An inhibitor of glycinamide ribonucleotide formyltransferase is selectively cytotoxic to cells that lack a functional G1 checkpoint

Purpose: We studied the effects of purine depletion on the cell cycle using a specific inhibitor of de novo purine biosynthesis, AG2034, an inhibitor of glycinamide ribonucleotide formyltransferase (GARFT). Methods: Cytotoxicity was determined by clonogenic assays, and cell cycle perturbations by flow cytometry. Ribonucleotide pools were measured by anion exchange high-pressure liquid chromatography, and DNA strand-breaks were determined by alkaline elution and by the TUNEL assay. Results: When cells were maintained in standard tissue culture medium, which contained 2.2 μM folic acid, AG2034 was cytostatic in all the cell lines tested. Under low-folate conditions (50 nM folic acid), AG2034 caused up to 50% cell death in cell lines that possessed a functional G1 checkpoint (A549, MCF-7), but was only cytostatic to the remaining cells, even at very high concentrations (100 μM ). In contrast, AG2034 at 10 nM or 100 nM killed all the cells in cultures of HeLa/S3 or SW480 cells, which lack a functional G1 checkpoint. Flow cytometry studies indicated that in G1 checkpoint-competent cells, AG2034 caused a G1 arrest. Those cells (up to 50%) that were already in S phase died, but the cells that were in G1 arrest maintained viability, based upon clonogenic assays, for many days. In G1 checkpoint-deficient cells, no G1 arrest was seen after AG2034 treatment, all cells progressed into S phase, and all cells died. Measurement of DNA strand-breaks, either by alkaline elution or by the dUTP end-labelling technique, indicated no DNA strand-breaks 24 h after AG2034 treatment, indicating that purine nucleotide depletion can trigger the G1 checkpoint in the absence of DNA damage. Conclusion: Purine depletion causes slow cell death in cells that have passed the G1 checkpoint, but cytostasis in cells that are arrested at the G1 checkpoint. The GARFT inhibitor, at physiological folate concentrations, thus causes selective cytotoxicity to cells lacking a functional G1 checkpoint. Received: 8 May 1997 / Accepted: 26 June 1997     

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.     

不同方法静注肾上腺素对心肺复苏的疗效观察

目的 :观察大剂量间歇给药与小剂量递增给药在冠心病猝死心肺复苏中的疗效。方法 :79例冠心病猝死患者常规施以心肺复苏 (CPR) ,电除颤 1～ 2次无效 ,分为 A、B两组分别以小剂量递增法及大剂量间歇法静注肾上腺素 ,观察用药至自主循环恢复 (Rosc)时间、累积用药量、自主循环恢复率及存活出院率。结果 :A、B两组肾上腺素累积用量相近 ,Rosc时间 A组 (5 .1± 2 .7) min,B组 (3.8± 2 .1) min(P <0 .0 5 ) ;自主循环恢复率 A组 31.6 % ,B组 5 8.5 % (P <0 .0 5 ) ;存活出院率 A组 14 .8% ,B组 2 4 .4 % (P >0 .0 5 )。结论 :大剂量间歇给药较小剂量递增给药可明显促进心脏骤停患者的自主循环恢复 ,但对预后无改善。     

经食道心脏起搏在救治口服有机磷农药中毒心跳骤停中的应用体会

目的探讨口服有机磷中毒患者心跳骤停时应用食道心脏调搏进行救治的疗效。方法15例心脏骤停患者,在常规治疗基础上应用食道心脏调搏方法抢救。结果15例中救治成功13例。结论经食道心脏起搏方法简便、操作速度快、无创、无需消毒、效果良好,适用于临床紧急起搏,尤其适用于基层医院。     

小剂量硝酸甘油在心肺复苏中的应用

目的 探讨小剂量硝酸甘油在心肺复苏术中的临床疗效.方法 将我院重症监护病房中发生心脏骤停的患者按随机数字表法分为对照组(127例)和试验组(132例),对照组按照《2010年国际心肺复苏和心血管急救指南》进行急救,胸外心脏按压,液体扩容,肾上腺素静脉注射,多巴胺、多巴酚丁胺升压,呼吸机辅助呼吸等综合治疗,试验组在对照组治疗的基础上,加用小剂量硝酸甘油(5 μg/min)微量泵泵入,比较两组患者心脏复苏成功率,复苏成功时间,有创动脉血压及24h存活率.结果 试验组患者心脏复苏成功率及24h存活率、复苏成功时间与对照组比较差异有统计学意义(P＜0.05)；两组患者有创动脉血压比较差异无统计学意义(P＞0.05).结论 心肺复苏术中应用小剂量硝酸甘油可明显提高心肺复苏成功率,提高患者存活率,并且对外周动脉血压无明显影响.     

AWARE—AWAreness during REsuscitation—A prospective study

BackgroundCardiac arrest (CA) survivors experience cognitive deficits including post-traumatic stress disorder (PTSD). It is unclear whether these are related to cognitive/mental experiences and awareness during CPR. Despite anecdotal reports the broad range of cognitive/mental experiences and awareness associated with CPR has not been systematically studied.MethodsThe incidence and validity of awareness together with the range, characteristics and themes relating to memories/cognitive processes during CA was investigated through a 4 year multi-center observational study using a three stage quantitative and qualitative interview system. The feasibility of objectively testing the accuracy of claims of visual and auditory awareness was examined using specific tests. The outcome measures were (1) awareness/memories during CA and (2) objective verification of claims of awareness using specific tests.ResultsAmong 2060 CA events, 140 survivors completed stage 1 interviews, while 101 of 140 patients completed stage 2 interviews. 46% had memories with 7 major cognitive themes: fear; animals/plants; bright light; violence/persecution; deja-vu; family; recalling events post-CA and 9% had NDEs, while 2% described awareness with explicit recall of ‘seeing’ and ‘hearing’ actual events related to their resuscitation. One had a verifiable period of conscious awareness during which time cerebral function was not expected.ConclusionsCA survivors commonly experience a broad range of cognitive themes, with 2% exhibiting full awareness. This supports other recent studies that have indicated consciousness may be present despite clinically undetectable consciousness. This together with fearful experiences may contribute to PTSD and other cognitive deficits post CA.     

Dependency of 2-methoxyestradiol-induced mitochondrial apoptosis on mitotic spindle network impairment and prometaphase arrest in human Jurkat T cells

The present study sought to determine the correlation between 2-methoxyestradiol (2-MeO-E₂)-induced cell cycle arrest and 2-MeO-E₂-induced apoptosis. Exposure of Jurkat T cell clone (JT/Neo) to 2-MeO-E₂ (0.5–1.0 μM) caused G₂/M arrest, Bak activation, Δψm loss, caspase-9 and -3 activation, PARP cleavage, intracellular ROS accumulation, and apoptotic DNA fragmentation, whereas none of these events except for G₂/M arrest were induced in Jurkat T cells overexpressing Bcl-2 (JT/Bcl-2). Under these conditions, Cdk1 phosphorylation at Thr-161 and dephosphorylation at Tyr-15, up-regulation of cyclin B1 expression, histone H1 phosphorylation, Cdc25C phosphorylation at Thr-48, Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation at Ser-159/Thr-163, and Bim phosphorylation were detected irrespective of Bcl-2 overexpression. Concomitant treatment of JT/Neo cells with 2-MeO-E₂ and the G₁/S blocking agent aphidicolin resulted in G₁/S arrest and abrogation of all apoptotic events, including Cdk1 activation, phosphorylation of Bcl-2, Mcl-1 and Bim, and ROS accumulation. The 2-MeO-E₂-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by a Cdk1 inhibitor, but not by an Aurora A kinase (AURKA), Aurora B kinase (AURKB), JNK, or p38 MAPK inhibitor. Immunofluorescence microscopic analysis revealed that 2-MeO-E₂-induced mitotic arrest was caused by mitotic spindle network impairment and prometaphase arrest. Whereas 10–20 μM 2-MeO-E₂ reduced the proportion of intracellular polymeric tubulin to monomeric tubulin, 0.5–5.0 μM 2-MeO-E₂ increased it. These results demonstrate that the apoptogenic effect of 2-MeO-E₂ (0.5–1.0 μM) was attributable to mitotic spindle defect-mediated prometaphase arrest, Cdk1 activation, phosphorylation of Bcl-2, Mcl-1, and Bim, and activation of Bak and mitochondria-dependent caspase cascade.