Continuous culture of novel mitochondrial cells lacking nuclei

We isolated stable cell lines, designated as mitochondrial cells, from cybrids obtained by fusing mitochondria-less HeLa cells with platelets from patients with Leigh syndrome, a subtype of mitochondrial encephalomyopathy. The cells contain a pathogenic point mutation, T9176C, in the mitochondrial DNA. Hematoxylin-eosin staining, confocal fluorescent microscopy and flow cytometry in fixed or living cells showed that the majority of these mitochondrial cells lack nuclear DNA and nuclei, but contain active mitochondria. Despite the absence of nuclear DNA, these cells can be continuously generated in culture. Therefore, it is likely that they arise from the minority of cells which possess a nucleus.     

Denaturation and condensation of DNA in situ induced by acridine orange in relation to chromatin changes during growth and differentiation of Friend erythroleukemia cells

DNA in situ is progressively denatured when the cells or nuclei are treated with increasing concentration of acridine orange (AO). This transition can be monitored by flow cytometry as a decrease in green fluorescence. The complexes of denatured DNA and AO undergo immediate condensation and aggregation; this step is manifested by appearance of red luminescence and formation of precipitates that can be detected by electron microscopy. The precipitates form preferentially in heterochromatin as well as in ribosomes and polysomes. Their formation and further aggregation affects cellular light scatter properties in both the forward and right-angle direction. The AO-induced DNA denaturation and condensation was studied in nuclei of Friend erythroleukemia cells from exponentially growing, differentiated or quiescent cells. The DNA in nuclei of quiescent cells, from plateau-phase cultures, was the most sensitive to denaturation; it denatured (measured by changes in luminescence) at an AO concentration between 50 and 80 microM with the midpoint of the transition (Cd) at 70 microM. DNA in nuclei of differentiated cells (dimethyl-sulfoxide-induced erythroid differentiation) was more resistant (Cd = 77-83 microM), whereas DNA in exponentially growing cells was the most resistant (Cd = 86 microM). Extraction of proteins with 0.1 M HCl at 0 degree C abolished the differences between the cells and shifted the transition to a lower AO concentration (Cd = 46 microM). For comparison, the midpoint transitions representing condensation of free, nucleic acids measured as light scatter changes occurred at 13, 22, 31 and 53 microM of AO, for rRNA, tRNA, and denatured and native-calf thymus DNA, respectively. Denaturation and condensation of DNA, which can be induced by AO either in isolated nuclei or viable permeabilized or fixed cells provides a new approach to discriminate cell subpopulations with different chromatin structure by flow cytometry. The molecular mechanisms of this phenomenon are discussed.     

Palmitate induces structural alterations in nuclei of cardiomyocytes

The objective of this study was to examine the hypothesis that the alterations of cardiac nuclei, that has been noted in some cardiomyopathies, can be produced by palmitate, a saturated fatty acid present in high circulating concentrations in patients with conditions associated with a high probability of developing cardiomyopathy. Cardiomyocytes isolated from embryonic chick ventricle were maintained in culture for 72 h and then treated with palmitate, 100 microM for 24 h. Cells were stained with acridine orange or Giemsa and examined microscopically. Cell size and nuclear size were examined by forward light scatter during flow cytometry. Cells were permeabilized and their nuclei were stained with propidium iodide and examined by flow cytometry on populations of 10,000 cells. Cardiomyocytes treated with palmitate displayed changes in nuclear appearance as nuclei were larger, relative to cell size, with more intense acridine orange staining in a peripheral location. Nucleoli were often disrupted. Palmitate produced a significant (P < 0.001) and 17% increase in nuclear size compared to untreated cells using flow cytometry analysing forward light scatter to estimate nuclear and whole cells size. There were no significant changes in the size of the whole cell and ratio of nucleus to whole cell was significantly (P < 0.01) increased compared to control cells. Fluorescent activating cell sorting analysis of propidium iodide stained nuclei demonstrated that the nuclear enlargement was not due to cell mitosis as the proportion of nuclei in Go/G1, S or M was not changed by palmitate. In summary, these studies identify that palmitate can induce structural abnormalities of cardiomyocytes nuclei by producing increased nuclear size and nucleolar destruction.     

Rearrangement of nuclear ribonucleoproteins and extrusion of nucleolus-like bodies during apoptosis induced by hypertonic stress

Short-term hypertonic (HT) stress induces apoptotic cell death in human EUE cells in culture, as observed by electron microscopy, agarose-gel electrophoresis of low-molecular-weight DNA, DNA flow cytometry and annexin-V-propidium iodide double-staining. During HT-induced apoptosis, nuclear ribonucleoprotein (RNP)-containing structures undergo rearrangement, with the formation of Heterogeneous Ectopic RNP-Derived Structures (HERDS) which pass into the cytoplasm, as already reported for other examples of spontaneous and drug-induced apoptosis. Of special interest was the observation that nucleolus-like bodies (NLBs) which resemble morphologically nuclear functional nucleoli may be extruded into the cytoplasm of apoptotic cells and are observed inside the cytoplasmic fragments blebbing-out at the cell surface; these NLBs still contain immunodetectable nucleolar proteins (such as fibrillarin). This is an additional example of RNP-containing structures of nuclear origin which are extruded from the nucleus, in an almost "native" form, during apoptosis.     

Cisplatin treatment of NIH/3T3 cultures induces a form of autophagic death in polyploid cells

The effects induced by different concentrations (50, 75, 100 microM) of the cytostatic drug cisplatin (cDDP) in NIH/3T3 cells were analyzed. Sub-confluent cultures of this mouse fibroblast line, obtained after serum deprivation, showed the presence of aneuploid/polyploid cells with ploidy values ranging from 4c to 24c. DNA content cytofluorometry demonstrated that 50 and 75 microM cDDP induced a cytostatic effect; 100 microM concentration showed lower antiproliferative action. All treatments caused a partial cell detachment and apoptosis, the incidence of which appeared to be cDDP concentration-dependent. Ultrastructural and fluorescence microscopy integrated analyses of the still adherent cells demonstrated the presence of alternative degeneration patterns, especially in polyploid cells, with extensive modifications at both nuclear and cytoplasmic levels. There were events of micronucleation and phenomena of multilobulation and furrows of the nucleus that preceded the formation of heterogeneous fragments. These events were correlated, at cytoplasmic level, with actin reorganization and the appearance of autophagocytotic processes. In our cell model, the same pharmacological treatment was able to induce different cell death phenomena relating to cell dimension and ploidy. More actively proliferating cells (2c-4c DNA content) die throughout canonical apoptosis, while polyploid cells prevailingly degenerate by mechanisms partly referable to autophagic cell death.     

Uptake and quantification of intracellular concentration of lipophilic pro-oligonucleotides in HeLa cells

Several lipophilic prodrugs of oligonucleotides (T12 and T20) bearing enzymolabile protecting groups and labeled with fluorescein were synthesized. Their cellular uptake was studied by three different approaches using fluorescence: fluorescence microscopy, flow cytometry and spectrofluorometry. The corresponding prooligonucleotides (pro-oligos) were rapidly and efficiently taken up by HeLa cells and were found homogeneously in the cytoplasm and in the nucleus. The uptake was proportional to their relative lipophilicity and likely proceeded through a passive diffusion mechanism. Uptake followed a dose-response curve. This prooligo approach led to a 2-log increase of uptake in comparison with a T20 phosphorothioate oligonucleotide. Finally, an intracellular concentration of pro-oligo was estimated between 4 and 6 microM for an external concentration of 1 microM and up to 27 microM for an incubation at 10 microM.     

Enhancing the cellular uptake of Py-Im polyamides through next-generation aryl turns

Pyrrole-imidazole (Py-Im) hairpin polyamides are a class of programmable, sequence-specific DNA binding oligomers capable of disrupting protein-DNA interactions and modulating gene expression in living cells. Methods to control the cellular uptake and nuclear localization of these compounds are essential to their application as molecular probes or therapeutic agents. Here, we explore modifications of the hairpin γ-aminobutyric acid turn unit as a means to enhance cellular uptake and biological activity. Remarkably, introduction of a simple aryl group at the turn potentiates the biological effects of a polyamide targeting the sequence 5'-WGWWCW-3' (W =A/T) by up to two orders of magnitude. Confocal microscopy and quantitative flow cytometry analysis suggest this enhanced potency is due to increased nuclear uptake. Finally, we explore the generality of this approach and find that aryl-turn modifications enhance the uptake of all polyamides tested, while having a variable effect on the upper limit of polyamide nuclear accumulation. Overall this provides a step forward for controlling the intracellular concentration of Py-Im polyamides that will prove valuable for future applications in which biological potency is essential.     

Thymocyte K+, Na+ and water balance during dexamethasone- and etoposide-induced apoptosis.

The mechanism of apoptotic cell volume decrease was studied in rat thymocytes treated with dexamethasone (Dex) or etoposide (Eto). Cell shrinkage, i.e. dehydration, was quantified by using buoyant density of the thymocytes in a continuous Percoll gradient. The K+ and Na+ content of cells from different density fractions were assayed by flame emission analysis. Apoptosis was tested by microscopy and flow cytometry of acridine orange stained cells as well as by flow DNA cytometry. Treatment of the thymocytes with 1 microM Dex for 4-5.5 h or 50 microM Eto for 5 h resulted in the appearance of a new distinct high-density cell subpopulation. The cells from this heavy subpopulation but not those with normal buoyant density had typical features of apoptosis. Apoptotic increase of cell density was accompanied by a decrease in cellular K+ content, which exceeded the simultaneous increase in cellular Na+ content. Cellular loss of K+ contributed to most of the estimated loss of cellular osmolytes, but owing to the parallel loss of cell water, the decrease in cytosolic K+ concentration was less than one third. Due to gain of Na+ and loss of cell water the cytosolic Na+ concentration in thymocytes rose following treatment with Dex (5.5 h) or Eto (5 h) by a factor of about 3.6 and 3.1, respectively.     

Inhibitors of arachidonic acid metabolism reduce DNA and nuclear fragmentation induced by TNF plus cycloheximide in U937 cells

U937 human myeloid leukemia cells are induced to apoptosis by tumour necrosis factor (TNF) plus cycloheximide (CHX). We have analysed the effect of various inhibitors of the arachidonic acid (AA) metabolism on several features of this process. The formation of high molecular weight and oligonucleosomal DNA fragments as well as nuclear fragmentation were reduced by inhibitors of 5-lipoxygenase (BWA4C and BWB70C), 5-LO activating protein (MK-886), and cytosolic PLA2 (AACOCF3). None of these agents blocked the morphological changes detected by microscopy or flow cytometry, phosphatidylserine exposure on the cell surface or Caspase 3-like activation. AA also induced nuclear fragmentation at a concentration of 1-20 microM. However, the mechanisms by which these inhibitors act, remain unexplained since there was no 5-LO expression in the U937 cells and no AA release followed their stimulation with TNF plus CHX.     

Detection of an early cytomegalovirus antigen with two-color quantitative flow cytometry

An early cytomegalovirus (CMV) antigen was detected with a monoclonal antibody by two-color fluorescent flow cytometry. With the aid of a human diploid fibroblast cell strain, FLOW 2000, infected with the AD169 strain of CMV, the viral antigen and the DNA content of infected or uninfected cells were measured. There was no evidence of change in the cell-cycle distribution of the infected cells. The viral antigen was detected within 30 minutes following virus adsorption at 0.1 and 1.0 plaque-forming units/cells; and the percentage of positive cells increased with time and viral dosage. All stages of the cell cycle were susceptible to viral infection and the average fluorescence was greater than the background fluorescence. Flow cytometry detected the viral antigen earlier than conventional immunofluorescent microscopy and cell culture for CMV cytopathological effect (CPE). Ten bronchoalveolar lavages assayed by flow cytometry and conventional diagnostic procedures demonstrated that flow cytometry might be useful in early diagnosis for CMV infection.     

镉诱导HEK293细胞凋亡及其线粒体凋亡途径

本课题研究了氯化镉(CdCl_2)诱导HEK293细胞(人胚胎肾细胞系)的凋亡,初步探讨了凋亡过程中Caspase-3、Bcl-2的变化和凋亡诱导因子(AIF)的转移以及它们的意义。MTT法检测CdCl_2对HEK293细胞增殖的抑制作用;通过倒置显微镜、电镜、琼脂糖凝胶电泳、流式细胞术、激光共聚焦观察细胞凋亡;应用Western blot法和荧光免疫法测定Caspase-3酶原、Bcl-2蛋白的变化以及检测AIF蛋白在细胞中的定位。结果显示:CdCl_2对HEK293细胞具有显著的生长抑制作用,并呈明显的剂量和时间依赖性。在琼脂糖凝胶电泳中,显示有凋亡细胞特有的DNA梯状条带,其中30μmol/L作用6-9h梯状条带最为清晰,时间过长或浓度过高则梯状条带逐渐模糊,表明镉浓度过高或处理时间过长,细胞有坏死。流式细胞仪检测也印证了这一结果。形态学观察可见明显的细胞凋亡特征。同时线粒体膜电位明显下降,发现Caspase-3酶原蛋白、Bcl-2蛋白含量减少,并具有时间依赖性;另外检测到线粒体AIF向细胞核转移。而Bcl-2转染后有一定的抑制凋亡作用。实验结果提示,CdCl_2能够诱导HEK293细胞凋亡,线粒体损伤导致AIF转移与细胞色素c释放,从而引发的非Caspases与Caspases凋亡途径可能在镉引发的细胞凋亡过程中起重要作用,而Caspase-3, Bcl-2起着重要的调控作用。     

Glutathione is recruited into the nucleus in early phases of cell proliferation

We have studied the possible correlation between nuclear glutathione distribution and the progression of the cell cycle. The former was studied by confocal microscopy using 5-chloromethyl fluorescein diacetate and the latter by flow cytometry and protein expression of Id2 and p107. In proliferating cells, when 41% of them were in the S+G(2)/M phase of the cell cycle GSH was located mainly in the nucleus. When cells reached confluence (G(0)/G(1)) GSH was localized in the cytoplasm with a perinuclear distribution. The nucleus/cytoplasm fluorescence ratio for GSH reached a maximal mean value of 4.2 +/- 0.8 at 6 h after cell plating. A ratio higher than 2 was maintained during exponential cell growth. In the G(0)/G(1) phase of the cell cycle, the nucleus/cytoplasm GSH ratio decreased to values close to 1. We report here that cells concentrate GSH in the nucleus in the early phases of cell growth, when most of the cells are in an active division phase, and that GSH redistributes uniformly between the nucleus and the cytoplasm when cells reach confluence.     

Fluorescence in situ hybridization to interphase cell nuclei in suspension allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organization

Summary Fluorescence hybridization to interphase nuclei in liquid suspension allows quantification of chromosome-specific DNA sequences using flow cytometry and the analysis of the three-dimensional positions of these sequences in the nucleus using fluorescence microscopy. The three-dimensional structure of nuclei is substantially intact after fluorescence hybridization in suspension, permitting the study of nuclear organization by optical sectioning. Images of the distribution of probe and total DNA fluroescence within a nucleus are collected at several focal planes by quantitative fluorescence microscopy and image processing. These images can be used to reconstruct the three-dimensional organization of the target sequences in the nucleus. We demonstrate here the simultaneous localization of two human chromosomes in an interphase nucleus using two probe labeling schemes (AAF and biotin). Alternatively, dual-beam flow cytometry is used to quantify the amount of bound probe and total DNA content. We demonstrate that the intensity of probe-linked fluorescence following hybridization is proportional to the amount of target DNA over a 100-fold range in target content. This was shown using four human/hamster somatic cell hybrids carrying different numbers of human chromosomes and diploid and tetraploid human cell lines hybridized with human genomic DNA. We also show that populations of male, female, and XYY nuclei can be discriminated by measuring their fluores-cence intensity following hybridization with a Y-chromosome-specific repetitive probe. The delay in the increase in Y-specific fluorescence until the end of S-phase is consistent with the results recorded in previous studies indicating that these sequences are among the last to replicate in the genome. A chromosome-17-specific repetitive probe is used to demonstrate that target sequences as small as one megabase (Mb) can be detected using fluorescence hybridization and flow cytometry.     

Effect of all-trans-retinoic acid on mRNA binding protein p62 in human gastric cancer cells

p62 is a cancer-associated antigen binding to mRNA encoding insulin-like growth factor II that was isolated by immunoscreening a cDNA expression library with autoantibodies from patients with hepatocellular carcinoma (HCC). In the present study, multiple methods including flow cytometry, confocal laser-scanning microscope, electron microscope were used to characterize the effect of ATRA on BGC-823 cells, which presented two phenotypes of differentiation and apoptosis in cells treated with 1.0 and 50 microM ATRA, respectively. Interestingly, we found that p62 was cytoplasmic in location, but it significantly decreased in cytoplasm and appeared in nucleus of cells when the cells were treated with 50 microM all-trans retinoic acid (ATRA) for 5 days. Furthermore, proteomics approach on differential nucleus proteins showed that the up-regulation and/or down-regulation of cell cycle proteins and IGF binding proteins were involved in the apoptosis of BGC-823 cells induced by ATRA. These results suggest that there is a significant association between expression and distribution of p62 and the growth arrest of tumor cells, in which p62 is associated with cell apoptosis induced by ATRA.     

Potassium and sodium balance in U937 cells during apoptosis with and without cell shrinkage.

Staurosporine (STS) and etoposide (Eto) induced apoptosis of the human histiocytic lymphoma cells U937 were studied to determine the role of monovalent ions in apoptotic cell shrinkage. Cell shrinkage, defined as cell dehydration, was assayed by measurement of buoyant density of cells in continuous Percoll gradient. The K+ and Na+ content in cells of different density fractions was estimated by flame emission analysis. Apoptosis was evaluated by confocal microscopy and flow cytometry of acridine orange stained cells, by flow DNA cytometry and by effector caspase activity. Apoptosis of U937 cells induced by 1 muM STS for 4 h was found to be paralleled by an increase in buoyant density indicating cell shrinkage. An increase in density was accompanied by a decrease in K+ content (from 1.1 to 0.78 mmol/g protein), which exceeded the increase in Na+ content (from 0.30 to 0.34 mmol/g) and resulted in a significant decrease of the total K+ and Na+ content (from 1.4 to 1.1 mmol/g). In contrast to STS, 50 microM Eto for 4 h or 0.8-8 microM Eto for 18-24 h induced apoptosis without triggering cell shrinkage. During apoptosis of U937 cells induced by Eto the intracellular K(+)/Na+ ratio decreased like in the cells treated with STS, but the total K+ and Na+ content remained virtually the same due to a decrease in K+ content being nearly the same as an increase in Na+ content. Apoptotic cell dehydration correlated with the shift of the total cellular K+ and Na+ content. There was no statistically significant decrease in K+ concentration per cell water during apoptosis induced by either Eto (by 13.5%) or STS (by 8%), whereas increase in Na+ concentration per cell water was statistically significant (by 27% and 47%, respectively). The data show that apoptosis can occur without cell shrinkage-dehydration, that apoptosis with shrinkage is mostly due to a decrease in cellular K+ content, and that this decrease is not accompanied by a significant decrease of K+ concentration in cell water.     

Controlling the intracellular localization of fluorescent polyamide analogues in cultured cells

The intracellular distribution of fluorescent-labeled polyamides was examined in live cells. We showed that BODIPY-labeled polyamides accumulate in acidic vesicles, mainly lysosomes, in the cytoplasm of HCT116 colon cancer cells and human rheumatoid synovial fibroblasts (RSF). Verapamil blocked vesicular accumulation and led to nuclear accumulation of the BODIPY-labeled polyamide in RSFs. We infer that the basic amine group commonly found at the end of synthetic polyamide chains is responsible for their accumulation in cytoplasmic vesicles in mammalian cells. Modifying the charge on a polyamide by replacing the BODIPY moiety with a fluorescein moiety on the amine tail allowed the polyamide to localize in the nucleus of the cell and bypass the cytoplasmic vesicles in HCT116 cells.     

Phosphorylation at threonine-18 in addition to phosphorylation at serine-19 on myosin-II regulatory light chain is a mitosis-specific event

BACKGROUND: Cell division is an inevitable and vitally indispensable event in cell life, when the nucleus and cytoskeleton undergo profound reorganization. Cytoplasmic division (cytokinesis) is known to occur immediately after the end of nuclear division, when the nuclear envelope breaks down, and chromosomes condense and segregate, but its driving mechanism remains enigmatic. Myosin, particularly myosin-II, is thought to be required for cytokinesis as a force-generating element, the activity of which is mainly regulated through phosphorylations on its 20-kDa regulatory light chains (RLCs). MATERIALS AND METHODS: Multiparameter flow cytometric analysis was performed on fixed HeLa S3 cells (suspension culture cells) sequentially stained with the polyclonal antibody (termed PP1) against both phosphorylated sites (serine-19 and threonine-18) on the RLC, and with propidium iodide for DNA. "Positive" cells were sorted, followed by their microscopic examination. Fluorescence microscopy was employed to visualize the cell-cycle-dependent distribution of immunolabeled diphosphorylated RLCs in both HeLa S3 and adherent HeLa cells. RESULTS AND CONCLUSIONS: Doubly phosphorylated myosin RLCs were highly expressed in mitotic cells, suggesting the positive regulatory role of diphosphorylation in the redistribution of RLCs between daughter cells and then in cytokinesis. The increased immunofluorescence signal from the phosphorylated forms of RLC, together with flow cytometry, provides a clue with which to investigate the mechanisms governing the function of nonmuscle myosins during various cell motile events, including cytokinesis.     

Cytological characterization of isolated sperm cells of perennial ryegrass (Lolium perenne L.)

Summary The cytoplasmic content and the distribution of intramembrane particles (IMPs) of the plasma membrane of isolated sperm cells of perennial ryegrass (Lolium perenne L.) have been characterized using flow cytometry, transmission electron microscopy, confocal scanning laser microscopy and freeze-fracture studies. The isolated haploid sperm cells contain a variety of cell organelles with the exception of microtubules. Proplastids and plastids with starch were observed, although only rarely. Vacuoles containing remnants of organelles and stacked lamellae of endoplasmic reticulum with cytoplasmic inclusions were observed frequently, indicating that autophagy takes place. The number of mitochondria varies from 11 to 26 with an average of 17. Generally, the nucleus has a lobed shape and displays various interphasic stages of chromatin condensation. The analysis of the number of mitochondria and the nuclear state did not show evidence of sperm cell dimorphism. The cytological variability observed, could be explained by differences in developmental stages already present in vivo at the moment of isolation. No correlation between the number of mitochondria and the nuclear cross-sectioned area and/or the condensation state of the chromatin could be found. The density of intramembrane particles of the plasma membrane on the exoplasmic fracture face is more than twice that on the protoplasmic fracture face. That is the opposite of what was found for sporophytic cells of perennial ryegrass. These results are discussed in relation to the potential use of these cells for biotechnology and developmental studies.     

Nanosecond electric pulses penetrate the nucleus and enhance speckle formation

Nanosecond electric pulses generate nanopores in the interior membranes of cells and modulate cellular functions. Here, we used confocal microscopy and flow cytometry to observe Smith antigen antibody (Y12) binding to nuclear speckles, known as small nuclear ribonucleoprotein particles (snRNPs) or intrachromatin granule clusters (IGCs), in Jurkat cells following one or five 10 ns, 150 kV/cm pulses. Using confocal microscopy and flow cytometry, we observed changes in nuclear speckle labeling that suggested a disruption of pre-messenger RNA splicing mechanisms. Pulse exposure increased the nuclear speckled substructures by ∼2.5-fold above basal levels while the propidium iodide (PI) uptake in pulsed cells was unchanged. The resulting nuclear speckle changes were also cell cycle dependent. These findings suggest that 10 ns pulses directly influenced nuclear processes, such as the changes in the nuclear RNA-protein complexes.