Cell cycle effects of antifolate antimetabolites: implications for cytotoxicity and cytostasis

 Purpose: Cell cycle-related events in CCRF-CEM lymphocytic leukemia cells were examined subsequent to inhibition of thymidylate synthase (TS) or GAR formyltransferase (GARFT) and prior to cell death or stasis. Methods: Cell populations were treated with the GARFT inhibitors 6R-5,10-dideazatetrahydrofolate (lometrexol) or LY309887, the TS inhibitor ZD1694, or the multitargeted antifolate LY231514. DNA content, nucleoside precursor incorporation and proliferating cell nuclear antigen (PCNA) expression as functions of drug treatment were assessed by multiparameter flow cytometry. Cellular respiration was measured by MTT analysis and apoptosis was detected by extraction of DNA fragments. Results: Cell populations treated for up to 96 h with lometrexol or LY309887 did not replicate and maintained a cell cycle distribution with distinct G₁, S and G₂/M regions. The number of S phase cells in treated populations was slightly elevated relative to control as measured by DNA content and PCNA. However, these cells were unable to incorporate 5-bromodeoxyuridine (BrdU). Throughout treatment, cells incubated with GARFT inhibitors maintained intact membranes and respired at a level comparable to untreated cells. In contrast, ZD1694 as well as LY231514, induced synchronization of the treatment population at the G₁/S interface within 12 h of drug addition. This was followed by synchronous entry of the population into S phase. After 24 h of treatment, more than 90% of the cells were capable of incorporating BrdU and stained positive for PCNA. DNA fragmentation occurred in cells treated with ZD1694 or LY231514 but not in those treated with GARFT inhibitors. In addition, the viable cells remaining after 24–48 h of treatment with ZD1694 or LY231514 were respiring at twice the level of untreated cells. Conclusion: These results demonstrate that the distinct endpoints of GARFT and TS inhibition are preceded by distinct cell cycle and metabolic alterations. Received: 1 April 1996 / Accepted: 5 September 1996     

CeReS-18, a novel cell surface sialoglycopeptide, induces cell cycle arrest and apoptosis in a calcium-sensitive manner

Very few growth inhibitors have been identified whichcan inhibit the proliferation of a broad spectrumof human breast cancer cell lines. CeReS-18, anovel cell surface sialoglycopeptide growth inhibitor, can reversiblyinhibit the proliferation of both estrogen receptor positive(MCF-7) and negative (BT-20) human breast cancer celllines. In addition, at concentrations above those requiredfor the reversible inhibition of cell proliferation, CeReS-18can also induce cell death in MCF-7 cells.Changes in nuclear and cytoplasmic morphology, characteristic ofapoptosis, were detected in MCF-7 cells treated witha cytotoxic concentration of CeReS-18, and internucleosomal DNAcleavage was also observed. The sensitivity of MCF-7and BT-20 cells to the biological properties ofCeReS-18 could be influenced by altering the calciumconcentration in the extracellular growth medium, such thatwhen the calcium concentration in the environment wasdecreased, an increased sensitivity to CeReS-18-induced growth inhibitionand cytotoxicity were observed. The addition of thecalcium chelating agent EGTA to MCF-7 cells, culturedin a normal calcium environment, could mimic theincreased sensitivity to the biological effects of CeReS-18observed under reduced calcium conditions.     

Enhancement of Radiation Response by Paclitaxel in Mice According to Different Treatment Schedules

Purpose: The aim of our study was to determine if paclitaxel could be used as a radiosensitizer in vivo.

Materials and methods: Paclitaxel was tested as a single agent and combined with an X-ray treatment. Paclitaxel was administered i.p. in doses from 30 to 120 mg/kg b.w. to (C3D2F1) mice bearing spontaneous mammary carcinoma. Tumor growth delay (TGD) or tumor control dose (TCD₅₀, radiation dose needed to induce local tumor control in 50% of irradiated animals) and moist desquamation dose (MDD₅₀, radiation dose needed to induce serious moist desquamation in 50% of the non-tumor-bearing feet) were the endpoints. DNA flow cytometric analysis was performed.

Results: DNA analysis demonstrated a G2/M block of tumor cells and a depletion of cells in S phase, with a maximum at 24 h from paclitaxel administration. Administering paclitaxel, in graded doses, 15 min before a 10-Gy X-ray treatment resulted in a linear regression line, almost parallel to that with paclitaxel alone, with a growth delay of about 6 days. In contrast, varying the X-ray dose with a constant paclitaxel injection (45 mg/kg b.w.) treatment showed some degree of synergism as the linear regression curves diverged. Interval time and sequence between paclitaxel administration and a 10 Gy X-ray treatment did not influence TGD. Protocols with paclitaxel at 30, 45, or 60 mg/kg were combined with radiation treatments at various doses (from 10 to 65 Gy). Values of TCD50 varied from 50.8 Gy for X-ray alone to 31.8 Gy for paclitaxel 60 mg/kg + X-ray. No differences were observed among MDD of different protocols.

Conclusions: These results suggest that, under some conditions, paclitaxel combined with radiation can show superadditive effects and this result combined with the lack of severe normal tissue damage indicate that a favorable therapeutic gain can be obtained.     

半边旗抗肿瘤有效成分6F对HL-60细胞周期的影响及体外增效作用

目的：研究半边旗抗肿瘤有效成分６Ｆ对ＨＬ－６０细胞周期的影响及对常用抗肿瘤药的体外增效作用。方法：应用流式细胞光度术（ＦＣＭ）测定细胞周期，应用噻唑蓝（ＭＴＴ）法测定药物对细胞的抑制率。结果：不同浓度６Ｆ作用６小时即可使ＨＬ－６０细胞Ｓ期及Ｇ２／Ｍ期比例升高，Ｇ１期比例下降，并呈一定的剂量效应关系，当作用到２４小时后，Ｓ期比例进一步升高，但Ｇ２／Ｍ期比例稍有回落。低浓度６Ｆ分别与２－氯代脱氧腺苷（２－ＣＬｄＡｄｏ），顺铂（ＣＤＤＰ），长春新碱（ＶＣＲ），氟尿嘧啶（５ＦＵ）合用可增强它们对ＨＬ－６０细胞的杀伤作用，ｑ值大于０．８５，与各药有相加或协同作用，６Ｆ对２－ＣｌｄＡｄｏ，ＣＤＤＰ，ＶＣＲ，５ＦＵ的增效倍数分别为１．５８，１．５３，１．５５，１．３８。结论：６Ｆ可明显阻断ＨＬ－６０细胞在Ｓ期及Ｇ２／Ｍ期；６Ｆ可增强上述药物对ＨＬ－６０细胞的杀伤作用。已知，２－ＣｌｄＡｄｏ阻断细胞在Ｓ期，ＣＤＤＰ和ＶＣＲ阻断Ｇ２／Ｍ期，５ＦＵ阻断Ｇ１期。鉴于所试药物对细胞周期的影响不同，提示６Ｆ的体外增效作用可能与此有关。     

维甲类化合物Ro13-7410对HL-60细胞的作用

目的：探讨合成的第三代维甲类化合物（Ｅ）４［２５，６，７，８ｔｅｔｒａｈｙｄｒｏ５，５，８，８ｔｅｔｒａｍｅｔｈｙｌ２ｎａｐｈｔｈａｌｅｎｙｌ１ｐｒｏｐｅｎｙｌ］ｂｅｎｚｏｉｃａｃｉｄ（Ｒｏｌ３７４１０）对白血病细胞ＨＬ６０的影响。方法：采用ＮＢＴ还原能力测定检测细胞分化，电镜和程序性细胞死亡检测试剂盒（ＰＣＤａｓａｙｋｉｔ）鉴别凋亡细胞，Ｆｕｒａ２／ＡＭ荧光法测定胞浆内Ｃａ２＋浓度，ＰＣＲＥＬＩＳＡ试剂盒检测端粒酶活性，流式细胞术测定细胞周期分布。结果：Ｒｏｌ３７４１０诱导ＨＬ６０细胞分化的同时伴随细胞凋亡的发生。在ＨＬ６０细胞凋亡和分化的过程中胞浆内Ｃａ２＋浓度增高，端粒酶活性明显受抑，多数细胞被阻止在Ｇ２／Ｍ期。结论：Ｒｏｌ３７４１０可诱导ＨＬ６０细胞凋亡和分化的同时发生。胞浆内Ｃａ２＋浓度的增高，端粒酶活性的降低以及细胞周期的阻止与细胞凋亡和分化的诱导有关     

三氧化二砷对体外人胶质瘤细胞株U251的影响

目的探讨三氧化二砷(As2O3)对体外培养的人类成神经胶质瘤细胞株U251MG的作用及其机制。方法应用倒置相差显微镜、四甲基偶氮唑蓝法、流式细胞仪,分别对不同浓度加药组及对照组U251细胞的存活、形态学改变、细胞DNA含量的分布进行观察和测定。结果0.5～4μmol/L的As2O3可明显抑制U251的增殖。流式细胞仪分析显示,加药组在G1期细胞前均出现亚二倍体峰,且G0/G1期细胞减少,诱导细胞凋亡。结论As2O3能抑制体外人胶质瘤细胞株U251的增殖,有望用于神经胶质瘤的治疗。     

双歧杆菌脂磷壁酸体外诱导胃癌细胞凋亡的研究

目的 探讨双歧杆菌表面分子脂磷壁酸（LTA）的抗肿瘤作用及其机制.方法 应用MTT法、透射电镜及流式细胞术等方法观察双歧杆菌LTA对胃癌细胞BGC823的增殖抑制作用及其对细胞形态和细胞周期的影响,并用免疫细胞化学染色法检测LTA处理前后细胞凋亡相关蛋白表达的变化.结果 双歧杆菌LTA对体外培养的BGC823细胞具有明显的生长抑制作用,且具有剂量和时间依赖性.经LTA处理的BGC823细胞超微结构改变,出现凋亡小体,同时细胞周期发生变化,细胞被阻滞于G0/G1期.免疫细胞化学染色结果显示,LTA能下调Bcl-2蛋白和c-myc蛋白的表达,同时上调p53蛋白和c-fos蛋白的表达.结论 双歧杆菌LTA能抑制胃癌细胞BGC823的增长,其机制可能是通过调节凋亡相关蛋白的表达变化,将细胞阻滞在G0/G1期并诱导细胞凋亡.     

人肺癌组织端粒酶活性与p16基因二者相关性探讨

目的:探讨非小细胞肺癌(NSCLC)组织中端粒酶活性与p16基因的相关性及二者与肺癌发生发展的关系.方法:肺癌标本48例,12例癌旁组织,7例非肿瘤病例的正常肺组织.以Telomerase PCR ELISA检测标本端粒酶活性,以RT-PCR的方法检测p16基因mRNA转录情况,以免疫组化方法检测组织标本p16蛋白表达.结果:1)肺癌组织标本36/48(75.00%)和1例癌旁组织检出端粒酶活性.2)48例NSCLC组织中32例进行了p16 mRNA及蛋白表达检测.16/32(50.00%)检测到p16mRNA转录,7例正常组织中均检测到p16 mRNA转录.NSCLC组织标本中17/32(53.13%)未检测到p16蛋白表达,7例正常肺组织中均检测到p16蛋白表达.3)24例端粒酶阳性NSCLC组织中15例p16 mRNA表达缺失,8例端粒酶阴性的NSCLC组织中仅1例p16mRNA表达缺失.相关系数为-0.433,P=0.013,具有显著负相关.结论:端粒酶、p16基因在NSCLC的发生发展中起重要作用,端粒酶活性有望成为预测肿瘤发生、肿瘤诊断的良好指标.端粒、端粒酶与p16基因可能成为抗肿瘤治疗的新靶点.     

Cyclin E、CDK2和p21WAF1在食管上皮癌变过程中的表达及意义

目的探讨食管上皮癌变过程中细胞周期调控因子cyclin E、CDK2和p21WAF1的表达状况及其意义.方法应用免疫组化SP法和原位杂交方法分别检测48例食管癌组织、31例非典型增生组织和17例正常食管粘膜中cyclin E、CDK2和p21WAF1蛋白及mRNA表达.应用半定量RT-PCR和Western blot检测22例新鲜食管癌及相应癌旁组织的mRNA和蛋白表达.结果从食管正常粘膜、非典型增生组织到癌组织,cyclin E和CDK2蛋白和mRNA阳性表达率逐渐上升,差异具有统计学意义(P＜0.01或P＜0.05).食管癌组织中cyclin E、CDK2和p21WAF1蛋白及mRNA高表达,与癌旁组织或切缘正常食管粘膜有显著性差异(P＜0.01).cyclin E、CDK2和p21WAF1基因表达显著正相关(P＜0.01或P＜0.05).结论食管上皮癌变过程中,细胞周期相关基因cyclin E和CDK2表达逐渐增强.cyclin E基因表达异常是食管癌变过程中的早期事件.p21WAF1基因在食管癌中高表达,可能与细胞周期调控的反馈机制有关.     

canstatin基因转染对肺癌A549细胞和血管内皮细胞增殖与凋亡的影响

背景与目的　肿瘤的生长和转移需要大量新血管的生成,人血管能抑素(canstatin)是新近发现的高效内源性血管生成抑制剂,其抑制血管内皮细胞的作用已引起人们广泛关注。本研究的目的是探讨can statin基因在人肺癌A549细胞和人脐静脉内皮细胞HUV ECC中的表达及意义。方法　将canstatin基因通过电穿孔的方法转染人肺癌细胞A549和人脐静脉内皮细胞HUV ECC,行G418筛选获得转基因细胞克隆。用SDS PAGE电泳检测canstatin蛋白在转基因细胞培养上清液中的表达,以流式细胞仪分析细胞周期,并比较转基因和未转基因细胞的生长特性。结果　canstatin在转染组A549 细胞和ECC细胞表达并分泌至上清液中。canstatin基因转染组ECC的凋亡率(16.04%)显著高于空载体组(0.43%)和亲代细胞组(2.92%)(P<0.01),转染细胞生长明显受抑(P<0.01)。而转染组A549细胞的凋亡率(0.19%)与空载体组(0.13%)及亲代细胞组(0.07%)比较无显著性差异(P>0.05),细胞生长也未受明显影响。结论　canstatin能特异地抑制内皮细胞增殖,并诱导内皮细胞凋亡。