradiation, whereas normal cells showed cell cycle arrest both in the G0G1 and G2M phases. The fraction of apoptotic cells after gamma-irradiation was smaller in the NBCCS cells. The level of p27 expression markedly decreased after gamma-irradiation in the NBCCS cells, although the effects of the irradiation on the expression profiles for p53, p21 and Rb did not differ in normal and NBCCS cells. These findings may provide a clue to the molecular mechanisms of tumorigenesis in NBCCS.     

Normal and c-Myc-promoted human keratinocyte differentiation both occur via a novel cell cycle involving cellular growth and endoreplication

The relationship between cell cycle and differentiation in human keratinocytes is poorly understood. It is believed that keratinocytes suppress DNA replication and cell cycle arrest in G0 before they initiate terminal differentiation. However, a temporal separation between both events has not been established. Moreover, c-Myc promotes keratinocyte differentiation without causing cell cycle arrest. To address these paradoxes we have analysed cell cycle control during normal and c-Myc-promoted differentiation. Continuous activation of c-Myc or initiation of terminal differentiation results in a block of G2/M, cellular growth, endoreplication and polyploidy. Keratinocytes abandon G1, continue replicating DNA as they differentiate terminally and become polyploid. In fact, simply blocking mitosis with nocodazole resulted in increased cell size, terminal differentiation and endoreplication. This indicates that terminal differentiation associates with defective cell cycle progression and provides a novel insight into c-Myc biology.     

电离辐射对胃癌细胞增殖和周期的影响

目的：研究电离辐射对体外胃腺癌细胞增殖、凋亡及周期的影响。方法：~（60）Coγ射线单次照射胃腺癌BGC-823细胞,照射剂量分别为0、2、4 Gy,利用MTT和克隆形成率实验检测各剂量组BGC-823的增殖能力;同时利用Annexin-V/PI双染法检测各剂量组BGC-823细胞的凋亡;用PI单染测定各剂量组BGC-823的细胞周期。结果：与0 Gy组相比,2、4Gy 60Coγ射线照射可显著抑制胃腺癌BGC-823细胞的增殖（P〈0.05）,诱导BGC-823细胞在照射后依次出现S期和G2/M期阻滞。4Gy 60Coγ射线照射后48h,凋亡细胞比例显著高于对照组（P〈0.05）。结论：60Coγ射线照射可抑制胃腺癌细胞BGC-823增殖、诱导胃癌细胞出现周期阻滞和细胞凋亡。     

F9 embryonal carcinoma cells fail to stop at G1/S boundary of the cell cycle after gamma-irradiation due to p21WAF1/CIP1 degradation

We studied the ability of F9 teratocarcinoma cells to arrest in G1/S and G2/M checkpoints after gamma-irradiation. Wild-type p53 protein was rapidly accumulated in F9 cells after gamma-irradiation, however, this was followed not by a G1/S arrest but by a short and reversible delay of the cell cycle in G2/M. In order to elucidate the reasons of the lack of G1/S arrest in F9 cells, we investigated the expression of p53 downstream target Cdk inhibitor p21WAF1/CIP1. In spite of p53-dependent activation of p21WAF1/CIP1 gene promoter and p21WAF1/CIP1 mRNA accumulation upon irradiation, the p21WAF1/CIP1 protein was not detected by either immunoblot or immunofluorescence techniques. However, the cells treated with a specific proteasome inhibitor lactacystin revealed the p21WAF1/CIP1 protein both in non-irradiated and irradiated cells. Therefore we suggest that p21WAF1/CIP1 protein is degraded by a proteasome-dependent mechanism in F9 cells and the lack of G1/S arrest after gamma-irradiation is due to this degradation. We also examined the expression and activity of cell cycle regulatory proteins: G1- and G2-cyclins and cyclin-dependent kinases. In the absence of functional p21WAF1/CIP1 inhibitor, the activity of G1 cyclin/Cdk complexes was insufficiently inhibited to cause a G1 arrest, whereas a decrease of cdc2 and cyclin B1-associated kinase activities was enough to contribute to a reversible G2 arrest following gamma-irradiation. After gamma-irradiation, the majority of F9 cells undergo apoptosis implying that wt-p53 likely triggers pro-apoptotic gene expression in DNA damaged cells. Elimination of defected cells might ensure maintenance of genome integrity in the remaining cell population.     

汉防己甲素对人肺腺癌细胞的放射增敏作用及其机制的实验研究

目的 探讨汉防己甲素（Tet）对人肺腺癌SPC-A1细胞的放射敏感性的影响及其作用机制。方法 MTT法检测Tet对SPC-A1细胞的增殖抑制作用,比较单纯照射组（4 Gy）、照射（4 Gy）+Tet（1 μmol／L）组、单用Tet组（1 μmol／L）及空白对照组间SPC-A1细胞增殖抑制率的差异。采用克隆形成实验来计算受照射后的细胞存活率,拟合细胞存活曲线,计算D0、Dq、SF2。流式细胞术检测照射前后SPC-A1细胞周期的分布情况。结果 Tet对SPC-A1细胞的24、48和72 h半数抑制浓度（IC50）分别为10.77、5.78、和3.89 μmol／L。照射+Tet组24、48、72 h的细胞增殖抑制率均高于单纯照射组,差异有统计学意义（P＜0.05）。克隆形成实验显示照射+Tet组的D0、Dq和SF2值分别为（1.551±0.045）Gy、（0.522±0.023）Gy和0.503±0.008,均低于单纯照射组。放射增敏比（SER）为1.48。流式细胞仪检测结果显示照射导致了SPC-A1细胞G2期阻滞（P＜0.05）。联合Tet后可以降低G2期阻滞细胞比例（P＜0.05）。结论 Tet可以有效增加人肺腺癌SPC-A1细胞的放射敏感性,其机制可能是通过降低放射导致的G2期阻滞细胞比例,从而使DNA的损伤固定,发生增殖性死亡。     

CDK1、CDK2 siRNA干扰对肿瘤细胞凋亡和细胞周期的影响

Objective: We investigated the influence of CDK1 and CDK2 expression inhibited by cotransfection of CDK1 and CDK2 siRNA on cell cycle and apoptosis, explored the exact role of cell cycle master regulator in tumor cell apoptosis process. Methods: The siRNA targeting the CDK1 and CDK2 genes were synthesized and simultaneously cotransfected into Hela cells by lipofectamine 2000.48 or 60 h after the cotransfection, CDK1 and CDK2 protein expressions were examined by Western blot. Cell cycle distribution was analyzed by flow cytometry. Cell apoptosis was detected by the Annexin V/PI method. The changes of the transfected cell morphological under a microscope after Wright-Giemsa Staining were studied. Results: CDK1 and CDK2 protein expression was decreased at 48 or 60 h after cotransfection. The accumulation of the G2/M and S phase population in cell cycle of the cotrensfected cells at 48 or 60 h after transfection was enhanced obviously compared with control. The ratio of apoptotic cell of cotransfected cells at 48 or 60 h after transfection was increased significantly compared with control. More binucleate or multinucleate cells among cotransfected cells were observed under the microscope. Conclu- sion: The decreased expression of CDK1 and CDK2 by cotransfection of CDK1 and CDK2 siRNA not only leads to tumor cell cycle arrest in S phase and G2/M phase, but also induces tumor cell apoptosis.     

Cytotoxicity of the bioreductive agent RH1 and its lack of interaction with radiation

BACKGROUND AND PURPOSE: RH1 is a new bioreductive agent that was developed as a cytotoxic agent with selectivity for tumour cells expressing high levels of the enzyme DT-diaphorase (DTD). The aim of the present study was to investigate the cytotoxicity of RH1 in relation to cellular levels of reducing enzymes and any interaction of RH1 with ionizing radiation under oxic and hypoxic conditions. PATIENTS AND METHODS: The MB-MDA231 human breast cancer cell line (WT) and WT cells transfected with the NQO1 gene encoding DTD (the D7 cell line) were used to examine the dependency of RH1's cytotoxicity on cellular DTD activity. The role of the 1-electron reducing enzyme P450 reductase was also studied using a P450 reductase-transfected isogenic cell line (R4). A clonogenic assay was used to investigate the cytotoxicity of RH1 with and without irradiation in air and in nitrogen. In all cases drug exposure was for 3 h. RESULTS: DTD levels were around 300-fold higher in D7 compared to WT and R4 cells. RH1 was cytotoxic at nanomolar concentrations to all the cell lines, and was 2-3 times more toxic in the D7 cells with high DTD than in the other two cell lines. Doses of RH1 was around 2-fold more effective in hypoxic than in oxic WT cells, but not by as much in D7 cells. RH1 did not radiosensitise the cells but showed an additive effect when combined with irradiation under oxic and hypoxic conditions. CONCLUSIONS: RH1 shows high clonogenic cytotoxicity to MDA231 cells with high DTD activity but its selectivity based on the presence of DTD is much less than as shown in previous reports. RH1 showed an additive cell killing effect when combined with irradiation under both oxic and hypoxic conditions.     

Different G2/M accumulation in M059J and M059K cells after exposure to DNA double-strand break-inducing agents

PURPOSE: To investigate and compare the cell cycle progression in relation to cell death in the human glioma cell lines, M059J and M059K, after exposure to DNA double-strand break-inducing agents. METHODS AND MATERIALS: The M059J and M059K cells, deficient and proficient in the catalytic subunit of the DNA-dependent protein kinase, respectively, were exposed to 1 and 4 Gy of photons or accelerated nitrogen ions. In addition, M059J and M059K cells were treated with 10 and 40 mug/mL of bleomycin for 30 min, respectively. Cell cycle progression, monitored by DNA flow cytometry, was measured up to 72 h after treatment. RESULTS: M059J, but not M059K, cells displayed G(2)/M accumulation after low linear energy transfer irradiation. High linear energy transfer radiation exposure however, resulted in a substantial increase of M059K cells in the G(2)/M phase detected at 48 h. At 72 h, the number of cells in the G(2)/M phase was equivalent to its control. M059J cells accumulated mainly in S phase after high linear energy transfer irradiation. In contrast to M059K, M059J cells were still blocked at 72 h. Bleomycin induced G(2)/M accumulation for both M059J and M059K cells detected 24 h after treatment. At 48 h, the percentage of bleomycin-treated M059J cells in G(2)/M phase remained high, and the number of M059K cells had decreased to control levels. Neither cell line showed cell cycle arrest (< or =10 h) after exposure to these agents. CONCLUSION: Distinct cell cycle block and release is dependent on the complexity of the induced DNA damage and the presence of the DNA-dependent protein kinase catalytic subunit.     

Secretory activity and cell cycle alteration of alveolar type II cells in the early and late phase after irradiation

PURPOSE: Type II cells and the surfactant system have been proposed to play a central role in pathogenesis of radiation pneumonitis. We analyzed the secretory function and proliferation parameters of alveolar type II cells in the early (until 24 h) and late phase (1-5 weeks) after irradiation (RT) in vitro and in vivo. METHODS AND MATERIALS: Type II cells were isolated from rats according to the method of Dobbs. Stimulation of secretion was induced with terbutaline, adenosine triphosphate (ATP), and 12-O-tetradecanoylphorbol-13-acetate (TPA) for a 2-h period. Determination of secretion was performed using (3)H-labeled phosphatidylcholine. For the early-phase analysis, freshly isolated and adherent type II cells were irradiated in vitro with 9-21 Gy (stepwise increase of 3 Gy). Secretion stimulation was initiated 1, 6, 24, and 48 h after RT. For late-phase analysis, type II cells were isolated 1-5 weeks after 18 Gy whole lung or sham RT. Each experiment was repeated at least fivefold. Flow cytometry was used to determine cell cycle distribution and proliferating cell nuclear antigen index. RESULTS: During the early-phase (in vitro) analysis, we found a normal stimulation of surfactant secretion in irradiated, as well as unirradiated, cells. No change in basal secretion and no dose effect were seen. During the late phase, 1-5 weeks after whole lung RT, we observed enhanced secretory activity for all secretagogues and a small increase in basal secretion in Weeks 3 and 4 (pneumonitis phase) compared with controls. The total number of isolated type II cells, as well as the rate of viable cells, decreased after the second post-RT week. Cell cycle alterations suggesting an irreversible G(2)/M block occurred in the second post-RT week and did not resolve during the observation period. The proliferating cell nuclear antigen index of type II cells from irradiated rats did not differ from that of controls. CONCLUSION: In contrast to literature data, we observed no direct effect of radiation on secretory activity in the early phase after RT. In our study of isolated type II cells, as well as in intact animals, RT did not result in an impaired surfactant secretion up to 5 weeks after RT. Our in vivo experiments even showed an increased response of phosphatidylcholine secretion to all known secretagogues at Weeks 3 and 4 after whole lung RT, possibly due to inflammatory cytokines. Cell cycle alterations with G(2)/M block and cell loss in the late post-RT period may contribute more to the manifestation of radiation-induced lung damage than functional impairment in type II cells.     

BML-210对脑胶质瘤U251细胞增殖、凋亡及细胞周期的影响

目的 探讨组蛋白去乙酰化酶抑制剂BML-210 对脑胶质瘤U251细胞增殖、凋亡及细胞周期的影响。方法 采用0、5、10、20 μmol／L BML-210 处理U251细胞,活细胞计数试剂盒（CCK-8）法检测不同浓度BML-210处理24、48、72和96 h的增殖抑制率,磷酯酰丝氨酸结合蛋白-异硫氢酸荧光素／碘化丙啶双染法（Annexin-FITC／PI）双染法检测不同浓度BML-210处理后的细胞凋亡率,流式细胞仪检测不同浓度BML-210处理48 h后的细胞周期分布情况,免疫印迹检测不同浓度BML-210处理48 h后凋亡相关基因（Bcl-2、Bax和Cleaved caspase-3）蛋白表达。结果 BML-210 可呈剂量和时间依赖的方式抑制U251细胞的增殖（P＜0.05）;除5 μmol／L组的晚期凋亡率外,BML-210处理组的早、晚期凋亡率均高于对照组（P＜0.05）,且BML-210处理组作用48 h后的凋亡率均高于24 h,组间差异均有统计学意义（P＜0.05）;与对照组比较,BML-210作用48 h后U251细胞的Bcl-2水平及S、G2／M期细胞比例降低,Bax和Cleaved caspase-3水平及G0／G1期细胞比例升高,差异均有统计学意义（P＜0.05）。结论 BML-210 可抑制脑胶质瘤U251细胞的增殖并诱导其凋亡和细胞周期阻滞,对脑胶质瘤的辅助治疗有一定价值。     

Cell cycle effects of vinflunine, the most recent promising Vinca alkaloid, and its interaction with radiation, in vitro

Purpose: Vinflunine (VFL) is a novel third generation Vinca alkaloid with superior antitumour activity in preclinical models and an anticipated more favourable toxicity profile compared to the other Vinca alkaloids. Method: We investigate the radiosensitising properties of VFL and its cell cycle effects in four human tumour cell lines (ECV304, MCF-7, H292, and CAL-27). The sulforhodamine B test was used to determine cell survival, and cell cycle analysis was performed by flow cytometry. Radiosensitisation (RS) was represented by dose enhancement factors (DEFs). Results: Twenty-four hours treatment with VFL before radiation caused dose-dependent RS in all cell lines. This was most pronounced in ECV304 cells with RS already at VFL concentrations that reduced cell survival by 10% (IC10). DEFs ranged from 1.57 to 2.29 in the different cell lines. A concentration-dependent G2/M block was observed (starting at 4 h of incubation). After maximal G2/M blockade cells started cycling again, mainly by mitosis, while a small portion of cells started a polyploid cell cycle. Also drug removal immediately caused recycling of cells and induction of a polyploid cell population. The polyploid cell population was most impressively noticeable after prolonged incubation with VFL (48 h), in particular in CAL-27 and ECV304. This was never observed in a tested normal fibroblast cell line (Fi 360). The fate of these cells is of particular interest, but yet uncertain. Conclusion: VFL has radiosensitising potential. The exact role of the cell cycle effects of VFL in its radiosensitising mechanism is still not fully elucidated and requires further study.Orally presented (in part) at the 95th annual meeting of the American Association for Cancer Research (AACR).