Effects of hyperthermia, irradiation, and cytotoxic drugs on fluorescein isothiocyanate staining intensity for flow cytofluorometry

Measurement of fluorescein isothiocyanate (FITC) staining intensity of cultured lymphoblastoid cells following hyperthermia showed large increases without concomitant increases in nuclear protein. Similar measurements of cells following incubation with cytotoxic drugs showed fluorescent intensity increases that exceeded the increases in nuclear protein that were due to the cell cycle blocking action of the drug. The reverse, however, was true for cells following irradiation. In contrast, FITC staining intensity and nuclear protein measurements of cells proceeding through the cell cycle after removal of the cycle blocking agent showed nearly parallel changes, although there were reproducible minor differences, especially following blocking with hydroxyurea. These results suggest that FITC staining intensity is a function not only of nuclear protein content but also of stain access to the reaction sites of the protein constituents of the chromatin. Thus, it is possible that FITC staining may be used as a probe of changes in chromatin structure following experimental manipulation of cells in vitro or treatment of tumors in vivo.     

Nuclear protein following heat shock: protein removal kinetics and cell cycle rearrangements

Nuclear protein and DNA content of HeLa cells was determined as a function of time following hyperthermia by staining isolated nuclei with two fluorescent dyes: fluorescein isothiocyanate (FITC) for protein content and propidium iodide (PI) for DNA content. Bivariate FITC and PI histograms were obtained by flow cytometry. Univariate flow cytometric analysis was shown to be inadequate for this study, because some of the nuclear protein changes were due to cell cycle redistribution. Posthyperthermia cell kinetics could be divided into two distinct phases: an early phase characterized by the removal of heat-induced excess nuclear proteins with little or no cell progression through the cell cycle; and a late phase characterized by a redistribution of cells in the cell cycle resulting in an accumulation of cells in G2. The duration of these phases was dependent upon the hyperthermia dose. In the early phase, the rate of removal of excess nuclear protein was found to vary with heating time and temperature for time-temperature combinations which resulted in the same amount of excess nuclear protein. In the late phase, the cells blocked in G2 did not reduce their nuclear protein levels back to control values.     

Reagentless and regenerable immunosensor for monitoring of immunoglobulin G based on non-separation immunoassay

Based on the enhancement of fluorescein isothiocyanate (FITC) fluorescence caused by reactions between proteins, we developed a reagentless, regenerable and rapid immunosensing system to determine immunoglobulin G (IgG). Fluorescence intensity of the immobilized FITC depends on IgG concentration, ranging from 10 to 50 microg/ml, specifically, even with co-existing proteins. The response time is 30 min during steady-state measurement and is less than a minute during transient measurement. When the FITC-labeled protein A binds to IgG, the surrounding atmosphere of FITC becomes hydrophobic. Since the fluorescence intensity of fluorescent substances generally increases at a hydrophobic environment, FITC fluorescence intensity increases with the concentration of protein A bonding to IgG. This system is regenerable because the fluorescence enhancement repeatedly occurs every time the immobilized fluorescent reagent is immersed in sample solutions.     

Binding and internalization of heparin by vascular smooth muscle cells

Previous work from our laboratory has demonstrated that heparin specifically inhibits the proliferation of vascular smooth muscle cells in vivo and in vitro. In this paper, we examine the binding and mode of internalization of heparin by smooth muscle cells. For these studies, radiolabeled and fluoresceinated (FITC) heparin probes were synthesized that retained their antiproliferative capacity. Binding of 3H-heparin to these cells occurs via specific, high-affinity binding sites (Kd = 10(-9) M, 100,000 binding sites per cell). Approximately 80% of the heparin bound to the cell surface was shed into the culture medium within 2 hr. The heparin that was left on the cell surface was internalized with biphasic kinetics. Approximately 50% of the bound material was internalized within 2 hr. After this initial rapid uptake, the rate slowed substantially, with the remaining heparin requiring 1-2 days to be internalized. Binding and uptake of FITC heparin was monitored using video image intensification fluorescence microscopy. When smooth muscle cells were exposed to FITC heparin at 4 degrees C, a diffuse surface staining pattern was observed. After warming the cells to 37 degrees C, intensely fluorescent vesicles were seen superimposed over the diffuse surface staining within 2 min. After 15 min at 37 degrees C, numerous large punctate vesicles were seen inside the cell. After 2 hr these vesicles had concentrated in the perinuclear region. This pattern of uptake, when considered along with the presence of specific, high-affinity binding sites and the initial rapid uptake of 3H-heparin, suggests that heparin enters smooth muscle cells by both receptor-mediated and other endocytic pathways.     

A note on the demonstration of nuclear acidic HCl insoluble proteins in peripheral blood smears.

The present study deals with the conditions necessary for the specific demonstration of nuclear acidic HCl insoluble proteins is smeared peripheral leucocytes by means of a simple cytochemical procedures. These proteins can be very easily selectively demonstrated by staining with Azure C or Toluidine blue above pH 6, after the fixation of smears with methanol, extraction of histones, nucleic acids (with HCl, TCA) and treatment with formaldehyde. The double fixation with methanol and formaldehyde facilitated specific extraction of nuclear acidic HCl insoluble proteins of smeared leucocytes with pepsin. The nuclear acidic HCl insoluble proteins are present in leucocytes in interchromatin areas and their isoelectric point determined by cytochemical procedure is pH 6.     

Excess protein in nuclei isolated from heat-shocked cells results from a reduced extractability of nuclear proteins

An excellent correlation has been established between the quantity of protein associated with nuclei isolated from heat-shocked cells and the level of hyperthermic cell killing. However, controversy remains about whether increases in nuclear-associated protein result from a heat-induced migration of cytoplasmic proteins into the nucleus or because hyperthermia reduces the solubility of nuclear proteins in the detergent buffers commonly used to isolate nuclei. To address this controversy, the nuclear protein content was measured in whole and detergent-extracted cells before and following hyperthermia. It was found that hyperthermia caused no significant change in the nuclear protein content of whole, unextracted cells, and when fluorescently labeled proteins were microinjected into the cytoplasm no gross change in the selective permeability of the nuclear membrane to soluble proteins was observed during or following hyperthermia. Measurements in extracted cells showed that the detergent buffers removed protein from both the nucleus and cytoplasm of control, nonheated cells and that hyperthermia reduced the extractability of both nuclear and cytoplasmic proteins. The amount of protein found in nuclei isolated from heated cells approached that observed in nuclei within nonheated whole cells as the hyperthermic exposure was increased. Thus, the dose-dependent, two- to threefold increase in the protein content of nuclei isolated from heated cells represents a heat-induced reduction in the extractability of proteins normally present within cell nuclei and does not result from a mass migration of cytoplasmic proteins into the nucleus, although some specific proteins (e.g., the 70 KDa heat shock protein) do migrate to the nucleus following heat shock. Differential scanning calorimetry (DSC) measurements of whole cells, isolated nuclei, cytoplasts, and karyoplasts supported these conclusions and suggested that most of the detergent-insoluble proteins remaining in the nuclei and cytoplasm of heated cells are in their native state. Thus, a relatively small amount of denatured protein may be sufficient to initiate and sustain insoluble protein aggregates comprised of mostly native proteins. Analyses of the DSC data also implied that the previously identified critical target proteins, predicted to have a Tm of 46.0°C, are present in both the nucleus and cytoplasm. © 1996 Wiley-Liss, Inc.     

A faithful double stain of proteins in the polyacrylamide gels with Coomassie blue and silver

The conditions for prior fixing of proteins in a gel in order to attain a greater degree of faithful silver staining and sensitivity were examined. Fixing with formaldehyde enhanced the retention of proteins in a gel, particularly basic proteins such as histones and ribosomal proteins. The gel, one stained with Coomassie blue and following the removal of the free dyes, is capable of undergoing silver staining, and, moreover, the prestain considerably enhanced the staining intensity of various proteins differing in basicity in subsequent silver staining. Coupling the formaldehyde fixation with Coomassie brilliant blue prestain afforded a reproducible and pronounced stainability of various proteins.     

Propidium iodide staining correlates with the extent of DNA degradation in isolated nuclei.

Gradual degradation of internucleosomal DNA is a hallmark of apoptosis and can be simulated by incubating isolated thymocyte nuclei in the presence of 5 mM Mg2+ and 5 mM Ca2+ at 37 degrees C. Staining of nuclei with the DNA binding fluorescent dye propidium iodide (PI) showed that intensity of fluorescence correlated with the extent of DNA degradation. PI fluorescence was increased in the presence of DNase I. Thus it seems that the cleavage of chromatin DNA by DNase 1 or by the endogenous enzyme increases the accessibility of DNA for the dye. No increase of fluorescence was observed in the presence of the known inhibitors of the endogenous endonuclease: Zn2+ and EGTA. However, the presence of Zn2+ led to decreased staining of the nuclei by PI and caused a shift in the scatter profile of the nuclei, suggesting that a conformational change of chromatin is induced by this ion. This correlation between intensity of PI staining and DNA degradation should be useful to compare endogenous nuclease levels in lymphocyte populations.     

Induced thermotolerance to apoptosis in a human T lymphocyte cell line.

A brief exposure to elevated temperatures elicits, in all organisms, a transient state of increased heat resistance known as thermotolerance. The mechanism for this thermotolerant state is unknown primarily because it is not clear how mild hyperthermia leads to cell death. The realization that cell death can occur through an active process of self destruction, known as apoptosis, led us to consider whether thermotolerance provides protection against this mode of cell death. Apoptosis is a common and essential form of cell death that occurs under both physiological and pathological conditions. This mode of cell death requires the active participation of the dying cell and in this way differs mechanistically from the alternative mode of cell death, necrosis. Here we show that mild hyperthermia induces apoptosis in a human leukemic T cell line. This is evidenced by chromatin condensation, nuclear fragmentation and the cleavage of DNA into oligonucleosome size units. DNA fragmentation is a biochemical hallmark of apoptosis and requires the activation of an endogenous endonuclease. The extent of DNA fragmentation was proportional to the severity of heat stress for cells heated at 43 degrees C from 30 to 90 minutes. A brief conditioning heat treatment induced a resistance to apoptosis. This was evident as a resistance to DNA fragmentation and a reduction in the number of apoptotic cells after a heat challenge. Resistance to DNA fragmentation developed during a recovery period at 37 degrees C and was correlated with enhanced heat shock protein (hsp) synthesis. This heat-induced resistance to apoptosis suggests that thermotolerant cells have gained the capacity to prevent the onset of this pathway of self-destruction. An examination of this process in heated cells should provide new insights into the molecular basis of cellular thermotolerance.     

Changes in cyclin/proliferating cell nuclear antigen distribution during DNA repair synthesis

UV irradiation of quiescent human fibroblasts immediately triggers the appearance of the nuclear protein cyclin/proliferating cell nuclear antigen (PCNA) as detected by indirect immunofluorescent staining after methanol fixation. This was found to be independent of new synthesis of cyclin/PCNA by two-dimensional gel analysis and cycloheximide treatment. The intensity of the immunofluorescent staining of cyclin/PCNA observed in UV-irradiated cells corresponded with the UV dose used and with the DNA repair synthesis detected by autoradiography. The nuclear staining remains as long as DNA repair activity is detected in the cells. By extracting the UV-irradiated quiescent cells with Triton X-100 and fixing with formaldehyde, it was possible to demonstrate by indirect immunofluorescence rapid changes in the cyclin/PCNA population after irradiation, a small proportion (5-10%) of which is tightly associated to the nucleus as determined by high salt extraction. By incubating at low temperature and depleting the ATP pools of the cells before UV irradiation, we have demonstrated that the changes in cyclin/PCNA distribution observed involve at least two different nuclear associations.     

Fluroescent characteristics of drumsticks, drumstick-like projections, and other nuclear bodies in human blood cells.

The fluorescent properties of drumsticks, drumstick-like appendages, and other nuclear bodies in the polymorphonuclear leukocytes from six human males and females were studied with the aid of the quinacrine-mustard staining technique. Both brightly and weakly fluorescent drumsticks (in females) and drumstick-like bodies (in males) were observed, and they were readily differentiated on the basis of size, shape and, usually, fluorescent intensity. An analysis of the correlation between the extent of nuclear lobulation of the polymorphs and the corresponding fluorescent patterns of the adjoining drumsticks and drumstick-like bodies indicated that a possible change in the state and/or condensation of chromatin in these nuclear bodies might occur with increasing age of the polymorphs. Although the brightly fluorescent regions of the nuclei usually corresponded to the areas darkly stained with Giemsa, much finer patterns of differential staining of drumsticks and other nuclear bodies were obtained only by the fluorescent method.     

Reduction of levels of nuclear-associated protein in heated cells by cycloheximide, D2O, and thermotolerance.

Hyperthermia increases levels of nuclear-associated proteins in a manner that correlates with cell killing. If the increase in nuclear-associated proteins represents a lethal lesion then treatments that protect against killing by heat should reduce and/or facilitate the recovery of levels of the proteins in heated cells. This hypothesis was tested using three heat protection treatments: cycloheximide, D2O, and thermotolerance. All three treatments reduced levels of the proteins measured immediately following hyperthermia at 43.0 or 45.5 degrees C, with the greatest reduction occurring at 43.0 degrees C. In addition to reducing the proteins, thermotolerance facilitated the recovery of the proteins to control levels following hyperthermia. Thus thermotolerance may protect cells by both reducing the initial heat damage and facilitating recovery from that damage. Cycloheximide and D2O did not facilitate recovery of nuclear-associated proteins, suggesting that their protection against cytotoxicity related to the proteins resulted solely from their reduction of increases in levels of the proteins. All three treatments have been shown to stabilize cellular proteins against thermal denaturation. The results of this study suggest that the increase in nuclear-associated proteins may result from thermally denatured proteins adhering to the nucleus and that it is the ability of cycloheximide, D2O, and thermotolerance to thermostabilize proteins that reduces the increase in levels of the proteins within heated cells.     

Feulgen-Naphthol Yellow S cytophotometry of liver cells. The effect of formaldehyde induced shrinkage on nuclear Naphthol Yellow S binding.

1. In isolated liver cells, fixed in 4 per cent formaldehyde (NFS) for Feulgen-Naphthol Yellow S (F-NYS) staining of DNA and protein, nuclear shrinkage increases the nuclear concentration of solids to 46 per cent (w/v) before the start of the NYS staining. 2. When a fixative mixture of methanol:acetic acid:formalin (85:5:10 by volume; MAF) is used, the concentration of nuclear solids during NYS staining remain at a physiological level of 19 per cent. 3. By exposing liver cells to NFS for 10 to 120 seconds before fixation in MAF, increasing nuclear shrinkage can be induced with increasing pretreatment in NFS. Nuclear NYS binding decreases in parallel with the decreasing nuclear volume in cells thus treated. As the shrinkage induced reduction in NYS binding may vary with the net charge of nuclear non-histone proteins, MAF fixation must be preferred for quantitative determinations of nuclear non-histone protein in F-NYS stained, isolated cells. 4. Fixation in MAF offers the same advantages as NFS fixation as regards the small loss of proteins during the Feulgen staining procedure and the excellent reproducibility of the F-NYS staining. Storage of MAF fixed cells in the fixative for a few days does not alter their F-NYS staining properties. 5. In MAF fixed, F-NYS stained cells there is no NYS binding to histone basic amino acid residues.     

Changes in nuclear phenotypes following cold shock in Panstrongylus megistus (Burmeister)

The nuclear phenotypes of Malpighian tubule epithelial cells of 5th instar male nymphs of the blood-sucking insect Panstrongylus megistus were studied immediately after a short (1 h) cold shock at 0 degrees C, and 10 and 30 days later. The objective was to compare the responses to a cold shock with those known to occur after hyperthermia in order to provide insight into the cellular effect of cold in this species. Nuclei which usually exhibited a conspicuous Y chromosome chromocenter were the most frequent phenotype in control and treated specimens. Phenotypes in which the heterochromatin was unravelled, or in which there was nuclear fusion or cell death were more abundant in the shocked specimens. Most of the changes detected have also been found in heat-shocked nymphs, except for nuclear fusion which generates giant nuclei and which appeared to be less effective or necessary than that elicited after heat shock. Since other studies showed that a short cold shock does not affect the survival of more than 14% of 5th instar nymphs of P. megistus with domestic habit and can induce tolerance to a prolonged cold shock, heat shock proteins proteins are probably the best candidates for effective protection of the cells and the insects from drastic damage caused by low temperature shocks.     

Fluorimetric measurements and chromatin condensation patterns of nuclei from 3T3 cells throughout G1

Using two cytological methods based on nuclear morphology, quinacrine dihydrochloride (QDH) staining and premature chromosome condensation (PCC), it has been possible to identify cell cyle positions within G1 of growing and arrested 3T3 cells. The fluorescent intensity of QDH-stained interphase cells appears to decrease as the cells pass from mitosis to S phase. Likewise, the length and thickness of prematurely condensed chromatids can be related to the cells' position within the G1 period. Data are presented that deal with three interrelated topics: (1) We determined by fluorometric measurements of nuclei from 3T3 cells that the visual observation of the decrease in QDH fluorescence during G1 reflects an actual decrease in total fluorescence and not a dispersion of the fluorescent chromatin in a larger nuclear area. (2) We correlated the results obtained by QDH staining with those of PCC on the same cell samples blocked in G1 by different conditions. Serum-starved and contact-inhibited cell nuclei had the highest intensity, hydroxyurea-treated ones had the lowest intensity, while that of isoleucine-deprived cells was in between. The same relative order of G1 positions was obtained based on PCC morphology. Thus, both methods monitor the state of chromatin condensation and can be used to identify cell cycle position within G1.(3) We showed with both methods that the states of chromatin resulting from the various G1 blocking conditions differ from each other.     

Cell-cycle dependence of heat-induced interphase death in mouse L5178Y cells.

Cell lysis and eosin staining were observed in L5178Y cells within the first 3 h of post-hyperthermia incubation at 37 degrees C, after which both leveled to a plateau. Lysis and eosin staining were proportional to the severity of heat in asynchronous cells, whereas it was maximum in the most heat-sensitive M phase, intermediate in S, and least in heat-resistant G1 for the same heat treatment. Further, leakage of labeled [3H]thymidine and a decrease in radioactivity retained within heated cells coincided with an increase in eosin staining, indicating that the dye uptake was due to membrane damage. It was presumed that the eosin-stained fraction represented dead cells. The percentage eosin-stained cells reached a plateau, and this level was used to determine survival; when the results were compared with those obtained by the colony formation method, they were identical. By comparing the two survival assay methods we concluded that cell death after hyperthermia in L5178Y cells is mainly by interphase death in all phases of the cell cycle. The reasons for this conclusion are that a reduction in survival could be detected within one generation of L5178Y cells by the eosin staining method, and the survival values obtained by this method were identical to those obtained by the colony formation method.     

Hyperthermia induces apoptosis in thymocytes.

Mild hyperthermia (43 degrees C for 1 h) induces extensive double-stranded DNA fragmentation and, at a later time, cell death in murine thymocytes. The cleavage of DNA into oligonucleosome-sized fragments resembles that observed in examples of apoptosis including radiation-induced death of thymocytes. Following hyperthermia, incubation at 37 degrees C is necessary to detect DNA fragmentation, although protein and RNA synthesis do not seem to be required. Two protein synthesis inhibitors, cycloheximide and emetine, and two RNA synthesis inhibitors, actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, do not inhibit DNA fragmentation or cell death in heated thymocytes at concentrations which significantly block these effects in irradiated thymocytes. We have used this difference in sensitivity to show that the DNA fragmentation induced in thymocytes which are irradiated and then heated seems to be caused only by the heating and not by the irradiation.     

Nature of the death of thymus lymphocytes as affected by different kinds of harmful factors

It was shown that thymus lymphocyte death caused by the effect of dithiobisdinitrobenzoic acid, hyperthermia, and osmotic shock is not accompanied by ordered chromatin degradation. Protein synthesis inhibitors do not prevent thymocyte death under these effects. The authors discuss the kinds of lymphoid cell death which depend upon the nature of the damaging factor and differ both in morphology and the character of nuclear material degradation.     

流感病毒感染诱导MDCK细胞凋亡的研究

用荧光染色、DNA凝胶电泳等方法检测了A型流感病毒株A1／京防86－1和B型流感病毒株B/沪防93－1诱导狗肾体代细胞(MDCKcells)的凋亡情况,并采用MTT法和流式细胞仪比较了这2株病毒对MMCK细胞的毒力和凋亡诱导能力水平。结果显示：病毒感染6h后,细胞DNA发生断裂,病毒感染12h后,可见明显的染色质凝聚;在一定范围内,细胞凋亡强度表现出明显的时间和剂量依赖关系;并且,A型流感病毒株的毒力和调亡诱导能力均强于B型流感病毒株。实验结果表明：流感病毒感染引起的细胞死亡主要是通过调亡实现的,毒力不同的流感病毒株诱导细胞调亡的能力不同。