HMBA对人舌癌Tca8113细胞增殖和KLF6及相关蛋白表达的影响

目的: 研究六亚甲基二乙酰胺(hexamethylene bisacetamide, HMBA)对人舌癌Tca8113细胞增殖和Kruppel样因子6(Kruppel-like factor 6, KLF6)及相关蛋白表达的影响。方法: HMBA处理Tca8113细胞后,观察细胞生长和细胞形态;流式细胞术观察细胞周期;RT-PCR检测KLF6 mRNA的表达;免疫细胞化学技术检测KLF6、p53、cyclin D1及c-Jun的蛋白表达。结果: HMBA处理后贴壁细胞的数量随浓度增加明显减少;MTT实验显示其生长抑制作用呈浓度/时间-效应关系;镜下观察显示处理后Tca8113细胞出现向正常细胞形态逆转演变的特征。流式细胞术检测结果显示,对照组Tca8113细胞G₁期比例为(52.00±0.02)%,S期为(34.00±0.08)%,G期为(14.00±0.10)%;HMBA处理后,随作用时间增加,G₁期细胞显著增加,S期细胞显著减少,G₂期细胞也减少,细胞明显被阻滞于G₁期(P<0.05);出现凋亡峰。 RT-PCR结果显示HMBA处理后KLF6 mRNA的表达上调(P<0.05); 免疫组化检测显示HMBA处理后KLF6和p53蛋白表达上调(P<0.05),cyclin D1和c-Jun表达下调(P<0.05)。结论: HMBA对Tca8113细胞增殖具有抑制作用;其作用机制一方面可能通过p53上调p21的表达和活性,另一方面可能通过上调表达的KLF6解除c-Jun对p21的抑制作用,下调cyclin D1,抑制CDK4/6和cyclin D1复合物活性,将Tca8113细胞阻滞于G₀/G₁期,诱导Tca8113细胞向正常细胞分化,恢复正常细胞的表型和功能,并促进细胞凋亡。     

洛伐他汀诱导人子宫内膜癌JEC细胞G1 期同步化的研究

目的: 探讨洛伐他汀(Lov)诱导人子宫内膜癌JEC细胞G₁期同步化的方法及JEC细胞解除G₁期同步化后的细胞周期进程。方法: CCK-8法测定JEC细胞的倍增时间,采用10 μmol/L、20 μmol/L、30 μmol/L和40 μmol/L的Lov,作用1×细胞倍增时间进行流式细胞术检测G₁期细胞的比例,获得G₁期同步化的最佳Lov浓度;采用最佳浓度作用JEC细胞,于作用后的0.5×倍增时间~2×倍增时间内,每隔4 h检测同步化程度,获得最佳浓度Lov作用下的最佳同步化时间;解除G₁期同步化,每隔4 h检测一次,研究JEC细胞解除G₁期同步化后的细胞周期进程。结果: CCK-8法检测显示JEC细胞的倍增时间约为24 h;JEC细胞的最佳G₁期同步化条件为:20 μmol/L Lov作用24 h,可获取G₁期细胞(85.00±0.79)%;JEC细胞于解除G₁期同步化后16 h,获得最大量的S期细胞,为(45.28±0.53)%;同时G₁期细胞比例达到最低,为(35.93±0.44)%。结论: 20 μmol/L Lov作用24 h,可获得大量G₁期细胞;解除G₁期同步化后16 h,S期细胞比例最高,G₁期细胞比例达到最低,达到满意的G₁期同步化释放后效果。     

辛伐他汀抑制胎牛血清及PDGF-BB诱导的血管平滑肌细胞增殖及对抑癌基因PTEN表达的影响

目的:观察辛伐他汀(simvastatin)对血清及血小板源生长因子(PDGF-BB)诱导的血管平滑肌细胞(VSMCs)增殖的抑制作用及simvastatin对细胞周期和G₁/S周期转换重要调节基因PTEN蛋白表达的影响。方法:[³H]-胸腺嘧啶核苷酸掺入测定VSMCsDNA合成,流式细胞仪检测细胞周期情况,Western印迹杂交法检测PTEN蛋白表达。结果:Simvastatin以剂量依赖关系抑制血清及PDGF-BB诱导的VSMCs[³H]-胸腺嘧啶核苷酸掺入,使G₀/G₁期细胞比例明显增多,S期细胞比例显著减少,并上调PTEN蛋白表达,而3羟3甲戊二酰辅酶A代谢产物甲羟戊酸能抑制simvastatin上调PTEN的表达。结论:Simvastatin抑制血清及PDGF-BB诱导的VSMCs增殖阻滞细胞周期可能与上调PTEN表达有关,且simvastatin调节PTEN的表达可能通过抑制甲羟戊酸的合成而实现。     

蝎毒多肽对肿瘤细胞的抑制作用研究

目的:观察蝎毒多肽(PBMV)对肿瘤细胞生长、细胞周期、P53表达和膜电位的影响。方法:MTT法、集落形成、细胞分裂指数、流式细胞仪和玻璃微电极细胞内记录法。结果:PBMV对数种肿瘤细胞具有毒性作用,细胞生长抑制、集落形成能力和分裂指数降低、多核细胞数减少;PBMV使进入S期的CNE-2Z细胞明显减少,G₁、S和G₂期细胞的百分比增多(P<0.01);PBMV处理后,CNE-2Z细胞P53蛋白表达量降低(P<0.01),细胞膜负电位明显减小,胞膜呈去极化状态。结论:PBMV可能具有膜毒素的作用,通过使CNE-2Z细胞膜电位降低,影响肿瘤细胞的生长增殖。     

人Gax基因转染对血管平滑肌细胞增殖、迁移和细胞周期的影响

目的: 探讨腺病毒介导人生长终止特异性同源异型盒基因(hGax基因)转染对血清刺激后兔血管平滑肌细胞(VSMCs)增殖、迁移和细胞周期的影响,为静脉桥再狭窄基因治疗新策略提供帮助。方法: 构建含 hGax 的重组腺病毒载体并感染兔VSMCs;应用RT-PCR、免疫荧光染色检测hGax mRNA和蛋白的表达; MTT法观察hGax基因过表达对血清刺激后VSMCs增殖的影响;划痕法测定细胞迁移;流式分析细胞周期。结果: 成功构建Ad5-hGax重组腺病毒载体。转染48 h后 RT-PCR和免疫荧光染色法示转染 hGax 基因的VSMCs中存在目的基因特异性片段(174 bp)和目的蛋白表达;MTT法示hGax蛋白显著抑制血清刺激后VSMCs增殖;划痕法示hGax基因过表达明显抑制血清刺激后VSMCs迁移;流式结果示血清刺激72 h后,Ad5-hGax转染组G₀/G₁期细胞比例明显增加,G₂/M+S期细胞减少。结论: 含hGax的重组腺病毒载体构建成功,并在细胞中过表达。hGax基因过表达能有效抑制血清刺激后兔VSMCs的增殖和迁移,并使细胞停滞于G₀/G₁期。     

亚硒酸钠对子宫内膜癌细胞增殖能力的影响

目的: 研究亚硒酸钠(Na₂SeO₃)对子宫内膜癌细胞增殖能力的影响,并探讨其作用机制。方法: 用Na₂SeO₃作用于子宫内膜癌Ishikawa细胞和HEC-1A细胞,MTT法检测细胞增殖能力,流式细胞法测定Na₂SeO₃作用后细胞周期的变化及凋亡情况,Western blotting检测周期蛋白cyclin A的表达。结果: Na₂SeO₃对子宫内膜癌Ishikawa细胞和HEC-1A细胞的增殖均有抑制作用,抑制率在一定浓度范围内与Na₂SeO₃浓度呈正相关,对Ishikawa细胞作用48 h的IC₅₀为3.26 μmol/L,对HEC-1A细胞作用48 h的IC₅₀为4.77 μmol/L。Na₂SeO₃作用后两种细胞G₀/G₁期减少,S期细胞增多,G₂/M期细胞有所增加。作用48 h后两种细胞凋亡率增加。Na₂SeO₃使子宫内膜癌Ishikawa细胞和HEC-1A细胞的cyclin A表达增加。结论: 亚硒酸钠对子宫内膜癌Ishikawa细胞和HEC-1A细胞增殖有抑制作用,其作用机制与上调cyclin A表达,引起细胞周期阻滞和诱导凋亡有关。     

胰岛素对自发型高血压大鼠血管平滑肌细胞TGF β及其受体的调节作用

目的:从细胞增生、转化生长因子β₁和受体表达等方面,探讨胰岛素对自发型高血压大鼠血管平滑肌细胞(SHR VSMC)和正常血压Wista-Kyoto大鼠血管平滑肌细胞(WKY VSMC)的影响异同。方法:采用组织移植法培养SHR和WKY大鼠胸主动脉VSMC|用细胞计数仪和流式细胞仪分别检测细胞增生情况|TGF β及其受体的mRNA水平利用定量RT-PCR技术进行检测。结果:(1)胰岛素加强SHR VSMC的增生,而不影响WKY VSMC的增生。胰岛素加强SHR VSMC的增生,且呈剂量依赖方式(2)以20%小牛血清培养基培养VSMC, SHR VSMC的S期百分率为31.44%,而WKY VSMC的S期百分率仅为19.86%|以2%小牛血清培养基培养VSMC,SHR VSMC的G₀／G₁和S期百分率分别为73.23%和9.35%,加入胰岛素后,SHR VSMC的G₀／G₁降低为67.58%,S期百分率则增加到15.64%。但是,加入胰岛素前后,WKY VSMC各期百分率无明显变化|(3)RT-PCR分析结果表明,生长静止的SHR VSMC和WKY VSMC均有TGF β₁、Ⅰ型和Ⅱ型TGF β受体的表达,但SHR VSMC的TGFβ₁、Ⅰ型TGF β受体的表达量高于WKY VSMC的TGF β₁、Ⅰ型TGF β受体的表达量,胰岛素加强了WKY VSMC的TGF β₁、Ⅰ型TGF β受体的表达(P＜0.01和P＜0.05),而削弱了SHR VSMC相应分子的表达(P＜0.01和P＜0.05),胰岛素不影响二株系大鼠VSMC Ⅱ型TGF β受体的表达(P＜0.05)。结论:胰岛素对SHR VSMC和WKY VSMC的TGF β和它的受体表达具有不同的调节作用,这种异常调节作用可能和胰岛素加强SHR VSMC的增生密切相关。     

c-sis反义寡核苷酸对肺血管平滑肌细胞增殖和细胞周期的影响

目的:探讨从血小板源生长因子-B链(PDGF-B)的基因水平阻断对肺血管平滑肌细胞增殖的影响。方法:给肺动脉平滑肌细胞(VSMC)分别施加不同剂量的c-sis癌基因反义寡核苷酸(ASON), 通过四甲基偶氮唑蓝(MTT)法观察细胞不同时相的增殖曲线;采用流式细胞术观察不同干预条件下VSMC细胞周期的变化。结果:中、大浓度ASON对细胞增殖有明显抑制作用。在中浓度的ASON作用下, 加入PDGF-BB能促进VSMC的增殖。ASON干预前后, VSMC增殖周期中各期细胞构成发生显著的变化, 以G₀＋G₁期细胞增多、S+G₂＋M期细胞减少为特征。各组的G₀＋G₁期细胞均显著多于对照组(P＜0.05)。小剂量与中剂量、中剂量与大剂量间G₀＋G₁期细胞有显著差异(P＜0.05)。结论:中、大剂量ASON能明显抑制肺VSMC的增殖, 可使G₀＋G₁期细胞数明显增多, 且与ASON有剂量依赖关系。     

HIF-1α基因沉默对大鼠肝癌CBRH-7919细胞p27和Ki67表达的影响

目的: 探讨沉默缺氧诱导因子1α(hypoxia-inducible factor 1α,HIF-1α)基因对大鼠肝癌CBRH-7919细胞在缺氧状态下p27和Ki67表达的影响。方法: 选用大鼠肝癌细胞株CBRH-7919作为研究对象,利用氯化钻(CoCl₂)模拟缺氧状态。构建HIF-1α siRNA特异性载体,利用小分子RNA干扰技术沉默肝癌细胞HIF-1α基因。应用real-time RT-PCR和Western blotting方法分别检测肝癌细胞HIF-1α mRNA和蛋白的表达变化,用Western blotting方法检测肝癌细胞 p27和Ki67 蛋白的表达变化。应用流式细胞术检测细胞周期的变化。结果: 在缺氧条件下,肝癌细胞HIF-1α mRNA及蛋白表达显著增多(P<0.05)。HIF-1α基因被沉默后,HIF-1α mRNA和蛋白表达以及Ki67蛋白表达量明显减少(P<0.05),p27蛋白表达量显著增多(P<0.05)。转染组G₀/G₁期的肝癌细胞数量明显多于对照组,S期细胞显著减少(P<0.05)。 结论: 缺氧可以诱导肝癌细胞HIF-1α的表达;特异性siRNA沉默HIF-1α,可能通过促进p27的表达和抑制Ki67的表达,在一定程度上抑制了肝癌细胞的增殖。     

人参单体皂甙Rh2对前列腺癌细胞株PC-3M细胞移行周期的影响

目的:探讨Rh₂对体外培养的PC-3M细胞移行周期的影响。方法:应用[³H]-TdR掺入法和流式细胞仪测定DNA含量及进行细胞周期的分析。结果:PC-3M细胞的[³H]-TdR掺入量明显少于对照组(P＜0.01);流式细胞仪测定G₁期细胞数百分比明显高于对照组。结论:Rh₂可使PC-3M细胞增殖周期阻滞在G₁期,肿瘤细胞增殖减慢。     

G1 phase dependent nuclear localization of relaxed-circular hepatitis B virus DNA and aphidicolin-induced accumulation of covalently closed circular DNA

During chronic hepatitis B virus (HBV) infection, virus persistence relies on the maintenance of a pool of covalently closed circular DNA (cccDNA) in the nuclei of infected hepatocytes. To achieve this, HBV DNA has to be transported from the cytoplasm to the nucleus. By carrying out subcellular fractionation experiment, both of the relaxed-circular (RC) and single-stranded (SS) HBV DNA were found in the cytoplasm whereas only RC form could be detected in the nucleus of a hepatoblastoma cell line (HepG2) stably producing HBV. This fraction of nuclear RC viral DNA was clearly demonstrated in the G₁ but not S phase of synchronized HepG2 cells. Conversely, the relative amount of cytoplasmic RC viral DNA in the S phase was larger than that in the G1 phase. Although no cccDNA could be detected in HepG2 cells without synchronization, an increasing amount of cccDNA in the nucleus was demonstrated after prolonged incubation of the cells in aphidicolin. Finally, by undertaking in situ hybridization using a probe specific to plus-strand HBV DNA, nuclear viral DNA was detected predominantly in the G1 phase of HepG2 cells. In summary, the results indicated that only RC but not SS form of HBV DNA was localized to the nuclei of HepG2 cells. The nuclear localization occurred preferentially in the G₁ but not S phase and prolonged treatment with aphidicolin resulted in accumulation of nuclear cccDNA. J. Med. Virol. 55:42–50, 1998. © 1998 Wiley-Liss, Inc.     

P53-mediated G1/S checkpoint dysfunction in lymphocytes from Alzheimer's disease patients

To date, Alzheimer disease (AD) is still difficult to be diagnosed in its earliest stage. The cell cycle aberrations may be the earliest neuropathological events detected in AD thus far. The cell cycle regulatory failure in AD occurring at “G₁/S transition checkpoint” which is mediated by the tumor suppressor protein p53 has been identified. Herein, we observed the response of activated lymphocytes to G₁/S transition blocker to assess the G₁/S checkpoint function and the p53 conformation state adopted in lymphocytes from AD patients and healthy non-AD controls. We found that the activated lymphocytes from AD patients were less sensitive to G₁/S transition blocker than those from controls, indicating that the G₁/S checkpoint failed to function well in AD lymphocytes. In addition, AD cells specifically expressed an anomalous conformationally mutant-like p53 that made these cells distinct from lymphocytes of controls. We speculated that the altered conformational p53 probably be responsible for G₁/S checkpoint dysfunction in AD cells. Our hypothesis was supported by the results that G₁/S checkpoint dysfunction was not restricted to neurons in AD patients, but also occurred in peripheral lymphocytes. Two potential biomarkers were indicated in blood lymphocytes from AD patients: the G₁/S checkpoint dysfunction and the conformationally mutant-like p53 protein.     

威麦宁对小鼠Lewis肺癌细胞周期的影响(英)

目的：探讨威麦宁对小鼠Lewis肺癌的抑制作用，并检测其对细胞周期的影响。 方法:采用流式细胞术分析、免疫组化法检测药物对模型鼠肿瘤细胞周期及对cyclin D1表达的影响。 结果:威麦宁浓度在100 mg·kg^-1·d^-1、150 mg·kg^-1·d^-1、200 mg·kg^-1·d^-1、250 mg·kg^-1·d^-1时，对小鼠Lewis肺癌的抑制率分别为19.14%、33.59%、40.63%、51.56%, 药物对肿瘤的抑制作用具有剂量依赖性；该药物可将肿瘤细胞阻滞在G₀-G₁期，并降低肿瘤细胞cyclin D1的表达（P<0.01）。 结论:威麦宁对小鼠Lewis肺癌抑制作用的机制之一可能是通过降低肿瘤细胞cyclin D1的表达，将肿瘤细胞阻止于G₀-G₁期，使细胞不能进入S期进行DNA复制，从而抑制肿瘤的生长。     

肿瘤坏死因子-α对肝星状细胞增殖与凋亡的影响

目的:探讨肿瘤坏死因子-α(TNF-α)在肝纤维化发病中的作用。方法:采用TNF-α对体外培养的HSC进行干预, 通过流式细胞术、电镜及TUNEL等方法, 对HSC的增殖与凋亡进行研究。结果:①流式细胞术显示:TNF-α各组的G₀/G₁期细胞比率明显高于对照组, 而S期细胞比率明显低于对照组, 各组的细胞增殖指数均明显低于对照组;在凋亡方面, TNF-α各组的HSC凋亡率明显高于对照组, TNF-α各组HSC中抗凋亡基因bcl-2的表达明显低于对照组, 而促凋亡基因bax的表达明显高于对照组。②TUNEL分析显示:TNF-α(2.0μg/L)组的HSC凋亡率为18.7%±2.5%, 而对照组为5.3%±1.2%, 两者之间有显著差异。结论:TNF-α能够阻止HSC从G₀/G₁期进入S期, 从而具有抑制HSC增殖的作用;TNF-α能够降低HSC中bcl-2的表达、提高bax的表达, 这可能与其促进HSC凋亡有关。     

JNK targets p53 ubiquitination and degradation in nonstressed cells

In this study we elucidated the role of nonactive JNK in regulating p53 stability. The amount of p53–JNK complex was inversely correlated with p53 level. A peptide corresponding to the JNK binding site on p53 efficiently blocked ubiquitination of p53. Similarly, p53 lacking the JNK binding site exhibits a longer half-life than p53^wt. Outcompeting JNK association with p53 increased the level of p53, whereas overexpression of a phosphorylation mutant form of JNK inhibited p53 accumulation. JNK–p53 and Mdm2–p53 complexes were preferentially found in G₀/G₁ and S/G₂M phases of the cell cycle, respectively. Altogether, these data indicate that JNK is an Mdm2-independent regulator of p53 stability in nonstressed cells.     

人着色性干皮病D组基因对白细胞介素-6促进人血管平滑肌细胞增殖作用的影响

目的: 探讨人着色性干皮病D组基因(XPD)对白细胞介素-6(IL-6)促进人血管平滑肌细胞(VSMCs)增殖作用的影响。方法: 用脂质体转染法将重组质粒pEGFP-N2/XPD和空质粒pEGFP-N2稳定转染VSMCs,然后给予1×10⁵ U/L IL-6孵育48 h。实验分为6组:(1)空白对照组;(2)pEGFP-N2组;(3)pEGFP-N2/XPD组;(4)IL-6组;(5)IL-6 + pEGFP-N2组;(6)IL-6 + pEGFP-N2/XPD组。用荧光显微镜观察绿色荧光蛋白报告基因表达情况;用MTT法观察细胞增殖活力;用流式细胞仪检测细胞周期和凋亡率;用RT-PCR和Western blotting检测XPD、Bcl-2、Bax和野生型P53(wt-P53)表达量的变化。结果: 在荧光显微镜下,可在转染了重组质粒pEGFP-N2/XPD或空质粒pEGFP-N2的细胞中观察到绿色荧光,即转染成功;MTT结果显示,重组质粒pEGFP-N2/XPD的转染抑制了细胞增殖活力(P<0.05),并能抑制IL-6促进VSMCs增殖的作用(P<0.05);流式细胞仪结果显示,重组质粒pEGFP-N2/XPD的转染引起了细胞G₀/G₁期增加(P<0.05)、S期减少(P<0.05)、凋亡率增加(P<0.01),并能抑制IL-6促进VSMCs G₀/G₁期减少、S期增加、凋亡率降低的作用(P<0.01);RT-PCR和Western blotting检测发现,重组质粒pEGFP-N2/XPD的转染使得XPD表达增高(P<0.05或P<0.01),同时Bcl-2表达降低,Bax和wt-P53表达增高(P<0.05或P<0.01),并能抑制IL-6促进VSMCs的Bcl-2表达增高、Bax和wt-P53表达降低的作用(P<0.05或P<0.01)。结论: XPD能抑制VSMCs增殖并促进其凋亡,并能抑制IL-6促进VSMCs增殖和降低其凋亡的作用,有望成为治疗动脉粥样硬化的靶点。     

Human lymphocyte proliferation II. Formation of activated (G1) cells

The kinetics of phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes in the early phases (G₀, G_1a, G_1b) of the first cell cycle have been analyzed in vitro by cytofluorometry and [3H] thymidine incorporation. Cells were detected in the G_1a G_1b and S phases 12-14, 20-22 and 24-26 h after stimulation, respectively. The total number of PHA-induced G₁ cells reached a plateau after 32 h of incubation, upon which a second increase followed, 10 h later. The latter increase was considered to be a result of cells initiating their second cell cycle, since it could be abolished by hydroxyurea. By examination of supernatants, interleukin 2 (IL 2) activities could be recovered 6 10 h after PHA stimulation. In contrast, the monokine IL 1 was already detected after 2 h. Both interleukins were present in the culture medium before the major G_1a, cell formation took place. The titer of free IL 2 increased to a maximum after 18-22 h, whereafter a decline was observed. This decline was less pronounced if cultures were treated with hydroxyurea. Finally, among individual donors, the total number of lymphocytes entering the G₁ phase and the time at which the G_1a-G_1b transition took place varied. Lower numbers of G₁ cells and delayed G_1a-G_1b transition coincided with lower IL 2 titers and delayed occurrence of maximal titers in the supernatants.     

Immunofluorescent detection of thermolability of chromatin in situ during the cell cycle using antithymine antibodies

Nuclear immunofluorescence was observed in cultured cells reacted with antithymine antibodies after heat denaturation of whole cells. The intensity of the nuclear fluorescence was measured quantitatively by a scanning densitometer. The intensity increased linearly by heating the cells up to 10 min. Using this procedure the thermolability of chromatin of ts AF8 cells was in vestigated in different phases of the cell cycle. The intensity of nuclear fluorescence in G₁ and S phase cells was significantly increased compared with G₀ phase cells when serum-quiescent cells were stimulated to proliferate by 10% serum. This increase of nuclear fluorescence was not observed when the cells were incubated at the nonpermissive temperature. The distribution pattern of nuclear fluorescence changed during S phase, although the intensity itself did not change significantly. This procedure may provide a possible application of the method for cytodiagnosis.     

软组织平滑肌肉瘤的流式细胞术研究

目的　研究软组织平滑肌肉瘤 (LMS)中DNA倍体与S期分数 (SPF)及二者与临床病理的联系。方法　应用流式细胞术 (FCM )检测 33例LMS的DNA指数及SPF。结果　LMS中异倍体率为 39 4 % ,平均SPF值为 12 2 36。低分化LMS组的异倍体率 (70 0 % )高于高分化LMS组 (2 6 1% ) (P <0 0 5 ) ;异倍体LMS组的SPF均值 (15 5 92 )高于二倍体组 (10 0 5 5 ) (P<0 0 5 ) ;瘤体最大径 >6cm组的SPF均值 (13 6 36 )明显高于 <6cm组 (9 4 36 ) (P <0 0 5 )。结论　LMS的SPF与DNA倍体和体积均呈现出相关关系。LMS的DNA倍体与肿瘤的分化程度具有相关性     

Studies on cell division in mammalian cells: VI. A temperature-sensitive mutant blocked in both G1 and G2 phases of the cell cycle

A temperature-sensitive mammalian cell cycle mutant with blocks in G ₁ and G₂ phases of the cell cycle has been isolated in culture. When shifted from the permissive temperature of 33 C to the nonpermissive temperature of 39 C, the fraction of cells initiating DNA synthesis as well as the fraction of cells entering mitosis decreased rapidly. Combined cytophotometric and autoradiographic analysis on the cells at 39 C showed that G₁ cells, with the exception of those in late G₁, were arrested in that phase. Cells in S phase at the time of temperature shift, together with the late G₁ cells which subsequently entered S, continued through S into G₂, but were blocked in that phase of the cell cycle and unable to initiate mitosis. Those cells already in mitosis completed cell division at 39 C. The G₁ block point of ts-550C was found to be located after the serum starvation and isoleucine deprivation arrest points, approximately 3 h before initiation of DNA synthesis.