c—myc表达对顺铂诱导人胶质瘤细胞系BT325凋亡的影响

研究c-myc在化疗药物顺铂诱导肿瘤细胞凋亡中的作用。在原有已构建好c-myc反义表达载体pcDNA3-MYC并成功转染BT325细胞的基因上,用顺铂诱导细胞凋亡。采用TUNEL技术并从光镜、电镜、荧光微镜及激光内共聚焦显微镜水平观察BT325细胞凋亡的形态学特征;流式细胞仪检测实验组（即pcDNA3-MYC转染的细胞）及对照组（包括未转染或仅转染空载体pcDNA3的细胞）凋亡率的异同。结果显示：与转染pcDNA3及未转染组的细胞相比,在转染pcDNA3-MYC的BT325细胞中,顺铂诱导的细胞凋亡作用明显受阻,结果表明：肿瘤细胞内异常表达的c-myc在顺铂诱导肿瘤细胞凋亡的过程中有重要作用。     

Genistein增加顺铂诱导的SKOV-3细胞的凋亡与活性氧的关系

目的:探讨Genistein增加顺铂诱导的耐药卵巢癌细胞SKOV-3凋亡的可能作用机制.方法:倒置相差显微镜下观察药物处理后细胞形态学的变化;MTT比色法检测不同药物处理后对SKOV-3细胞增殖的影响;流式细胞仪检测药物处理后细胞的凋亡情况;流式细胞仪和荧光显微镜检测细胞内活性氧(ROS)的水平.结果:10ug/ml的Genistein和2.5ug/ml的顺铂联用24h后,引起了细胞内ROS的增加,细胞的凋亡率也显著增高,与单用顺铂组相比差异有显著性(P＜0.05);用NAC预处理细胞2h后,有效抑制了ROS的产生,并增加了细胞的活性,降低了细胞的凋亡率,与未加NAC组相比差异有显著性(P＜0.05).结论:Genistein增加顺铂诱导的耐药卵巢癌细胞SKOV一3的凋亡与细胞内ROS水平的升高有关,这可能是Genistein增加顺铂诱导的耐药卵巢癌细胞SKOV-3凋亡的作用机制之一.     

外周血淋巴细胞凋亡依赖于细胞周期运行

目的以培养的人类正常外周血淋巴细胞（peripheral blood lymphocytes,PBLs）为模型,模拟细胞周期运行的不同状态,研究细胞凋亡与细胞周期运行的关系。方法将PHA及咖啡因处理前后的PBLs,分别用喜树碱（CPT）、X-射线、抗Fas抗体和肿瘤坏死因子TNF-α诱导,流式细胞术方法对细胞凋亡进行定量检测。结果PBLs经PHA刺激后进入细胞周期并发生凋亡,在凋亡因子打击作用下凋亡明显增加,用咖啡因废除细胞周期检测点后对凋亡打击敏感性降低。未经PHA刺激的PBLs对凋亡打击无应答。结论细胞进入细胞周期总是伴随细胞凋亡的发生,细胞不进入周期,则不会发生凋亡,细胞周期不停止,也不会凋亡。细胞周期检测点是联结细胞周期和细胞凋亡的重要位点。     

三苯氧胺联合顺铂及紫杉醇抑制MCF-7细胞增殖的研究

目的:研究三苯氧胺联合不同浓度的紫杉醇及顺铂对MCF-7细胞株增殖的影响.方法:流式细胞仪检测IC30及IC10两种不同剂量紫杉醇及顺铂与2×10-8MOL/L三苯氧胺联用对MCF-7细胞周期的影响,MTT法测定不同方法处理后MCF-7细胞的倍增时间.Annexin Ⅴ/PI双标法检测细胞凋亡.结果:未经处理的MCF-7细胞株G2/M期比例分别为14.2±11.4%,细胞倍增时间分别为24±4小时,凋亡率2±1.2%,死亡细胞比例1±0.5%;2×10-8MOL/L三苯氧胺与IC10剂量强度的紫杉醇+顺铂联用(L3-10)与IC10剂量的紫杉醇+顺铂(L2-10)组比较,处理后MCF-7细胞G2/M期比例分别为48.2±6.4%、47.3±8.6%,细胞倍增时间分别为42±5小时及41±5小时,凋亡率5±1.7%及6.8±2.6%.死亡细胞比例4±2.3%、2±1.8%.2×10-8MOL/L三苯氧胺与IC30剂量强度的紫杉醇+顺铂联用(L3-30)与IC30剂量强度的紫杉醇+顺铂(L2-30)比较,处理后MCF-7细胞G2/M期比例分别为50.9±8.1%、56.4±8.9%,细胞倍增时间分别为65±12小时及66±9小时,凋亡率13±3.6%及13±4.3%,死亡细胞比例4±3.0%、5±2.9%.细胞倍增时间及G2/M期比例与未处理组比较差别有统计学意义(p<0.05),但不同剂量强度的紫杉醇+顺铂组与相应联用2×10-8MOL/L三苯氧胺组比较细胞倍增时间及G2/M期比例均无统计学意义(p>0.05.结论:不同剂量顺铂、紫杉醇联合及二者与2×10-8MOL/>三苯氧胺联合均导致MCF-7细胞G2/M期阻滞及明显延长细胞倍增时间,但2×10-8MOL/L三苯氧胺与不同剂量紫杉醇+顺铂联用对抑制MCF-7的增殖抑制无明显的协同作用.     

姜黄素联合顺铂对人骨肉瘤细胞增殖和凋亡的影响

目的:观察姜黄素联合顺铂对人骨肉瘤细胞MG-63增殖和凋亡的影响。方法:采用不同浓度的姜黄素、顺铂和姜黄素联合顺铂处理人骨肉瘤细胞MG-63不同时间,通过MTT法检测其对MG-63细胞生长的抑制作用;平板克隆实验检测其对MG-63细胞克隆形成能力的影响;流式细胞仪检测其对MG-63细胞凋亡的影响。结果:单用姜黄素或顺铂均可以时间和浓度依赖性方式抑制骨肉瘤细胞MG-63的增殖。与空白对照组相比,单用10μmol/L姜黄素和2、4μmol/L顺铂可抑制MG-63细胞的增殖,降低其克隆形成率并提高细胞凋亡率(P<0.05);而与姜黄素和顺铂单用处理相比,10μmol/L姜黄素分别与2、4μmol/L顺铂联合应用,可更显著抑制MG-63细胞的增殖,降低其克隆形成率并提高细胞凋亡率(P<0.01)。结论:姜黄素联合顺铂与单药相比能够显著抑制人骨肉瘤细胞MG-63细胞的增值,促进其凋亡,两药对骨肉瘤细胞的杀伤效应具有一定的协同性。     

单宁酸联合顺铂增强肝癌HepG2细胞IRE1-XBP1通路的激活水平

本文主要研究单宁酸(TA)联合顺铂(CDDP)对肝癌HepG2细胞内质网应激IRE1-XBP1通路的影响。用180μM单宁酸、0.9μg/m L顺铂单独用药或者联合用药处理肝癌HepG2细胞24 h或48 h后,应用流式细胞技术测定HepG2细胞的凋亡率,实时荧光定量PCR技术(q-RT-PCR)、蛋白免疫印迹(western blot)技术检测IRE1α和XBP-1分子的表达水平。MTT结果显示,单宁酸和顺铂均能显著抑制HepG2细胞的生长,且均呈剂量性依赖;二者联合用药能够显著增加HepG2细胞的生长抑制率;流式细胞术结果显示,单宁酸与顺铂联合用药能够显著抑制HepG2细胞的增殖,并诱导细胞凋亡的发生;q-RT-PCR及Western blot结果显示,单宁酸与顺铂联合用药能显著上调细胞IRE1α和XBP-1的表达水平。结果表明单宁酸能够联合顺铂增强肝癌HepG2细胞内质网应激IRE1-XBP1通路的激活水平,提示IRE1-XBP1通路可能是单宁酸和顺铂协同抗肝癌HepG2细胞的分子机制之一。     

c-myc表达对顺铂诱导人胶质瘤细胞系BT_(325)凋亡的影响

研究 c- m yc在化疗药物顺铂诱导肿瘤细胞凋亡中的作用。在原有已构建好 c- myc反义表达载体 pc DNA3- MYC并成功转染 BT32 5 细胞的基础上 ,用顺铂诱导细胞凋亡。采用 TUNEL技术并从光镜、电镜、荧光显微镜及激光共聚焦显微镜水平观察 BT32 5 细胞凋亡的形态学特征 ;流式细胞仪检测实验组 (即 pc DNA3- MYC转染的细胞 )及对照组 (包括未转染或仅转染空载体 pc DNA 3的细胞 )凋亡率的异同。结果显示 :与转染空载体 pc DNA3及未转染组的细胞相比 ,在转染 pc DNA 3-MYC的 BT32 5 细胞中 ,顺铂诱导的细胞凋亡作用明显受阻 ,结果表明 :肿瘤细胞内异常表达的 c- myc在顺铂诱导肿瘤细胞凋亡的过程中有重要作用。     

顺铂诱导非洲绿猴肾细胞凋亡模型的建立

目的:建立常见凋亡诱导剂顺铂(Cisplatin)诱导非洲绿猴肾细胞(Vero)凋亡的模型,为进一步研究抗凋亡基因在细胞凋亡中的分子机理打下基础。方法:分别以不同浓度的顺铂处理Vero细胞48h,用噻唑蓝(MTT)比色法、Gimesa染色、流式细胞术检测,观察处理后细胞的生长活力和凋亡情况。结果:以未处理的细胞作为对照组,1、2、3、4、5μg/mL的顺铂处理的Vero细胞生存率分别为(79.02±6.10)%、(68.84±4.42)%、(56.66±4.07)%、(46.83±3.76)%、(29.04±5.93)%(P<0.01);经顺铂诱导后细胞形态学发生明显改变,出现膜小泡和凋亡小体形成等凋亡细胞特征;流式细胞仪检测,0、1、2、3、4、5μg/mL的顺铂处理的Vero细胞后凋亡率分别为1.66%±0.19%、16.65%±1.26%、24.82%±1.03%、36.22%±1.04%、48.49%±1.24%、43.34%±1.17%(P<0.01)。结论:本实验成功建立顺铂诱导非洲绿猴肾细胞凋亡模型,将有助于进一步探讨目的基因在Vero细胞凋亡作用的的分子机制。     

纳秒脉冲电场对人卵巢癌顺铂敏感及耐药细胞的凋亡诱导效应及机制研究

利用纳秒脉冲电场(nanosecond pulsed electric fields,ns PEFs)治疗肿瘤具有潜在优势。该研究探讨了纳秒脉冲电场对体外培养的人卵巢癌顺铂敏感细胞COC1及顺铂耐药细胞COC1/DDP的生长抑制、凋亡诱导效应及其机制。CCK-8法分析显示,纳秒脉冲电场降低COC1及COC1/DDP细胞存活率,而且抑制效应呈明显时间依赖效应。流式细胞术检测结果结合Hoechst 33342荧光染色结果表明,纳秒脉冲电场可诱导COC1及COC1/DDP细胞发生凋亡。JC-1荧光染色及caspase-3蛋白酶活性检测表明,纳秒脉冲电场处理可引起COC1及COC1/DDP细胞线粒体膜电位降低及caspase-3活性增强。流式细胞术检测细胞周期显示,纳秒脉冲电场可将COC1细胞周期阻滞于S期。以上研究结果揭示,纳秒脉冲电场可以抑制COC1及COC1/DDP细胞增殖,诱导细胞凋亡,线粒体途径凋亡为其可能机制。     

LRP16影响宫颈癌Siha细胞对化疗药物的敏感性

目的:探讨抑制LRP16的表达对宫颈癌Siha细胞的化疗药物敏感性的影响。方法:将抑制LRP16表达的小干扰RNA:negativecontrol-si RNA(NC)、si RNA-374(si374)转染入Siha宫颈鳞癌细胞系中,通过顺铂(DDP)和紫杉醇(TAX)的处理后,采用CCK-8检测不同浓度紫杉醇、顺铂作用宫颈癌细胞系Siha48 h后,计算出细胞被抑制一半时顺铂、紫杉醇的药物浓度(IC50);使用Hoechst33342染色观察细胞凋亡,采用流式细胞仪检测顺铂IC50作用Siha细胞48小时后的细胞凋亡情况,紫杉醇IC50作用Siha细胞之后的细胞周期分布情况。结果:CCK-8检测转染的Siha细胞增殖活性受到抑制,Hoechst33342染色观察转染的Siha细胞凋亡明显增加,流式细胞仪检测凋亡显示,si374+顺铂的早期凋亡率22.15±2.24,NC+顺铂12.45±2.72,流式细胞仪检测周期显示G2/M(%),si374+紫杉醇29.94±1.87,NC+紫杉醇17.66±2.32。结论:LRP16基因表达下调之后,抑制Siha细胞的增殖、促进其凋亡,使细胞周期滞留于G2/M期,从而提高Siha细胞的化疗敏感性。     

Caffeine promotes apoptosis in mitotic spindle checkpoint-arrested cells

The spindle assembly checkpoint arrests cells in mitosis when defects in mitotic spindle assembly or partitioning of the replicated genome are detected. This checkpoint blocks exit from mitosis until the defect is rectified or the cell initiates apoptosis. In this study we have used caffeine to identify components of the mechanism that signals apoptosis in mitotic checkpoint-arrested cells. Addition of caffeine to spindle checkpoint-arrested cells induced >40% apoptosis within 5 h. It also caused proteasome-mediated destruction of cyclin B1, a corresponding reduction in cyclin B1/cdk1 activity, and reduction in MPM-2 reactivity. However, cells retained MAD2 staining at the kinetochores, an indication of continued spindle checkpoint function. Blocking proteasome activity did not block apoptosis, but continued spindle checkpoint function was essential for apoptosis. After systematically eliminating all known targets, we have identified p21-activated kinase PAK1, which has an anti-apoptotic function in spindle checkpoint-arrested cells, as a target for caffeine inhibition. Knockdown of PAK1 also increased apoptosis in spindle checkpoint-arrested cells. This study demonstrates that the spindle checkpoint not only regulates mitotic exit but apoptosis in mitosis through the activity of PAK1.     

Activation of the G2 cell cycle checkpoint enhances survival of epithelial cells exposed to hyperoxia

Reactive oxygen species produced during hyperoxia damage DNA, inhibit proliferation in G1- through p53-dependent activation of p21(Cip1/WAF1/Sdi1), and kill cells. Because checkpoint activation protects cells from genotoxic stress, we investigated cell proliferation and survival of the murine type II epithelial cell line MLE15 during hyperoxia. These cells were chosen for study because they express Simian large and small-T antigens, which transform cells in part by disrupting the p53-dependent G1 checkpoint. Cell counts, 5-bromo-2'-deoxyuridine labeling, and flow cytometry revealed that hyperoxia slowed cell cycle progression after one replication, resulting in a pronounced G2 arrest by 72 h. Addition of caffeine, which inactivates the G2 checkpoint, diminished the percentage of hyperoxic cells in G2 and increased the percentage in sub-G1 and G1. Abrogation of the G2 checkpoint was associated with enhanced oxygen-induced DNA strand breaks and cell death. Caffeine did not affect DNA integrity or viability of cells exposed to room air. Similarly, caffeine abrogated the G2 checkpoint in hyperoxic A549 epithelial cells and enhanced oxygen-induced toxicity. These data indicate that hyperoxia rapidly inhibits proliferation after one cell cycle and that the G2 checkpoint is critical for limiting DNA damage and cell death.     

Centrosome misorientation mediates slowing of the cell cycle under limited nutrient conditions in Drosophila male germline stem cells

Drosophila male germline stem cells (GSCs) divide asymmetrically, balancing self-renewal and differentiation. Although asymmetric stem cell division balances between self-renewal and differentiation, it does not dictate how frequently differentiating cells must be produced. In male GSCs, asymmetric GSC division is achieved by stereotyped positioning of the centrosome with respect to the stem cell niche. Recently we showed that the centrosome orientation checkpoint monitors the correct centrosome orientation to ensure an asymmetric outcome of the GSC division. When GSC centrosomes are not correctly oriented with respect to the niche, GSC cell cycle is arrested/delayed until the correct centrosome orientation is reacquired. Here we show that induction of centrosome misorientation upon culture in poor nutrient conditions mediates slowing of GSC cell proliferation via activation of the centrosome orientation checkpoint. Consistently, inactivation of the centrosome orientation checkpoint leads to lack of cell cycle slowdown even under poor nutrient conditions. We propose that centrosome misorientation serves as a mediator that transduces nutrient information into stem cell proliferation, providing a previously unappreciated mechanism of stem cell regulation in response to nutrient conditions.     

G2/M-phase arrest and death by apoptosis of HL60 cells irradiated with exponentially decreasing low-dose-rate gamma radiation.

Cells of the TP53-deficient human leukemia cell line HL60 continue to progress throughout the cell cycle and arrest in the G2/M phase during protracted exposure to exponentially decreasing low-dose-rate radiation. We have hypothesized that G2/M-phase arrest contributes to the extent of radiation-induced cell death by apoptosis as well as to overall cell killing. To test this hypothesis, we used caffeine and nocodazole to alter the duration of G2/M-phase arrest of HL60 cells exposed to exponentially decreasing low-dose-rate irradiation and measured the activity of G2/M-phase checkpoint proteins, redistribution of cells in the phases of the cell cycle, cell death by apoptosis, and overall survival after irradiation. The results from these experiments demonstrate that concomitant exposure of HL60 cells to caffeine (2 mM) during irradiation inhibited radiation-induced tyrosine 15 phosphorylation of the G2/M-phase transition checkpoint protein CDC2/p34 kinase and reduced G2/M-phase arrest by 40-46% compared to cells irradiated without caffeine. Radiation-induced apoptosis also decreased by 36-50% in cells treated with caffeine and radiation compared to cells treated with radiation alone. Radiation survival was significantly increased by exposure to caffeine. In contrast, prolongation of G2/M-phase arrest by pre-incubation with nocodazole enhanced radiation-induced apoptosis and overall radiation-induced cell killing. To further study the role of cell death by apoptosis in the response to exponentially decreasing low-dose-rate irradiation, HL60 cells were transfected with the BCL2 proto-oncogene. The extent of G2/M-phase arrest was similar for parental, neomycin-transfected control and BCL2-transfected cells during and after exponentially decreasing low-dose-rate irradiation. However, there were significant differences (P < 0.01) in the extent of radiation-induced apoptosis of parental and neomycin- and BCL2-transfected cells after irradiation, with significantly less radiation-induced apoptosis and higher overall survival in BCL2-transfected cells than similarly irradiated control cells. These data demonstrate that radiation-induced G2/M-phase arrest and subsequent induction of apoptosis play an important role in the response of HL60 cells to low-dose-rate irradiation and suggest that it may be possible to increase radiation-induced apoptosis by altering the extent of G2/M-phase arrest. These findings are clinically relevant and suggest a novel therapeutic strategy for increasing the efficacy of brachytherapy and radioimmunotherapy.     

Cisplatin DNA cross-links do not inhibit S-phase and cause only a G2/M arrest in Saccharomyces cerevisiae.

Cisplatin (CDDP) has been used as a DNA cross-linking agent to evaluate whether there is a specific cell cycle checkpoint response to such damage in Saccharomyces cerevisiae (S. cerevisiae). Fluorescent-activated cell sorting (FACS) analysis showed only a G2/M checkpoint, normal exit from G1 and progression through S-phase following alpha-factor arrest and CDDP treatment. Of the checkpoint mutants tested, rad9, rad17 and rad24, did not show increased sensitivity to CDDP compared to isogenic wild-type cells. However, other checkpoint mutants tested (mec1, mec3 and rad53) showed increased sensitivity to CDDP, as did controls with a defect in excision repair (rad1 and rad14) or a defect in recombination (rad51 and rad52). Thus, by survival and cell cycle kinetics, it appears that DNA cross-links do not inhibit entry into S-phase or slow DNA replication and that replication continues after cisplatin treatment in yeast.     

Caffeine inhibits checkpoint responses without inhibiting the ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases

The ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) kinases regulate cell cycle checkpoints by phosphorylating multiple substrates including the CHK1 and -2 protein kinases and p53. Caffeine has been widely used to study ATM and ATR signaling because it inhibits these kinases in vitro and overcomes cell cycle checkpoint responses in vivo. Thus, caffeine has been thought to overcome the checkpoint through its ability to prevent phosphorylation of ATM and ATR substrates. Surprisingly, I have found that multiple ATM-ATR substrates including CHK1 and -2 are hyperphosphorylated in cells treated with caffeine and genotoxic agents such as hydroxyurea or ionizing radiation. ATM autophosphorylation in cells is also increased when caffeine is used in combination with inhibitors of replication suggesting that ATM activity is not inhibited in vivo by caffeine. Furthermore, CHK1 hyperphosphorylation induced by caffeine in combination with hydroxyurea is ATR-dependent suggesting that ATR activity is stimulated by caffeine. Finally, the G2/M checkpoint in response to ionizing radiation or hydroxyurea is abrogated by caffeine treatment without a corresponding decrease in ATM-ATR-dependent signaling. This data suggests that although caffeine is an inhibitor of ATM-ATR kinase activity in vitro, it can block checkpoints without inhibiting ATM-ATR activation in vivo.     

Poly(ADP-ribose) polymerase inhibition enhances p53-dependent and -independent DNA damage responses induced by DNA damaging agent

Targeting DNA repair with poly(ADP-ribose) polymerase (PARP) inhibitors has shown a broad range of anti-tumor activity in patients with advanced malignancies with and without BRCA deficiency. It remains unclear what role p53 plays in response to PARP inhibition in BRCA-proficient cancer cells treated with DNA damaging agents. Using gene expression microarray analysis, we find that DNA damage response (DDR) pathways elicited by veliparib (ABT-888), a PARP inhibitor, plus topotecan comprise the G1/S checkpoint, ATM, and p53 signaling pathways in p53-wildtype cancer cell lines and BRCA1, BRCA2 and ATR pathway in p53-mutant lines. In contrast, topotecan alone induces the G1/S checkpoint pathway in p53-wildtype lines and not in p53-mutant cells. These responses are coupled with G2/G1 checkpoint effectors p21CDKN1A upregulation, and Chk1 and Chk2 activation. The drug combination enhances G2 cell cycle arrest, apoptosis and a marked increase in cell death relative to topotecan alone in p53-wildtype and p53-mutant or -null cells. We also show that the checkpoint kinase inhibitor UCN-01 abolishes the G2 arrest induced by the veliparib and topotecan combination and further increases cell death in both p53-wildtype and -mutant cells. Collectively, PARP inhibition by veliparib enhances DDR and cell death in BRCA-proficient cancer cells in a p53-dependent and -independent fashion. Abrogating the cell-cycle arrest induced by PARP inhibition plus chemotherapeutics may be a strategy in the treatment of BRCA-proficient cancer.     

Regulating mammalian checkpoints through Cdc25 inactivation

Precise monitoring of DNA replication and chromosome segregation ensures that there is accurate transmission of genetic information from a cell to its daughters. Eukaryotic cells have developed a complex network of checkpoint pathways that sense DNA lesions and defects in chromosome segregation, spindle assembly and the centrosome cycle, leading to an inhibition of cell-cycle progression for the time required to remove the defect and thus preventing genomic instability. The activation of checkpoints that are responsive to DNA damage or incomplete DNA replication ultimately results in the inhibition of cyclin-dependent kinases. This review focuses on our understanding of the biochemical mechanisms that specifically inactivate Cdc25 (cell division cycle 25) phosphatases to achieve this. The evidence for links between checkpoint deregulation and oncogenesis is discussed.     

Cell-cycle fate-monitoring distinguishes individual chemosensitive and chemoresistant cancer cells in drug-treated heterogeneous populations demonstrated by real-time FUCCI imaging

Essentially every population of cancer cells within a tumor is heterogeneous, especially with regard to chemosensitivity and resistance. In the present study, we utilized the fluorescence ubiquitination-based cell cycle indicator (FUCCI) imaging system to investigate the correlation between cell-cycle behavior and apoptosis after treatment of cancer cells with chemotherapeutic drugs. HeLa cells expressing FUCCI were treated with doxorubicin (DOX) (5 μM) or cisplatinum (CDDP) (5 μM) for 3 h. Cell-cycle progression and apoptosis were monitored by time-lapse FUCCI imaging for 72 h. Time-lapse FUCCI imaging demonstrated that both DOX and CDDP could induce cell cycle arrest in S/G₂/M in almost all the cells, but a subpopulation of the cells could escape the block and undergo mitosis. The subpopulation which went through mitosis subsequently underwent apoptosis, while the cells arrested in S/G₂/M survived. The present results demonstrate that chemoresistant cells can be readily identified in a heterogeneous population of cancer cells by S/G₂/M arrest, which can serve in future studies as a visible target for novel agents that kill cell-cycle-arrested cells.