Staining with hoechst 33342 and rhodamine 123: An attempt to detect multidrug resistant phenotype cells in leukemia

Development of resistance is the major cause of failure in chemotherapeutic treatments. We have previously shown that the level of labeling with Hoechst 33342 and rhodamine 123 in established cell lines was decreased in cells with ‘classic’ MDR phenotype. This functional test was carried out using fluorescence image cytometry on living cells. We applied this protocol to patients with chronic lymphocytic leukemia. Although a large variability of the labeling is observed in cells from healthy donors, this approach seems to be useful for early detection of P-gp-dependent resistance in leukemia cells and for identification of new reversing agents on patient lymphocytes.     

Stable hybrid myotubes: A new model for studying re-expression of enzymatic activities in vitro

Heterokaryons represent a stable and reproducible model system for the study of biochemical and molecular aspects responsible for muscle gene activation. Previous experiments have used this fusion system to demonstrate human gene activation in hybrids formed between human and non-human cells. The aim of this research was to apply this experimental model to the correction of a cytoplasmic activity, namely glucose-6-phosphate dehydrogenase (G6PD), in vitro, in hybrid myotubes formed between G6PD-negative and positive myoblasts. Different identification methods were used (Hoechst stain and Fluorescent Latex Microspheres, FLMs) to identify hybrid myotubes formed. We demonstrated the restoration of G6PD activity in all hybrid myotubes formed; we then tried to elucidate the mechanisms underlying the restoration of this specific activity and apply the results obtained to the understanding of more complex mechanisms involved in muscle gene activation. Paper presented at the National Congress at Sorrento in 1991 and selected by the Editorial Board of the Journal     

Reversible synchronization of cultured rodent and human diploid fibroblast cells in G2 phase

This paper describes a three-step procedure for induction of reversible cell synchrony in the G₂ phase of the cell cycle of Chinese hamster ovary (line CHO) cells and non-transformed, human skin fibroblast (HSF) cells. The CHO cells are presynchronized in early S phase by isoleucine deficiency and hydroxyurea blockades. The culture is transferred to medium supplemented with the DNA topoisomerase II inhibitor, Hoechst 33342 for 12 hours after which 95% of the cells are arrested in G₂ phase. When G₂ synchronized cells are transferred to Hoechst-free, complete medium, they divide as a highly synchronous cohort within 3 hours. The HSF cells are initially cultured in low serum to arrest them in G₀ and then transferred to complete medium containing aphidicolin to arrest them in early S phase. The culture is then transferred to aphidicolin-free, complete medium with Hoechst 33342 (0.1 g/ml) for 10 hours after which up to 85% of the cells arrest in G₂ phase. Synchronous cell division occurs 3.5 hours after transfer of cells to complete, drug-free medium. The synchrony techniques are useful for studying G₂/M biochemical events in mammalian cells.Abbreviations -MEM minimum essential medium alpha medium - BCS bovine calf serum - DMSO dimethyl sulfoxide - EDTA ethylenediaminetetraacetic acid - FCM flow cytometry - PBS phosphate buffered saline - Top II DNA topoisomerase II     

PKH26和Hoechst 33258联合示踪Uncv无毛小鼠iRhom2及其突变体蛋白

目的利用PKH26和Hoechst 33258联合示踪Uncv无毛小鼠iRhom2及其突变体蛋白在Vero细胞中的定位。方法用PKH26对细胞膜进行染色,同时用Hoechst 33258染料对细胞核进行染色,置于激光共聚焦显微镜下观察。结果通过激光共聚焦荧光显微镜观察目的蛋白,发现野生型iRhom2分布在细胞的胞浆,而iRhom2mut于细胞浆内和细胞核内都存在。结论亚细胞定位的不同推测到iRhom2的N末端缺失突变可能对其细胞内的定位有影响。     

Colchicine-induced cytoskeletal collapse and apoptosis in N-18 neuroblastoma cultures is rapidly reversed by applied S-100β

Brain connections depend on a stable association between dendrites and axons whose cytoskeleton is stabilized by the proteins MAP-2 and tau, respectively. The glial protein S-100β inhibits the phosphorylation by PKC of these two microtubule-associated proteins. In order to determine if exogenous S-100β can directly influence the cytoskeleton of living cells, cultures of N-18 cells (neuroblastoma clonal cell line) are treated for 30 min in serum-free medium with 10⁻⁶ M colchicine. In normal media, colchicine induces a rapid retraction of processes, membrane blebbing, nuclear collapse, and cell death. The observed cellular changes, due to cytoskeletal collapse after exposure to colchicine, are similar and consistent with the loss of processes and cytoplasmic blebbing seen in cells undergoing apoptosis. The addition of 20 ng/ml of S-100β after the initial 30-min exposure to colchicine prevents apoptosis, nuclear collapse and induces the regrowth of retracted processes. Cells were treated with the Hoechst Stain, a fluorescent marker that binds to nuclear material, to determine the occurrence of apoptosis in our cultures. In our control cultures, receiving no drugs, we found that 15.1% of the cells were apoptotic. When colchicine was added to the culture medium we found that 31.6% of the cells became apoptotic. However, when colchicine was followed by exposure to S-100β we found that only 5.4% of the cells were apoptotic. Our results suggest that extracellular application of the glial protein S-100β is sufficient to reverse colchicine-induced cytoskeletal collapse and prevent the resultant apoptosis of the cells. The increased levels of S-100β seen after brain injury and in certain neurological and psychiatric disorders may be considered as beneficial for brain recovery.     

异常黑胆质成熟剂与清除剂对H2O2诱导的淋巴细胞凋亡的影响

目的观察异常黑胆质成熟剂及清除剂对低浓度H2O2诱导的淋巴细胞凋亡的作用.方法不同浓度H2O2诱导体外培养的淋巴细胞,运用Hoechst33258染色法观察凋亡发生的凋亡指数,以确定H2O2诱导凋亡的剂量及作用时间;随后运用不同浓度异常黑胆质成熟剂及清除剂进行干预,观察其对淋巴细胞凋亡指数的影响.结果高浓度的异常黑胆质成熟剂及清除剂均能降低H2O2所致的淋巴细胞凋亡的凋亡指数. 结论异常黑胆质成熟剂及清除剂能够抑制H2O2诱导的淋巴细胞的凋亡.     

CD40 ligand inhibits Fas/CD95-mediated apoptosis of human blood-derived dendritic cells

Dendritic cells (DC) are considered to be the most potent antigen-presenting cells (APC) in the immune system. In this study, we analyzed the regulation of apoptosis of human peripheral blood-derived DC. DC were generated from adherent peripheral blood mononuclear cells that had been cultured for 7 days with granulocyte-macrophage colony-stimulating factor and interleukin-4. These cells displayed phenotypic properties of DC, including dendritic processes, expression of CD1a and lack of expression of CD14, and were very potent at presenting soluble antigens to T cells. Blood-derived DC were demonstrated to express the Fas/CD95 antigen and an agonist antibody to CD95 strongly induced apoptotic cell death in these cells. Soluble trimeric CD40 ligand potently inhibited both CD95-mediated and spontaneous apoptosis in DC. The data suggest that interactions between members of the tumor necrosis factor family of ligands expressed by T cells with their receptors on DC play an important role in the regulation of apoptosis in DC during antigen presentation and may, therefore, regulate the duration of T cell expansion and cytokine production.     

H33342释放法检测NK细胞和单核细胞的杀伤活性

本文采用DNA特异性荧光染料Hoechst dye No 33342(H33342)标记肿瘤细胞技术,建立了自动微量荧光分析法,检测NK细胞和单核细胞介导的杀伤效应。结果表明,损伤的靶细胞其DNA断裂后释放到上清液中,采用微量荧光仪可定量测定上清液中的荧光强度。效应细胞的杀伤活性与靶细胞释放的荧光强度呈正相关性。此外,该法的检测敏感性与经典的~(51)Cr释放法基本相似,是检测细胞杀伤活性的一种新型方法。     

PI3K/mTOR信号通路参与胰腺肿瘤干细胞样SP细胞生存增殖的调控

目的 分离鉴定胰腺癌中肿瘤干细胞样的侧群(SP)细胞亚群并探讨磷脂酰肌醇3-激酶/雷帕霉素靶蛋白(PI3K/mTOR)信号通路对其生存与增殖的调控.方法 应用流式分析检测5个胰腺癌细胞系中SP细胞的含量.观察加入PI3K/mTOR)R信号通路特异性抑制剂LY294002或雷帕霉素培养后胰腺癌细胞PANC-1中SP细胞的含量变化.通过平板集落形成试验,NOD-SCID小鼠异种移植成瘤试验和移植瘤的SP再次分析评价PANC-1 SP细胞的自我更新和分化潜能.采用 MTT试验和集落形成试验检测LY294002或雷帕霉素对分选的SP细胞和非SP细胞的抑制作用.结果 除了BXPC-3,其他胰腺癌细胞系都存在维拉帕米敏感的SP细胞.SP细胞具有较高的集落形成能力(SP细胞:43.7%±3.1%,非SP细胞8.3%±1.6%,P=0.000)和成瘤能力(至少100倍于非sP细胞),并能够发生不对称分裂生成非SP细胞.加入LY294002或雷帕霉素培养后PANC-1中sP细胞的含量明显降低(LY294002,7.60%±0.27% vs 1.90%±0.22%,P=0.000;雷帕霉素,7.60%±0.27%vs 1.14%±0.20%,P=0.000).与非SP细胞相比,LY294002及雷帕霉素均优先抑制sP细胞.结论 SP细胞富集胰腺癌肿瘤干细胞.PI3K/mT0R信号通路参与对其生长增殖的调控,可能成为根治胰腺癌的治疗新靶点.     

Sclerosing granuloma after short-term administration of Depot-Insulin Hoechst

Summary A 41-year-old male diabetic patient is described who exhibited firm infiltrative lesions on the abdomen and both buttocks after six injections of Depot-Insulin Hoechst. Initially there were no indications of an allergic or infectious origin. Histologic examination showed a sclerosing granuloma, suggesting a foreign body reaction. Oily substances could not be detected in a skin lesion or in the contents remaining in the insulin bottle. Skin tests suggested an allergic reaction to surfen. This side effect of insulin must be very rare. We could find only one report in the literature describing similar cases; no case was found in the files of Hoechst Limited. Data from literature concerning reactions to Depot-Insulin Hoechst and to surfen, one of its components, are reviewed.