Reverse differential staining of sister chromatids induced by Hoechst plus black light and endonuclease

A differential Giemsa staining between sister chromatids was obtained by treating chromosomes replicated twice in medium containing 5-bromodeoxyuridine (BrdU) with Hoechst 33258 plus black light at 55 degrees C (HB pretreatment) and deoxyribonuclease (DNase) I, II, or micrococcal nuclease. In this staining pattern the BrdU bifilarly substituted chromatids were darkly and the unifilarly substituted chromatids lightly stained. This staining pattern was obtained only by staining the HB-DNase I-treated chromosomes with Giemsa and methylene blue, not by several other dyes tested. Relatively more DNA labelling was removed from the non-BrdU-substituted than the BrdU-substituted chromosomes, when the HB-pretreated chromosomes were digested with DNase I. But the protein labelling was not removed appreciably in the same treatment. The differential DNase I sensitivity between the non-BrdU-substituted and BrdU-substituted chromosomes disappeared when the HB-pretreated chromosomes were incubated with proteinase K before The DNase I digestion. Moreover, no differential DNase I sensitivity was found between the HB-pretreated isolated DNA containing and not containing BrdU. We propose that during the HB pretreatment, more DNA-protein cross-linkings are induced in BrdU bifilarly substituted than the unifilarly substituted chromatids. This structure protects the chromosomal DNA against the DNase I digestion. Thus, a reverse differential Giemsa staining between sister chromatids is obtained by the HB-DNase I treatment.     

Involvement of sulfhydryl groups of chromosomal proteins in sister chromatid differentiation

The fluorescence of human lymphocyte chromosomes stained with sulfhydryl group-specific fluorochromes is markedly enhanced by a mild near-ultraviolet irradiation pretreatment, indicating breakage of protein disulfide bonds. When metaphase preparations of cells cultured in the presence of BrdU during two cell cycles are irradiated and subsequently stained with the sulfhydryl group-specific fluorescent reagents used in this study, a differential fluorescence of sister chromatids is observed. After staining with the DNA-specific fluorochrome DAPI an opposite pattern of lateral differentiation appears. It can be concluded that the chromatid containing bifilarly BrdU-substituted DNA has a higher content of sulfhydryl groups than the chromatid containing unifilarly BrdU-substituted DNA. This implies a more pronounced effect of breakage of disulfide bonds in the chromatid with the higher degree of BrdU-substitution. BrdU-containing chromosomes pretreated with the mild near-ultraviolet irradiation procedure used by us, do not show any differentiation of sister chromatids after Feulgen staining. Using sulfhydryl group-specific reagents, differential fluorescence of sister chromatids could still be induced by irradiation with near-ultraviolet light after the complete removal of DNA from the chromosomes by incubation with DNase I. Thus, the protein effect of irradiation of BrdU-containing chromosomes takes place independently of what occurs to DNA.Our results indicate that subsequent to the primary alteration of chromatin structure caused by the incorporation of BrdU into DNA, breakage of disulfide bonds of chromosomal proteins might play an important role in bringing about differential staining of sister chromatids, at least for those procedures that use irradiation as a pretreatment or prolonged illumination during microscopic examination.     

The mechanism of differential sister chromatid fluorescence as studied with the GC-specific DNA-ligand mithramycin

Chromosomes of human blood cells exposed to BUdR for two cell cycles showed an R-band pattern of fluorescence without lateral differentiation after staining with the GC-specific DNA-fluorochrome mithramycin. Differential sister chromatid fluorescence could be induced by a mild near-ultraviolet irradiation pretreatment which was without effect in Feulgen staining. Thus, besides the primary alteration of DNA structure caused by incorporation of BUdR, secondary structural alterations, probably mediated via chromosomal proteins, are required in order to obtain differential mithramycin-fluorescence of sister chromatids. The differential staining pattern was similar to that achieved with the AT-specific DNA-fluorochrome DAPI. Therefore, it may be concluded that the base specificity of fluorochromes does not play any part in the production of differential fluorescence of sister chromatids by this method.     

Simultaneous estimation of sister chromatid exchange and evaluation of cell cycle delay

The frequency of sister chromatid exchange and cell cycle duration were evaluated simultaneously. This approach is based on the analysis of distribution of cells with differential staining of sister chromatids after treatment of cells with 5-bromodeoxyuridine. The treatment of cells with thiotepa caused no changes in cell cycle duration, while the combination of thiotepa and hydroxyurea (HU) or cytosine-beta-D-arabinofuranoside (ARA-C) was observed to prolong cell cycle duration. Furthermore, it has been shown that caffeine, HU, ARA-C do not increase frequency of sister chromatid exchange in control cells and in cell treated with thiotepa.     

Differential inhibition of sister chromatid condensation induced by 5-azadeoxycytidine in human chromosomes

The deoxycytidine analogue 5-azadeoxycytidine (5-aza-dC) induces differential inhibition of sister chromatid condensation when cells are treated with this substance for two replication cycles, as the subsequent staining of metaphase chromosomes with Giemsa shows. The bifilarly substituted chromatid is dramatically longer than the unifilar one. A percentage of the metaphases treated with 5-azad-C even show a complete undercondensation of the bifilarly substituted chromatid. The optimum conditions for inducing sister chromatid differentiation were determined. No method has been developed as yet to permit enhancement of the differential staining in 5-aza-dC-treated preparations. The interactions between 5-aza-dC and chromosomal DNA as well as the factors involved in the differential staining of sister chromatids are discussed.     

Simple differential Giemsa staining of sister chromatids after treatment with photosensitive dyes and exposure to light and the mechanism of staining

The essential steps of the 33258 Hoechst-Giemsa method for differential chromatid staining consist of (1) 33258 Hoechst treatment, (2) exposure to light, and (3) Giemsa staining. The staining was shown to be a function of the concentration of 33258 Hoechst and the light exposure. The dye was successfully replaced by various metachromatic dyes such as thionine. Two simple methods are proposed. Failure of the pale stained chromatids to restore Giemsa affinity with urea and trypsin and the diminished Feulgen reaction after light exposure suggest that not masking proteins but photolysis of the BrdU-incorporation chromatid components in the present of photosensitive dyes play a role in the differential staining.     

Behaviour of chromosomes in potato leaf tissue cultured in vitro as studied by BrdC-Giemsa labelling

Summary Cells of leaf explants of a monohaploid potato (Solanum tuberosum) were stimulated to mitosis on a medium with 5-bromodeoxycytidine during a period of 7 days. The cells cycled with mono- or diplochromosomes which showed differential staining of the sister chromatids and sister chromatid exchanges by the fluorescent plus Giemsa technique after two rounds of BrdC incorporation. Through the staining pattern the course of the first three cell cycles could be traced and the duration of the cycles estimated. Polyploidisation was enhanced by selective stimulation of polyploid cells and by endoreduplication of G2-phase cells. The percentage of polyploid mitoses increased from 10 to 70.     

D-β-Hydroxybutyrate Prevents MPP+-Induced Neurotoxicity in PC12 Cells

Numerous studies show that D-β-Hydroxybutyrate (DβHB) is neuroprotective. The present study was to explore the neuroprotective effects of DβHB against the cell death and apoptosis induced by 1-methyl-4-phenylpyridinium ion (MPP+) in PC12 cells. PC12 cells were pretreated with DβHB and followed by MPP+ exposure. The cell viability was determined by MTT assay. The morphological characteristics of apoptosis was observed by Acridine Orange (AO) staining and apoptotic rates were measured by flow cytometer. The product of lipid peroxidation, malondialdehyde (MDA), was measured using thiobarbituric acid method. The mitochondrial membrane potential (MMP), intracellular ROS and total glutathione were detected by microplate reader. In PC12 cells, pretreatment with DβHB significantly reduced MPP+-induced the decrease of cell viability. AO staining and flow cytometric analysis found DβHB inhibited MPP+-induced apoptosis. The measurement of MDA formation showed that DβHB alleviated lipid peroxidation induced by MPP+. The loss of MMP induced by MPP+ was preventive by DβHB. The changes of intracellular ROS and total glutathione induced by MPP+ were reversed by DβHB. DβHB protected PC12 cells against MPP+-induced death and apoptosis.     

Influence of cysteamine on differential staining of BUdR-substituted human chromosomes.

In 5-bromodeoxyuridine (BUdR)-substituted human chromsomes stained with 4'-6-diamidino-2-phenylindole (DAPI) differential staining is suppressed totally by the H+-donor cysteamine (concentration 0.08 M). We propose that differential staining appears because the double BUdR-substituted chromatid will be disintegrated via a photosensitive dye-visible light system. It is suggested that cysteamine prevents the production of strand breaks in DNA and, consequently, differential staining in BUdR-substituted chromosomes. Furthermore it is shown that differential staining with DAPI causes irreversible changes in the double BUdR-substituted chromatid. This finding can be explained with the above mentioned mechanism.     

Analysis of the Length of S-Phase Required to Show Sister Chromatid Differential Staining

Abstract. In vitro studies of BrdU-dependent sister chromatid differential staining typically employ two cycles of BrdU incorporation. Experiments are described which determined the actual fraction of both S-phases that the rat embryonic fibroblasts (Rat-1) cells had to traverse in order to show distinctive differential staining. Following synchronization of cells by a combination of serum deprivation and hydroxyurea blockage, sister chromatid differential staining, labelling index, mitotic index, and per cent DNA replication are determined. Results indicate that only ≤50% of the first S-phase is necessary in order to show distinctive differential staining. the importance of this finding to studies of cellular proliferation using BrdU incorporation is discussed.     

Selective differential staining of sister chromatids of the facultative heterochromatic X chromosome in the female mouse

Selective differential staining of sister chromatids for the facultative heterochromatic X chromosome in the female mouse has been achieved by the combination of two differential staining techniques; one for the heterochromatic X chromosome and the other for sister chromatids. Thermal hypotonic treatment moderately destroyed the chromosome structure except for the heterochromatic X in BrdU labelled metaphase cells, resulting in the selective sister chromatid differentiation of this X with Giemsa stain. This technique enables us to know the exact frequency of the spontaneous sister chromatid exchanges in the heterochromatic X without using ³H-TdR labelling for detecting the late DNA replication. The results indicate that the sister chromatid exchange frequency of the heterochromatic X chromosome is not affected by its late DNA replication during S phase, or by the genetic inactivation and the resulting heterochromatinization.     

Analysis of the length of S-phase required to show sister chromatid differential staining

In vitro studies of BrdU-dependent sister chromatid differential staining typically employ two cycles of BrdU incorporation. Experiments are described which determined the actual fraction of both S-phases that the rat embryonic fibroblasts (Rat-1) cells had to traverse in order to show distinctive differential staining. Following synchronization of cells by a combination of serum deprivation and hydroxyurea blockage, sister chromatid differential staining, labelling index, mitotic index, and per cent DNA replication are determined. Results indicate that only approximately 50% of the first S-phase is necessary in order to show distinctive differential staining. The importance of this finding to studies of cellular proliferation using BrdU incorporation is discussed.     

Different effects of 33258 Hoechst and DAPI in fluorescent staining of sister chromatids differentially substituted with bromodeoxyuridine

Summary After substitution with 5-bromodeoxyuridine (BrdUrd) for two rounds of replication, chromosomes in cytological preparations stained with 33258 Hoechst show upon epiluminescence an immediate differential sister chromatid fluorescence. When stained with DAPI, however, which has a structural resemblance to part of the 33258 Hoechst molecule, such a differential pattern of fluorescence was only induced after some delay. Upon restaining with the same dye the differential fluorescence appeared instantly. In preparations double stained with ethidium bromide and 33258 Hoechst the induction of a differential staining of sister chromatids with 33258 Hoechst was not accompanied by a differential staining with ethidium bromide. Once a differential staining was obtained with DAPI in preparations double stained with ethidium bromide and DAPI, the ethidium bromide pattern also appeared to be differential upon subsequent observation. No differentiation could be obtained with ethidium bromide alone. The observations described in the case of 33258 Hoechst staining are in agreement with a molecular quenching by BrdUrd without gross structural consequences for the DNA. In the case of DAPI staining, however, there occurs a differential photolysis of BrdUrd-substituted DNA. Besides the nature, most likely the size, of the fluorochrome molecules themselves, the state of the fixed chromatin appeared also to play a role in determining the mechanism of the sister chromatid differentiation: after prolonged incubation in buffer, BrdUrd-containing chromosomes stained with 33258 Hoechst showed a differential staining evidently caused by photolysis, indicating that they had become more susceptible to light.     

Autoradiographic evidence of differential loss of BUdR-substituted DNA after UV exposure in FPG harlequin staining

Autoradiographic analysis of CHO cells labelled with [³H]TdR or [³H]BUdR shows that only [³H]BUdR label is removed from metaphase chromosomes after FPG staining. [³H]BUdR is differentially removed from the bifilary labelled chromatid (BB) compared with the unifilary labelled chromatid (TB). UV treatment alone removes the label and produces harlequin staining and if the UV step is omitted from the FPG staining technique no loss of label or harlequin staining occurs. The heat treatment step (60 °C in 2 × SSC) removes further label, reducing the ratio of grains BB/TB to 0.8:1.0 and improving the differential staining. Over-treatment with heat produces paler staining chromatids without altering this ratio. The differential loss of BUdR-substituted DNA through UV photolysis and extraction in solution appears to be the cause of the differential harlequin staining of chromatids in this technique.     

Rapid irradiation procedure for obtaining permanent differential staining of sister chromatids and aspects of its underlying mechanism

Summary When fixed metaphase preparations of lymphocytes cultured in the presence of BrdU during two cell cycles are subjected to a 1-min simple irradiation treatment with near-ultraviolet light (radiation dose 3×10⁵ J/m²), subsequent Giemsa staining produces differential staining of sister chromatids irrespective of previous exposure to a photosensitizer. The effects of this procedure were analyzed by irradiating single metaphases under the microscope, thus allowing precise dosage of radiation: Metaphase were subsequently stained with Giemsa and then subjected to the Feulgen-Schiff procedure. Whereas in the presence of DAPI as a photosensitizer a differential breakdown of BrdU-containing DNA in the chromatids under the influence of irradiation appeared to be the cause of sister chromatid differentiation, alterations presumably in the higher oeder structure of chromatin, not accompanied by removal of DNA, induced sister chromatid differentiation without DAPI.     

Induction of endoreduplication by hydrazine in Chinese hamster V 79 cells and reduced incidence of sister chromatid exchanges in endoreduplicated mitoses

Hydrazine in high concentrations very effectively induces endoreduplication in Chinese hamster V 79 cells. The addition of 5-bromodeoxyuridine (BrdU) for the duration of one cell cycle prior to the induction of endoreduplication produces diplochromosomes with sister chromatid differentiation (SCD) after differential chromatid staining. The fact that diplochromosomes with complete SCD are obtained shows that endoreduplication was induced in cells that were in G2-phase. The analysis of sister chromatid exchanges (SCEs) showed that hydrazine treatment rarely led to increased SCE frequencies in mitoses after endoreduplication, but that it caused a strong SCE induction in diploid second division metaphases in the same culture. Neither catalase nor cysteine had an effect on the induction of endoreduplication or the incidence of SCEs. Treatment of the cells with mitomycin C prior to addition of BrdU led to increased SCE frequencies. Compared with the normal mitoses from the same preparation, the mitoses after endoreduplication showed a significantly reduced induction of SCEs. In contrast to these findings, SCE induction was not reduced in the common tetraploid V 79 cells after colcemid-induced polyploidization.     

Modified method of differential staining of sister chromatids]

A modified method of obtaining differential staining of sister chromatids is described. It is simple, rapid, and effective, and at the same time inexpensive and accessible, since it allows one to use available reagents. When 5-bromdeoxyuridine was administered 24 hours before fixation into the Chinese hamster cell culture the percentage of metaphases with a differential chromatid staining constituted 95--98, and when this substance was administered 28 hours before fixation into the human lymphocyte culture this percentage varied from 75 to 92, depending on the individual. The mean number of sister chromatid exchanges in human lymphocytes failed to depend on the time of fixation.     

Sister chromatid exchanges in Tradescantia paludosa

A modified fluorescence-plus-Giemsa technique is described that allows differential staining of sister chromatids in root tip cells from cuttings of Tradescantia paludosa. With this staining technique, chromatids with both DNA strands unsubstituted are differentiated from chromatids containing 5-bromouracil in place of thymine in one of the strands of the DNA duplex. The baseline level of sister chromatid exchanges was shown to be dependent on the concentration of 5-bromodeoxyuridine in the treatment solution, the mean frequency being 43.5 sister chromatid exchanges per cell for the experimental protocol suggested.     

Silver-staining specificity in metaphases after incorporation of 5-bromodeoxyuridine (BUDR)

Summary Silver staining is reported to be reduced in chromatin substituted by BUDR. This quenching effect allows for the demonstration of replication patterns and differential chromatid staining. Though the differentiation, as compared to other staining techniques, is of inferior quality, it is of theoretical importance concerning the effect of BUDR incorporation into chromatin.     

Topographic examination of sister chromatid differential staining by nomarski interference microscopy and scanning electron microscopy

BrdU-substituted Chinese hamster chromosomes were treated with a hot Na₂HPO₄ solution and stained with Giemsa to produce sister chromatid differential staining (SCD). The process of SCD was examined with the Nomarski differential interference microscope and the scanning electron microscope. After the Na₂HPO₄ treatment alone, unifilarly BrdU-substituted (TB) chromatids appeared somewhat more severely collapsed than the bifilarly substituted (BB) chromatids. Subsequent Giemsa staining, however, brought about pronounced piling up of the Giemsa dye on the TB-chromatids but not on the BB-ones, causing highly distinct differential Giemsa staining as well as a marked differentiation in surface topography between the sister chromatids. Removal of the Giemsa dye from the differentially Giemsa stained chromosomes resulted in a disappearance of such a pronounced topographic differentiation.