Radiation and the cell cycle, revisited

The cell cycle has been inextricably linked to the cellular response to radiation for many years. However, it is only in the past decade that the concept of a coordinated DNA damage response integrating damage recognition, cell cycle checkpoints and DNA repair has begun to be elucidated. The ATM protein is emerging as a key orchestrator of the damage response activating a wide variety of effectors involved in cell cycle arrest and DNA repair to elicit a concerted effort to prevent genome instability caused by unwanted changes in DNA sequence. The key proteins involved in cell cycle checkpoints in different phases of the cell cycle, and their interaction, is a fertile and rapidly developing area of research. This review summarizes the current state of knowledge of cellular checkpoints in response to radiation-induced double-strand breaks in mammalian cells and how this impacts on radiosensitivity.     

塞来昔布对胃癌SGC7901细胞放射敏感性的实验研究

目的:探讨环氧合酶2(COX-2)抑制剂塞来昔布对胃癌SGC7901细胞株的放射增敏作用及其机制。方法:MTT法检测塞来昔布对胃癌SGC7901细胞株的抑制作用,计算出塞来昔布的半数抑制浓度(IC50);克隆形成实验用于检测20%IC50这个浓度的塞来昔布对胃癌SGC细胞是否具有放射敏感性;流式细胞术(FCM)分析细胞周期的分布情况。结果:MTT实验显示塞来昔布对SGC7901细胞株的抑制率随浓度的升高而升高,48 h的IC50是34.38μmol/L;克隆形成实验显示,照射组+药物组与单纯照射组相比,反映放射敏感性指标的存活分数(SF2)、平均致死剂量(D0)及准阈剂量(Dq)均下降,放射增敏比(SER)升高。FCM检测细胞周期G2和M期细胞比例增加,S期细胞比例减少。结论:塞来昔布能增加胃癌SGC7901细胞的放射敏感性,其机制可能与其抑制肿瘤细胞亚致死性损伤修复能力和促进肿瘤细胞周期再分布有关。     

三氧化二砷对纤维肉瘤细胞放射增敏作用的研究

目的 研究和探讨三氧化二砷（As2O3）是否对纤维肉瘤细胞有放射增敏作用。方法以人纤维肉瘤细胞HTl080为实验对象，首先检测As2O3的单药毒性，确定IC10、IC50和IC90。放射增敏作用的实验分为空白对照组、单纯给药组、单纯照射组（包括1、2、4、6、8、10Gy剂量）、照射前加药组（于照射前24h加入设定浓度的As2O3，药物作用24h后进行照射）和照射后加药组（于照射后即刻加入设定浓度的As2O3，药物作用24h）。所有实验均重复3次。采用克隆形成分析法观察单纯照射和照射联合As2O3对细胞的杀伤作用。计算细胞的存活分数，用多靶单击模型进行拟合并做图。结果 HT1080细胞的IC10、IC50和IC90剂量分别为0．57、3．67和12．0μmol／L。无毒剂量的As2O3照射前给药增敏比（SER）为0．86（Do值比）、0．98（SF2值比），照射后给药SER为0．99（Do值比）、1．09（SF2值比）。IC50剂量的As2O3照射前给药SER为0．90（Do值比）、0．87（SF2值比），照射后给药SER为1．14（Do值比）、1．08（SF2值比）。IC90剂量的As2O3照射前给药和照射后给药的SER均为1．14（Do值比）、3．20（SF2值比），As2O3对低剂量照射的放射增敏作用好于高剂量照射（SERSF2〉SERDo）。结论 As2O3对HT1080纤维肉瘤细胞具有一定的放射增敏作用，为临床放疗和As2O3联合应用提供了实验依据。     

Thermal enhancement of oxaliplatin-induced inhibition of cell proliferation and cell cycle progression in human carcinoma cell lines

Hyperthermia is used to treat intraperitoneal colorectal carcinomatosis. In this setting, the molecular effects of oxaliplatin and hyperthermia, in combination and alone, were deciphered in ovarian and colon cancer cells. The combined antiproliferative effects of hyperthermia and oxaliplatin (Eloxatine?;) on human IGROV-1 ovarian carcinoma, Caco-2 and HT-29 colon carcinoma cell lines were investigated by cell viability test, cell cycle analysis and modulation of expression of cell cycle-related proteins. Oxaliplatin inhibited growth of all cell lines in a dose-dependent manner. The efficacy of the drug was markedly enhanced by concurrent exposure to mild heat shock (1?h, 42°C). In IGROV-1 cells, a low concentration (15?µg/ml) of oxaliplatin in combination with hyperthermia induced a transient G2/M arrest. In both colon carcinoma cell lines, a G1/S arrest with a reduction of the G0/G1 population occurred. In IGROV-1 and Caco-2 cells, growth arrest was accompanied by apoptosis as suggested by the appearance of sub-G1 population. Time-course changes of cell cycle regulatory proteins levels revealed accumulation of cyclins A and B as well as of cdc2 and cdk2 upon exposure of IGROV-1 cells to hyperthermia and oxaliplatin. In this cell line, p53 appeared to be implicated in both G2/M arrest and apoptosis. G1/S arrest of HT-29 cells was linked to up-regulation of cyclin E and p27^Kip1 and accumulation of the hypophosphorylated form of pRB, whereas in Caco-2 cells only the hyperphosphorylated form was detected as well as a down-regulation of the proto-oncogene c-myc. Taken together, the results of these in vitro studies suggest that hyperthermia and oxaliplatin might elicit antiproliferative effects by modulating the expression of cell cycle regulatory proteins through different signalling pathways.     

Thermal enhancement of oxaliplatin-induced inhibition of cell proliferation and cell cycle progression in human carcinoma cell lines.

Hyperthermia is used to treat intraperitoneal colorectal carcinomatosis. In this setting, the molecular effects of oxaliplatin and hyperthermia, in combination and alone, were deciphered in ovarian and colon cancer cells. The combined antiproliferative effects of hyperthermia and oxaliplatin (Eloxatine) on human IGROV-1 ovarian carcinoma, Caco-2 and HT-29 colon carcinoma cell lines were investigated by cell viability test, cell cycle analysis and modulation of expression of cell cycle-related proteins. Oxaliplatin inhibited growth of all cell lines in a dose-dependent manner. The efficacy of the drug was markedly enhanced by concurrent exposure to mild heat shock (1 h, 42 degree C). In IGROV-1 cells, a low concentration (15 microg/ml) of oxaliplatin in combination with hyperthermia induced a transient G2/M arrest. In both colon carcinoma cell lines, a G1/S arrest with a reduction of the G0/G1 population occurred. In IGROV-1 and Caco-2 cells, growth arrest was accompanied by apoptosis as suggested by the appearance of sub-G1 population. Time-course changes of cell cycle regulatory proteins levels revealed accumulation of cyclins A and B as well as of cdc2 and cdk2 upon exposure of IGROV-1 cells to hyperthermia and oxaliplatin. In this cell line, p53 appeared to be implicated in both G2/M arrest and apoptosis. G1/S arrest of HT-29 cells was linked to up-regulation of cyclin E and p27(Kip1) and accumulation of the hypophosphorylated form of pRB, whereas in Caco-2 cells only the hyperphosphorylated form was detected as well as a down-regulation of the proto-oncogene c-myc. Taken together, the results of these in vitro studies suggest that hyperthermia and oxaliplatin might elicit antiproliferative effects by modulating the expression of cell cycle regulatory proteins through different signalling pathways.     

三氧化二砷对卵巢癌细胞的放射增强作用

目的：探讨三氧化二砷（As2O3）联合放射肿瘤细胞杀灭的影响，方法：以人卵巢癌细胞SKOV－3为实验对象，用四氮唑盐（MTT）比色法和流式细胞仪磷酯酰丝氨酸Ⅴ／PI双重染色法观察不同浓度As2O3与放射联合和单纯As2O3对SKOV－3的杀伤和诱导凋亡作用。采用成克隆分析法观察5mol/L As2O3的放射增强作用。结果：（1）细胞生长抑制随着As2O3剂量的增加而增强；（2）As2O3＋放射组的细胞存活率均低于单纯As2O3组，但随着As2O3剂量的加大，放射组和未放射组的存活率越来越接近；（3）在2Gy下，随着As2O3剂量的加大，凋亡的比例增加；（4）细胞存活曲线显示，放射＋As2O3组的Dq值小于单纯照射组（1．44Gy，2．78Gy），D0值也小于单纯照射组（0．85Gy，1．30Gy，SF2也小于单纯照射（0．42，0．87），根据D0值求出的增强比为1．53，根据SF2求出的增强比为2．07。结论As2O3在临床应用剂量范围内，与常规分割剂量放射治疗联合时，有望对肿瘤有较好的放射增强作用。     

三氧化二砷对人宫颈癌Hela细胞系的放射增敏作用

目的 探讨三氧化二砷（As2O3）对人宫颈癌Hela细胞系的放射增敏作用。方法 MTT法确定As2O3对Hela细胞的半数致死浓度（LD30），采用集落形成法观察20％该浓度的As2O3对Hela细胞的放射增敏作用。结果（1）细胞生长抑制随As2O3浓度的增加而增强；（2）As2O3＋照射组的细胞存活率低于单纯照射组，耽值小于单纯照射组（1．58Gy、2．11Gy），Dq值也小于单纯照射组（0．27Gv、0．64Gv），存活曲线肩区（Dq）变小，放射增敏比（SER）1．34。结论 As2O3对宫颈癌细胞有明显的放射增敏作用，其作用机理有待进一步研究。     

Radiation clastogenesis and cell cycle checkpoint function as functional markers of breast cancer risk

BACKGROUND: Familial breast cancer is associated with mutations in several genes (BRCA1, BRCA2, p53, ATM) whose protein products protect against radiation-induced genotoxicity. This study tested whether sporadic breast cancer was associated with constitutive radiation hypersensitivity. METHODS: Blood lymphocytes and EBV-transformed lymphoblasts from patients with newly diagnosed breast cancer and controls without cancer were evaluated for ionizing radiation (IR)-induced chromosomal aberrations and cell cycle delays. Lymphoblasts from patients with ataxia telangiectasia (AT) and heterozygous AT carriers were tested as positive controls for radiation hypersensitivity. RESULTS: Lymphoblasts from AT patients and AT carriers displayed G2-irradiation, chromosomal hypersensitivity (GICH). Irradiated G2 phase lymphocytes from breast cancer cases and controls displayed 3-fold inter-individual variation in frequencies of chromatid damage. However, the percentage of breast cancer cases with damage frequencies in excess of 2 SD of the control mean (8/102 or 8%) was not significantly elevated compared to controls (2/48 or 4%, P=0.5). Lymphoblasts sampled 24 h after 3 Gy of IR also varied in the ratios of cells with 4N and 2N DNA content (4N/2N ratio), as a measure of cell cycle checkpoint function. 4N/2N ratios in irradiated lymphoblasts were strongly correlated with the fractions of S phase cells in un-irradiated control cultures (Pearson's correlation coefficient, r=0.87). After normalization to S fraction, the radiation-induced increment in the 4N/2N ratio was significantly elevated in AT lymphoblasts but not in lymphoblasts from AT carriers. The fraction of breast cancer cases with reduced checkpoint function (2/45 or 4%) was equal to the control fraction (2/45 or 4%). For breast cancer cases and controls, GICH in primary lymphocytes was not associated with reduced cell cycle checkpoint function in lymphoblasts. CONCLUSION: Constitutive radiation hypersensitivity in blood lymphocytes and lymphoblasts was not a useful biomarker for identifying women at increased risk of breast cancer.     

Radiation enhancement of radiolabelled antibody deposition in tumors

Recent clinical observations led to the use of external radiation to increase tumor targeting by radiolabelled 131-I antiferritin. Examination of increased uptake of 131-I labelled antiferritin following external radiation was carried out in syngeneic implanted hepatomas (H4IIE, 3924A, 7800, and 7777). Exposure to 10 Gy increased the tumor: liver uptake ratio from 1.55 to 1.86 for H4IIE; from 1.56 to 2.0 for 7800; from 1.34 to 1.97 for 7777; and from 1.05 to 1.19 for 3924A. The pattern of uptake varied among the different tumor types, reflecting their inherent differences in vascularity, tumor permeability, antigen density and growth rate, all of which influence antibody targeting of the tumors. When tumor and liver were irradiated, the tumor showed increased differential uptake of labelled antibody compared to normal liver. 51-Cr labelled erythrocytes were used to study the relative vascularity and blood pooling in H4IIE hepatoma and normal tissue. External radiation to the tumor did not increase the uptake of 51-Cr labelled erythrocytes in any site. These studies provide an insight into the role of external radiation as a modality that increases radiolabelled antibody targeting in hepatoma.     

致瘤病毒编码产物对细胞周期的调控

细胞周期失调是肿瘤形成的重要原因.致瘤病毒通过干扰、模拟、失活许多重要的细胞周期调节蛋白,使宿主细胞周期紊乱,诱导细胞不断增殖,发生癌变.     

Regulation of cell cycle checkpoints by polo-like kinases

Protein kinases play a pivotal role in execution of cell division. Polo and Polo-like kinases have emerged as major regulators for various cell cycle checkpoints. Early genetic studies have demonstrated that CDC5, a budding yeast counterpart of vertebrate Plks, is essential for successful mitotic progression. Mammalian Plks localize primarily to the centrosome during interphase and the mitotic apparatus during mitosis. Many key cell cycle regulators such as p53, Cdc25C, cyclin B, components of the anaphase-promoting complex, and mitotic motor proteins are directly targeted by Plks. Although the exact mechanism of action of these protein kinases in vivo remains to be elucidated, Plks are important mediators for various cell cycle checkpoints that monitor centrosome duplication, DNA replication, formation of bipolar mitotic spindle, segregation of chromosomes, and mitotic exit, thus protecting cells against genetic instability during cell division.     

The mechanism of cell cycle regulation by v-Src

The tyrosine kinase oncoprotein v-Src can overcome the requirements for serum growth factors and anchorage which restrain normal cell growth. Here we investigated the biochemical mechanisms whereby v-Src induces quiescent cells to enter S phase in the absence of serum mitogens. Activating a temperature sensitive v-Src in quiescent cells sequentially induced cyclins D1, E and A and also down regulated p27. We addressed whether p27 down regulation was required to activate cyclin D1/CDK4/6 or cyclin E/CDK2 by engineering cells with inducible p27. Both S phase entry and activation of cyclin/CDKs were inhibited by over expression of p27. Using cells engineered with inducible p16 we showed that Cyclin D/CDK4/6 activity was required for v-Src to increase expression of cyclin A but not cyclin E. To determine which downstream kinases mediated these effects of v-Src we added pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3-K), LY294002 or mitogen activated protein kinase kinase (MEK), U0126. PI3-K was required for v-Src to activate MEK and MEK was required for v-Src to increase expression of cyclins D1 and E. However, the MEK inhibitor prevented p27 protein down regulation whereas the PI3-K inhibitor did not. This was because reduced PI3-K activity lead to proteolytic degradation of p27.     

Regulation of cell cycle duration by c-myc levels

Early passage murine fibroblasts infected with retroviral vectors carrying human c-myc 'minigenes' express high levels of c-myc and have a dramatically shortened G1-phase of the cell cycle. Cells infected with viruses where c-myc is expressed from the viral LTR (MSN-4 virus) express more c-myc protein than cells infected with viruses where c-myc is expressed from the SV40 early promoter (NSM-7 virus). Populations of cells were infected with high titre viruses, selected for drug-resistance, pulse labelled with bromodeoxyuridine (BrdUrd) and chased in BrdUrd free media. This allows accurate, simultaneous, measurement of the rate of exit of unlabelled cells from G1 and progression of BrdUrd-labelled cells through S-phase. The length of the G1-phase in cell populations infected with the MSN-4 virus is 4.65 h, a reduction of nearly 30% compared to the G1-phase length of 6.50 h seen in cells infected with the VSN-2 control virus. Cells infected with NSM-7 virus show an intermediate phenotype and have a G1-phase of 5.25 h. The lengths of the S-phase (4.50 to 4.75 h) and G2 + M phases (2.75 h) were not significantly altered by exogenous c-myc expression. When chases are performed in growth-factor free media, the G1-phase of infected and non-infected cells is extended by approximately 2 h. Cells infected with the c-myc viruses continue to cycle more rapidly than uninfected cells. Growth factor-deprived cells, restimulated with serum, show similar alterations of the cell cycle kinetics. MSN-4 and NSM-7 infected cells, expressing high levels of c-myc, enter S-phase 2 to 4 h earlier, but less synchronously, than control cells, and sustain subsequent rounds of DNA synthesis, while control cells do not. However, cells carrying activated c-myc genes have nearly-normal morphologies and are not tumourgenic in syngenic mice. These results demonstrate that c-myc levels are rate limiting for events in G1, and the length of G1 varies proportionally with the level of exogenous c-myc expression.     

Radiation enhancement by 9-aminocamptothecin: the effect of fractionation and timing of administration.

PURPOSE: The camptothecins (CPTs) are potent radiation sensitizers in vivo but the optimal schedule of administration is unknown. In this article, the effects of irradiation combined with 9-aminocamptothecin (9AC) on a mouse mammary cancer and the gastrointestinal tract were compared for single and fractionated treatment. We also examined the circadian dependency for cytotoxicity, radiation sensitization, and acute toxicity after single doses of 9AC given at six different times over 24 h. MATERIALS AND METHODS: 9AC was administered intramuscularly to C3Hf/Kam mice with and without an 8 mm mouse mammary carcinoma (MCa-4). Acute toxicity was assessed by examination of body weight loss, peripheral blood counts, clinical assessment of diarrhea, and survival. Radiation sensitization was assessed using the tumor regrowth delay model. RESULTS: Regrowth delay of MCa-4 tumor after a single treatment of 15 Gy is comparable to 28 Gy given in 14 fractions (absolute regrowth delays of 7.1 days and 6.6 days, respectively). With 9AC alone, comparable tumor regrowth was obtained with a total dose of 4 mg/kg given intramuscularly repeated twice weekly (1 mg/kg doses X 4), or as a single injection of 4 mg/kg (2.9 days and 3.8 days, respectively). 9-AC and irradiation together in single doses of 15 Gy and 4 mg/kg resulted in little radiation sensitization compared to the repeated 9AC schedule combined with fractionated irradiation [Dose Modifying Factors (DMF) of 1.12 vs. 2.8, respectively]. Acute normal tissue toxicity after single or fractionated 9AC treatment was assessed at six times over a 24-h period (6 A.M., 10 A.M., 2 P.M., 6 P.M., 10 P.M., and 2 A.M.) and was highest at 2 A.M. after either single or multiple doses. A single dose of 9AC administered with single fraction irradiation could be escalated by 33% when given at the best-tolerated time. CONCLUSION: The frequency and timing of CPT administration with irradiation are important factors to be considered in the design of clinical protocols. CPTs are S-phase inhibitors that are better tolerated by the mouse when given during the rest phase when intestinal mucosal proliferation is relatively low. A modest increase in CPT dosage was possible by choosing the best tolerated time to administer the radiation sensitizer. This concept could potentially be evaluated in clinical trials with this class of agents.     

磷烯菌素诱导HL-60细胞凋亡及其与增殖周期的关系

目的　探讨一种新型的DNA拓扑酶 Ⅱ抑制剂磷烯菌素 (FST)对人急性粒细胞白血病细胞株HL 60的杀伤机理。方法　采用流式细胞术 (FCM )、TdT联合FCM及LadderDNA三种方法研究FST对HL 60细胞凋亡的诱导作用及其与增殖周期的关系。结果 :FCM (乙醇抽提法 )显示 :1 0 μmolFST作用 6h未引起HL 60凋亡 ,1 0 0 μmol/L时出现明显“亚G1 峰” ,但1 0 0 0 μmol/L诱导时 ,却不出现凋亡而呈“冰冻”现象。TdT联合FCM表明 :1 0 μmol/LFST作用 6h ,即有明显细胞凋亡 ,且凋亡发生在整个细胞周期。LadderDNA测定提示 :1 0 0 μmol/LFST作用HL 60细胞 6h ,其DNA凝胶电泳出现典型梯状图谱。结论　 1 FST诱导HL 60细胞死亡具有异质性 :低剂量触发凋亡 ,高剂量诱导坏死。 2 TdT联合FCM是一种灵敏的凋亡检测手段 ,它能同时测定细胞DNA含量及断裂 ,故能用以研究细胞凋亡及其与增殖周期的关系 ;3 FST诱导的凋亡似乎没有细胞周期特异性     

Mechanisms of cell cycle arrest by methylseleninic acid.

Methylseleninic acid (MSA) is a monomethylated form of selenium effective in inhibiting cell growth in vitro and experimental mammary carcinogenesis in vivo. MSA offers particular advantage in cell culture experiments because it is stable in solution and provides a monomethylated form of selenium that can be reduced by cellular reducing systems and released nonenzymatically within a cell. In the present study, MSA was used to elucidate the mechanisms of cell growth inhibition by selenium. These studies were performed using a mouse mammary hyperplastic epithelial cell line, TM6. MSA induced a rapid arrest of synchronized cells in the G(1) phase of the cell cycle. This effect was accompanied by a reduction in total cellular levels of cyclin D1. Whereas MSA had no effect on total levels of the cyclin-dependent kinase (CDK)4, the amount of CDK4 immunoprecipitated with cyclin D1 in MSA-treated cells was decreased as was the kinase activity of the immunoprecipitated complex. MSA did not significantly affect cyclin E or associated regulatory molecules. Treatment with MSA suppressed the hyperphosphorylated form of retinoblastoma (Rb) with a commensurate increase in the hypophosphorylated form. Levels of E2F-1 bound to Rb also were elevated. Levels of insulin-like growth factor-I receptor and phosphorylated Akt were reduced by MSA. It is concluded that MSA induces a G(1) arrest in the cell cycle. This effect may be induced by MSA via its modulation of insulin-like growth factor-I-mediated signal transduction leading to inhibition of Akt activation and limitation of cyclin D1-CDK4-mediated phosphorylation of Rb.     

Regulation of normal cell cycle progression by flavin-containing oxidases

Mechanisms underlying the role of reactive oxygen species (ROS) generated by flavin-containing oxidases in regulating cell cycle progression were examined in human and rodent fibroblasts. Incubation of confluent cell cultures with nontoxic/nonclastogenic concentrations of the flavoprotein inhibitor, diphenyleneiodonium (DPI), reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase activity and basal ROS levels, but increased proteolysis of cyclin D1, p21(Waf1) and phospho-p38(MAPK). When these cells were allowed to proliferate by subculture in DPI-free medium, an extensive G(1) delay was observed with concomitant activation of p53/p21(Waf1) signaling and reduced phosphorylation of mitogen-activated kinases. Compensation for decreased oxidant generation by simultaneous exposure to DPI and nontoxic doses of the ROS generators, gamma-radiation or t-butyl-hydroperoxide, attenuated the G(1) delay. Whereas the DPI-induced G(1) checkpoint was completely dependent on PHOX91, ATM and WAF1, it was only partially dependent on P53. Interestingly, G(1) to S progression was not affected when another flavin-containing enzyme, nitric oxide synthase, was inhibited nor was it associated with changes in mitochondrial membrane potential. Proliferating cells treated with DPI also experienced a significant but attenuated delay in G(2). We propose that ATM performs a critical function in mediating normal cellular proliferation that is regulated by nonphagocytic NAD(P)H oxidase enzymes activity, which may serve as a novel target for arresting cancer cells in G(1).