Optical studies of the interaction of 33258 Hoechst with DNA,chromatin, and metaphase chromosomes

The interaction of the bisbenzimidazole dye 33258 Hoechst with DNA and chromatin is characterized by changes in absorption, fluorescence, and circular dichroism measurements. At low dye/phosphate ratios, dye binding is accompanied by intense fluorescence and circular dichroism and exhibits little sensitivity to ionic strength. At higher dye/phosphate ratios, additional dye binding can be detected by further changes in absorptivity. This secondary binding is suppressed by increasing the ionic strength. A-T rich DNA sequences enhance both dye binding and fluorescence quantum yield, while chromosomal proteins apparently exclude the dye from approximately half of the sites available with DNA. Fluorescence of the free dye is sensitive to pH and, below pH 8, to quenching by iodide ion. Substitution of 5-bromodeoxyuridine (BrdU) for thymidine in synthetic polynucleotides, DNA, or unfixed chromatin quenches the fluorescence of bound dye. This suppression of dye fluorescence permits optical detection of BrdU incorporation associated with DNA synthesis in cytological chromosome preparations. Quenching of 33258 Hoechst fluorescence by BrdU can be abolished by appropriate alterations in solvent conditions, thereby revealing changes in dye fluorescence of microscopic specimens specifically due to BrdU incorporation.     

Mechanism of radiosensitization by halogenated pyrimidines: bromodeoxyuridine and beta-arabinofuranosyladenine affect similar subsets of radiation-induced potentially lethal lesions in plateau-phase Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells were grown to plateau phase in the presence of various amounts of bromodeoxyuridine (BrdU) and treated after irradiation with beta-arabinofuranosyladenine (ara-A), an inhibitor of DNA and potentially lethal damage (PLD) repair, in order to investigate the importance of repair reactions in general and of PLD repair, in particular, on the mechanism of radiosensitization by halogenated pyrimidines. The degree of BrdU-mediated radiosensitization observed in ara-A-treated cells was compared to that of cells incubated after irradiation in the absence of ara-A. A substantial reduction in BrdU-mediated radiosensitization was observed in cells treated with ara-A at concentrations that, when given alone, produced maximum potentiation in cell killing (500-1500 microM). The residual BrdU-mediated radiosensitization observed at high levels of thymidine replacement could be explained by a BrdU-mediated increase in DNA and chromosome damage induction per gray. These findings are similar to those reported previously for a repair-deficient mutant of CHO cells, the xrs-5 cell line, and consistent with the hypothesis that BrdU-mediated radiosensitization has two distinct components, one that derives from an increase in damage induction per gray, and a second one that derives from an effect of BrdU on the repair of radiation-induced damage. It is proposed that the reduction in BrdU-mediated radiosensitization observed in ara-A-treated cells is the result of ara-A-mediated expression of radiation damage, the repair of which would have been otherwise modulated by BrdU. Since ara-A is known to act by fixing a form of radiation-induced PLD (alpha-PLD), we further propose that BrdU acts by fixing alpha-PLD. A synergistic effect in the potentiation of cell killing was observed between ara-A and BrdU when ara-A was given at concentrations below 100 microM. This result suggests that a benefit may be expected in the clinic from the combined application of halogenated pyrimidines with repair inhibitors, if administered at a carefully screened range of concentrations.     

Microfluorometric analysis of cellular DNA following incorporation of bromodeoxyuridine.

Bromodeoxyuridine (BrdU) incorporation into cellular DNA has a differential effect on the cell-associated fluorescence of several DNA-specific dyes. After cells were treated with BrdU, flow microfluorometry was used to study the relative increase or decrease influorescence of stained cells. Bromodeoxyuridine incorporation into CHO cells increased the fluorescence of mithramycin-, olivomycin-, or chromomycin-stained cells, decreased that of propidium iodide-stained cells, and had little, if any, effect on the fluorescence of acriflavine Feulgen-stained cells. Changes in relative fluorescence of cell associated dyes are due to changes in the amounts of dye bound to cells with BrdU-substituted DNA. Colorimetric and absorbance measurement of DNA content showed that BrdU does not alter the diploid DNA content of CHO cells; however, BrdU induces perturbations in the distribution of cells about the cell cycle which cause an increase in average DNA content.     

Flow cytometric analysis of chromosomes and cells using a modified BrdU-Hoechst method

Chromosomes and interphase cells were harvested from cultures of the Chinese hamster line B14 F28 grown in medium containing BrdU up to four cell cycles and stained with the fluorescent dye 33342 Hoechst for flow cytometry. The newly synthetized BrdU-DNA is not stainable by the Hoechst dye which is highly specific for thymidine. The temporal development of the DNA fluorescence after addition of BrdU to the growth medium has been investigated. The chromosomal fluorescence intensity is reduced one step per generation. The extent of the intensity decrease by BrdU incorporation is proportional to the amount of new DNA and it is realized by repeated measurement following an UV-exposure. This UV-illumination stops the quenching by BrdU of the Hoechst stain induced DNA fluorescence. Therefore, the entire DNA content of these chromosomes now becomes measurable. The obtained intensity gain serves as a measure of the extent of the previous BrdU caused intensity shift. In this way we could establish 3 successive mitoses. Principally, this method is suitable also for measurement of whole cells in order to obtain both the number of generations in the experimental period and the phase distribution of the cell cycle.     

Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells

BACKGROUND: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. METHODS: Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. RESULTS: Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. CONCLUSIONS: Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability.     

Identification of late DNA-replicating regions in Chinese hamster chromosomes using a BrdU-giemsa technique

Asynchronous Chinese hamster cells were labelled with BrdU for 3 h prior to harvesting the metaphase cells. The late DNA replicating sites became unifilarly BrdU-substituted as compared to the earlier replicating sites having a normal DNA constitution. Those late replicating sites were identified by pale coloration or dot formation after treatment with 1.0 M Na-phosphate solution (adjusted to pH 9.0 with supersaturated amount of NaHCO₃ and at a temperature of 69–75° C) and staining with Giemsa dye. Using this technique, nuclei with incorporated BrdU could be distinguished from nuclei that had not incorporated BrdU. — One of the advantages of using this technique for identification of late DNA replicating sites is that cells are treated continuously with BrdU for a short period of time before harvesting and only one sampling, rather than a series of samplings, is required to achieve a clear-cut result.     

Optical studies of the interaction of 4′-6-diamidino-2-phenylindole with DNA and metaphase chromosomes

The optical absorption and fluorescence characteristics of 4-6-diamidino-2-phenylindole (DAPI) with DNA and chromosomes were studied. There is a decrease in extinction coefficient and shift in the absorption spectra to a higher wavelength when the dye binds to DNA. The fluorescence of DAPI is enhanced by both A-T and G-C base-pairs. The enhancement by A-T rich is significantly greater than by G-C rich DNA. The dye produces a localized bright fluorescence in centromeric regions of mouse chromosomes and the constrictions of human chromosomes 1 and 16; these regions are known to contain A-T rich DNA and show dull fluorescence when treated with quinacrine. This dye may be useful for identifying A-T rich region in chromosomes. The fluorescence of DAPI bound to polynucleotides or chromosomes is partially quenched by the introduction of BrdU. This suppression of dye fluorescence allows optical detection of sister chromatid exchanges and chromosome region containing DNA with an unequal distribution of thymidine between polynucleotide chains after BrdU incorporation.     

UV dose-dependent increase in the Hoechst fluorescence intensity of both normal and BrdU-DNA

If the DNA nucleoside thymidine is replaced by bromodeoxyuridine, the fluorescence of the nuclei of Hoechst-stained cells is quenched. The decrease of fluorescence intensity determined by flow cytometry and fluorometry is neutralized independent of the degree of BrdU substitution by an UV-exposure with a dose of 5-10 kJ/m2 to the unfiltered spectrum of a 100 W mercury high-pressure lamp. This dose is equivalent to that obtained in fluorescence microscopy after exposure for about 1 s. We suppose that this approximate matching of the intensities both of normal and BrdU in the DNA resulting in no further quenching. However, the fluorescence intensity of normal Hoechst-stained DNA also is increased by a previous exposure to UV light. We explain the time pattern of the Hoechst fluorescence in the course of an exposure with constant dose rate, by the superimposition of the well-known bleaching by an additional increase of the fluorescence intensity. Our results suggest that the UV-exposure of Hoechst dye creates a brightly fluorescing photoproduct which differs spectroscopically from the original dye. This product is stable in the dark and seems to fluorochrome DNA only if it is formed when the Hoechst dye is bound to DNA, thus increasing the nuclear fluorescence. Phosphorescence was not found.     

On the mechanism of differential giemsa staining of BrdU-substituted chromatids

This paper analyses the effect of acid hydrolysis on the differential Giemsa staining of 5-bromo-2deoxyuridine (BrdU) substituted chromatids in human and plant chromosomes, after treatment with a fluorochrome and light. Human lymphocytes and Allium cepa L. root tips were grown in BrdU for two or three cell cycles. Lymphocyte spreadings and meristem squashes were treated with fluorochrome Hoechst 33258, exposed to sunlight, hydrolysed with 5N HCl and stained with Giemsa. This acid hydrolysis improves the differential staining of BrdU substituted and non-substituted chromatin. It also allows the differentiation of sister chromatids with the DNA specific dye azure-A.     

Most Anti-BrdU Antibodies React with 2′-Deoxy-5-Ethynyluridine — The Method for the Effective Suppression of This Cross-Reactivity

5-Bromo-2′-deoxyuridine (BrdU) and 2′-deoxy-5-ethynyluridine (EdU) are widely used as markers of replicated DNA. While BrdU is detected using antibodies, the click reaction typically with fluorescent azido-dyes is used for EdU localisation. We have performed an analysis of ten samples of antibodies against BrdU with respect to their reactivity with EdU. Except for one sample all the others evinced reactivity with EdU. A high level of EdU persists in nuclear DNA even after the reaction of EdU with fluorescent azido-dyes if the common concentration of dye is used. Although a ten-time increase of azido-dye concentration resulted in a decrease of the signal provided by anti-BrdU antibodies, it also resulted in a substantial increase of the non-specific signal. We have shown that this unwanted reactivity is effectively suppressed by non-fluorescent azido molecules. In this respect, we have tested two protocols of the simultaneous localisation of incorporated BrdU and EdU. They differ in the mechanism of the revelation of incorporated BrdU for the reaction with antibodies. The first one was based on the use of hydrochloric acid, the second one on the incubation of samples with copper(I) ions. The use of hydrochloric acid resulted in a significant increase of the non-specific signal. In the case of the second method, no such effect was observed.     

4-Thio-5-bromo-2'-deoxyuridine: chemical synthesis and therapeutic potential of UVA-induced DNA damage

4-Thio-5-bromo-2'-deoxyuridine (3a) is prepared from 5-bromo-2'-deoxyuridine (BrdU) and its key properties are explored. The thionucleoside (3a) can react readily with monobromobimane and produces high fluorescence. 3a has UV maximum absorption at 340 nm and can be incorporated into cellular DNA. The cells containing 3a become sensitive to UVA light, offering therapeutic potential for UVA-induced cell killing.     

UV dose-dependent increase in the hoechst fluorescence intensity of both normal and BrdU-DNA

Summary If the DNA nucleoside thymidine is replaced by bromodeoxyuridine, the fluorescence of the nuclei of Hoechst-stained cells is quenched. The decrease of fluorescence intensity determined by flow cytometry and fluorometry is neutralized independent of the degree of BrdU substitution by an UV-exposure with a dose of 5–10 kJ/m² to the unfiltered spectrum of a 100 W mercury high-pressure lamp. This dose is equivalent to that obtained in fluorescence microscopy after exposure for about 1 s. We suppose that this approximate matching of the intensities both of normal and BrdU-substituted cells is caused by the splitting-off of bromine from BrdU in the DNA resulting in no further quenching. However, the fluorescence intensity of normal Hoechst-stained DNA also is increased by a previous exposure to UV light. We explain the time pattern of the Hoechst fluorescence in the course of an exposure with constant dose rate, by the superimposition of the well-known bleaching by an additional increase of the fluorescence intensity. Our results suggest that the UV-exposure of Hoechst dye creates a brightly fluorescing photoproduct which differs spectroscopically from the original dye. This product is stable in the dark and seems to fluorochrome DNA only if it is formed when the Hoechst dye is bound to DNA, thus increasing the nuclear fluorescence. Phosphorescence was not found.Non-standard abbreviations BrU-cells 5-bromodeoxyuridine holding S-180 cells - T-cells S-180 cells with normal (thymine) DNA - ICP pulse cytophotometer     

Quantification of DNA synthesis in multicellular organisms by a combined DAPI and BrdU technique

The development of a novel method to detect and quantify mitotic activity in multicellular organisms is reported. The method is based on the combinatorial use of 4',6-diamidino-2-phenylindole (DAPI) as a dye for the specific staining of DNA and the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) as a marker for DNA synthesis. It is shown that on nitrocellulose filters, the amount of DNA can be determined by DAPI as a prerequisite for the subsequent quantification of mitotic activity by BrdU. As a model system to prove the applicability of this technique, the blood fluke Schistosoma mansoni has been used. It is demonstrated that the DNA synthesis rate is higher in adult female schistosomes than in adult males. Furthermore, dimethyl sulfoxide, a widely used solvent for many mitogens and inhibitors of mitosis, has no influence on mitotic activity in adult schistosomes.     

The action of caffeine on X-irradiated HeLa cells. X. Depressed recovery from potentially lethal damage in cells containing 5-bromodeoxyuridine

HeLa S3 cells were sensitized to the lethal action of 220-kV X rays by partially replacing the thymidine in their DNA with 5-bromodeoxyuridine (BrdU). To examine the expression of and recovery from potentially lethal radiation damage (PLD), both BrdU-grown and control cells were treated with 4 mM caffeine for increasing times up to 2 days, either immediately after irradiation or after increasing delays up to 28 h. When the same dose of X rays (3 Gy) was applied to BrdU-grown and control cells, the difference in survival that is found in the absence of caffeine disappeared after about 30 h of incubation in its presence; when isosurvival doses were applied (BrdU-grown cells, 2.5 Gy; control cells, 4 Gy), the control cells suffered more killing. When treatment with caffeine was delayed for progressively longer times after both groups of cells received 3 Gy, the control cells achieved a higher level of survival. These results indicate that the increased radiation sensitivity of cells containing BrdU derives from a decreased ability to repair PLD.     

Assessment of cell proliferation and muscular structure following surgical tongue volume reduction in pigs

Objectives: Tongue volume reduction is an adjunct treatment in several orofacial orthopaedic procedures for various craniofacial deformities; it may affect structural reconstitution and functional recovery as a result of the repair process. The aim of this study was to investigate myogenic regeneration and structural alteration of the tongue following surgical tongue volume reduction. Materials and methods: Five 12‐week‐old sibling pairs of Yucatan minipigs (three males and two females) were used. Midline uniform glossectomy was performed on one of each pair (reduction); siblings had identical incisions without tissue removal (sham). All pigs were raised for a further 4 weeks and received 5‐bromo‐2‐deoxyuridine (BrdU) injection intravenously 1 day before killing. Tissue sections of tongues were stained with anti‐BrdU antibody to evaluate numbers of replicating cells. Haematoxylin and eosin plus trichrome staining were performed to assess muscular structure. Results: Reduction tongues contained significantly more BrdU+ cells compared to sham tongues (P < 0.01). However, these BrdU+ cells were mostly identified in reparative connective tissues (fibroblasts) rather than in regenerating muscle tissue (myoblasts). Trichrome‐stained sections showed disorganized collagen fibres linked to few intermittent muscle fibres in the reduction tongues. These myofibres presented signs of atrophy with reduced perimysium and endomysium. Matrix between reduced perimysium and endomysium was filled with fibrous tissue. Conclusions: Fibrosis without predominant myogenic regeneration was the major histological consequence of surgical tongue volume reduction.     

Cell cycle progression in denV-transfected murine fibroblasts exposed to ultraviolet radiation.

Repair-proficient murine fibroblasts transfected with the denV gene of bacteriophage T4 repaired 70-80% of pyrimidine dimers within 24 h after exposure to 150 J/m2 ultraviolet radiation (UVR) from an FS-40 sunlamp. Under the same conditions, control cells repaired only about 20% of UVR-induced pyrimidine dimers. After UVR exposure, both control and denV-transfected cells exhibited some degree of DNA-synthesis inhibition, as determined by flow cytometric analysis of cell-cycle kinetics in propidium iodide-stained cells. DenV-transfected cells had a longer and more profound S phase arrest than control cells, but both control and denV-transfected cells had largely recovered from UVR effects on cell-cycle kinetics by 48 h after UVR exposure. Inhibition of DNA synthesis by UVR was also measured by determining post-UVR incorporation of bromodeoxyuridine (BrdU). The amount of BrdU incorporated was quantitated by determining with flow cytometry the quenching of Hoechst dye 33342 by BrdU incorporated in cellular DNA. DenV-transfected cells showed more marked inhibition of BrdU incorporation after low fluences of UVR than control cells. Differences between denV-transfected and control cells in cell-cycle kinetics following UVR exposure may be related to differences in mechanisms of repair when excision repair of pyrimidine dimers is initiated by endonuclease V instead of cellular repair enzymes.     

NK cell-induced cytotoxicity is dependent on a Ca2+ increase in the target

In previous work we showed that programmed cell death (PCD) in thymocytes is mediated by a sustained increase in cytosolic Ca2+ concentration, resulting in the activation of an endogenous endonuclease, DNA fragmentation, and cell death. In this study we investigated the roles of Ca2+ and DNA fragmentation in target cell killing by natural killer (NK) cells. The effector cells induced a rapid, sustained increase in cytosolic Ca2+ concentration in Jurkat target cells. Buffering the target cell cytosolic Ca2+ with the Ca2(+)-selective dye, quin-2, prevented target cell killing. Extensive DNA fragmentation was associated with killing in every target tested, and this response was also blocked by quin-2. The endonuclease inhibitor, aurintricarboxylic acid, inhibited both DNA fragmentation and killing without influencing the Ca2+ increase in target cells. Thus, it is concluded that NK cell killing depends on a Ca2+ increase and appears to involve endogenous endonuclease activation in target cells.     

Simultaneous production of R-bands and either replication patterns or sister chromatid differentiation

Summary The dye triplet chromomycin/methyl green/DAPI and some related dye combinations were applied to bromodeoxyuridine (BrdU)-substituted and non-substituted chromosomes. In both kinds of chromosome preparations, tristaining followed by observation at pH 7 resulted in well defined R-bands (excitation wavelength 436 nm) and a mixed DA-DAPI/Q-banding pattern (360 nm). Two approaches have given satisfactory expression both of reverse bands and of the differential BrdU-substitution present in metaphase chromosomes: (1) direct tri-staining at pH 7 and mounting of the preparations at pH 11; or (2) pretreatment of the preparations with Hoechst 33258 plus UV and hot buffer followed by tri-staining and mounting at pH 7. These methods should prove useful for routine chromosome analysis and, in combination with BrdU-labelling, in studies of chromosome structure and replication.     

Mechanisms of contact-mediated killing of yeast cells on dry metallic copper surfaces

Surfaces made of copper or its alloys have strong antimicrobial properties against a wide variety of microorganisms. However, the molecular mode of action responsible for the antimicrobial efficacy of metallic copper is not known. Here, we show that dry copper surfaces inactivate Candida albicans and Saccharomyces cerevisiae within minutes in a process called contact-mediated killing. Cellular copper ion homeostasis systems influenced the kinetics of contact-mediated killing in both organisms. Deregulated copper ion uptake through a hyperactive S. cerevisiae Ctr1p (ScCtr1p) copper uptake transporter in Saccharomyces resulted in faster inactivation of mutant cells than of wild-type cells. Similarly, lack of the C. albicans Crp1p (CaCrp1p) copper-efflux P-type ATPase or the metallothionein CaCup1p caused more-rapid killing of Candida mutant cells than of wild-type cells. Candida and Saccharomyces took up large quantities of copper ions as soon as they were in contact with copper surfaces, as indicated by inductively coupled plasma mass spectroscopy (ICP-MS) analysis and by the intracellular copper ion-reporting dye coppersensor-1. Exposure to metallic copper did not cause lethality through genotoxicity, deleterious action on a cell's genetic material, as indicated by a mutation assay with Saccharomyces. Instead, toxicity mediated by metallic copper surfaces targeted membranes in both yeast species. With the use of Live/Dead staining, onset of rapid and extensive cytoplasmic membrane damage was observed in cells from copper surfaces. Fluorescence microscopy using the indicator dye DiSBaC(2)(3) indicated that cell membranes were depolarized. Also, during contact-mediated killing, vacuoles first became enlarged and then disappeared from the cells. Lastly, in metallic copper-stressed yeasts, oxidative stress in the cytoplasm and in mitochondria was elevated.     

Simultaneous detection of DNA synthesis, activation and cytokine secretion in collagen II (250-270)-activated T lymphocytes by flow cytometry

T cell activation and secretion of cytokines from activated peripheral blood mononuclear cells (PBMC) in culture have traditionally been measured by 3H-thymidine incorporation for assessment of cell proliferation. However, this method has many disadvantages that limit its usage in analyzing antigen-specific T responses, because of the low specific frequencies of the cells. Collagen II (250-270) may be an important autoantigen involved in the pathology of rheumatoid arthritis (RA). To further study the specific T cells response to CII 250-270, we developed an improved method for measuring lymphocyte proliferation and activation, and intracellular cytokine production, by flow cytometry at the single cell level. BrdU, an analog of thymidine, was incorporated into cellular DNA as a marker of individual cell proliferation. The cells were fixed and permeabilized, and a monoclonal antibody against BrdU conjugated with a fluorescent dye was used to measure BrdU incorporation. A Tris staining technique for the simultaneous determination of cell surface activation markers (CD69 or CD25) and intracellular cytokine production was also used and the parameters were assessed by 3-color flow cytometry. Optimal conditions were selected to improve the sensitivity and specificity of the assays. This method allowed simultaneous detection of lymphocytic DNA synthesis, phenotype analysis and cytokine production at the single cell level, and thus it may be a useful tool for analyzing immune responses.