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.     

In vivo BrdU-33258 Hoechst analysis of DNA replication kinetics and sister chromatid exchange formation in mouse somatic and meiotic cells

BrdU (5-bromodeoxyuridine)-33258 Hoechst methods have been adapted for in vivo analyses of replication kinetics, sister chromatid differentiation and sister chromatid exchange (SCE) formation in mice. Sufficient in vivo BrdU substitution for cytological detection was effected with multiple intraperitoneal injections of the analogue. The combination of centromere staining asymmetry and sister chromatid differentiation at metaphase permits unambiguous determination of the number of replications in BrdU and dT (deoxythymidine) undergone by individual cells. Late-replicating regions in marrow and spermatogonial chromosomes are highlighted by bright fluorescence after sequential incorporation of BrdU followed by dT during a single DNA synthesis period. SCEs are analyzed in marrow and spermatogonial metaphases after successive complete cycles of BrdU and dT incorporation. Significant induction of SCE was observed with both mitomycin C and cyclophosphamide; the latter drug requires host-mediated activation to be effective. In meiotic metaphase cells harvested two weeks after BrdU incorporation, satellite DNA asymmetry, sister chromatid differentiation and SCE could be detected in a few chromosomes, most frequently the X and the Y.     

Proliferative characteristics of progeny derived from normal human hemopoietic progenitor cells

Cell cycle times of cells derived from human hemopoietic progenitor cells were measured directly by using 5-bromodeoxyuridine (BrdU) incorporation and sister chromatid differential staining. Umbilical cord cells were cultured in the mixed-colony assay for 96 h prior to the addition of BrdU and then harvested at 6-12 h intervals. Individual cell cycle times ranging from 8.5-23.4 h were observed while population mean cell cycle times ranged from 17.5-24 h. These results show that some early progeny of hemopoietic progenitor cells have short cell cycle times and that there is considerable heterogeneity within the proliferating cell population.     

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.     

Proliferation of PHA- and PWM-stimulated lymphocytes measured by sister chromatid differential staining.

Lymphocyte proliferation in PHA- and PWM-stimulated cultures was measured by BrdU incorporation followed by sister chromatid differential staining. Both the PHA and PWM cultures showed a steady increase with time in the number of second- and third-division metaphases, accompanied by a corresponding decrease in the number of first-division metaphases. There was no difference between the PHA and PWM cultures in the number of first-, second-, and third-division cells, but the mitotic index was lower in the PWM cultures. During the 96-hr culture period the mitotic index of the PHA cultures decreased, whereas the PWM cultures showed an increase. It is suggested that the BrdU-Giemsa technique may prove to be a useful adjunct in the study of lymphocyte proliferation in response to mitogens.     

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.     

Generation time (GT) of human bone marrow cells cultured in the CFC-gm assay

Generation time (GT) of normal human bone marrow cells cultured in the CFC-gm assay was measured by using bromodeoxyuridine (BrdU) incorporation and sister chromatid differential staining. Cells were cultured in methylcellulose for 72 hr prior to the addition of BrdU and then harvested at 6-12 hr intervals for up to 72 hr. The time interval between the appearance of second and third division metaphases at the 50% level gave mean GTs which ranged from 32 to 43 hr. These values are longer than those reported for myeloblasts and promyelocytes but shorter than those previously reported for myelocytes.     

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.     

A high resolution study of the DNA replication patterns of Chinese hamster chromosomes using sister chromatid differential staining technique

Chinese hamster cells were grown for 1+ and 2+ cell cycles in the presence of BrdU and then treated by the sister chromatid differential staining technique (SCD). Those regions of a chromosome which had replicated twice in the presence of BrdU were pale staining and by selecting appropriate metaphase cells an accurate reconstruction of the DNA synthetic patterns was possible. A direct correlation between the staining intensity of the G bands and the order in which they replicate was found. Dark staining G bands were always the last region of a chromosome to replicate while G negative bands were first. It is concluded that each G band may be a cluster of replicons capable of initiating DNA synthesis simultaneously.     

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.     

Sister chromatid exchange in the Mongolian gerbil, Meriones unguiculatus

Sister chromatid exchange (SCE) studies have been performed in a variety of animals, both in vitro and in vivo, as well as in plants. To date, no such studies have been performed in any member of the gerbil sub-family (Gerbillinae). A new sister chromatid differential staining method was used with mice in vivo and has now been applied to the Mongolian gerbil, Meriones unguiculatus. With some modifications, this in vivo method was found to be highly reproducible in gerbils, and had consistently produced many metaphase cells with clear sister chromatid differentiation. The method involves subcutaneous implantation of a 50 mg slow-release BrdU pellet, the fluorochrome Hoechst 33258, phosphate buffering at pH 6.8, and incandescent light exposure.     

On the mechanism of differential Giemsa staining of bromodeoxyuridine-substituted chromosomes

Summary Experiments were performed to find out whether different mechanisms are involved in FPG-(fluorescent plus Giemsa) staining for the demonstration of replication patterns and sister chromatid differentiation (SCD) after bromodeoxyuridine (BrdU)-substitution of V79 Chinese hamster chromosomes. The influence of variations of the staining procedure on the quality of both SCD and replication patterns was comparatively investigated and differences in the demonstration of these two phenomena within the same chromosome were studied using various BrdU-labeling protocols. The results show that at least graduated differences exist. For a good differentiation of replication patterns a stronger FPG-treatment is necessary than it is for SCD. Partial BrdU substitution only leads to replication patterns in the next mitosis. A further round of replication either in the presence or absence of BrdU causes a reduced staining of the complete chromatid and three-way differentiation is seen in third generation mitoses. These results support the view that alterations of chromosomal proteins during BrdU-incorporation and replication of BrdU-substituted DNA are decisive for differential staining.     

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.     

Labelling of DNA and differential sister chromatid staining after BrdU treatment in vivo

A method of labelling DNA in vivo with 5-bromodeoxyuridine (BrdU) is described. After 6 h permanent subcutaneous infusion of BrdU in rodents (adult Microtus agrestis, pregnant NMRI-mice), cell nuclei which have undergone DNA synthesis during the BrdU treatment can be differentiated from the nuclei of other cycle stages by means of their altered staining behaviour after Giemsa. 24 h after the BrdU treatment, mitoses from both bone marrow of the adult animals and tissues from the fetuses showed a differential sister chromatid staining. In male M. agrestis, sister chromatid exchanges were most frequently found in the euchromatic part of the X and in the constitutive heterochromatin of both sex chromosomes.     

Proliferation kinetics of human B- and T-lymphocytes

Human peripheral blood B- and T-lymphocytes, highly purified by immunological methods, were supplemented with gamma-irradiated unseparated autologous mononuclear cells to restore helper functions and stimulated with pokeweed mitogen and phytohaemagglutinin, respectively. Proliferation kinetics of the cell populations were investigated using 5-bromodeoxyuridine (BrdU) labelling of the cell cultures and chromosome preparation at different times after stimulation. The percentages of metaphase cells having replicated for one, two or three generations in the presence of BrdU were determined following sister chromatid differential staining. In all donors, the changes in these percentages were faster in B- than in T-lymphocytes, indicating a longer cell cycle time in the latter population.     

Differential giemsa staining of sister chromatids after extraction with acids

Chromosomes of Chinese hamster strain cells were air-dried on slides after BrdU substitution for two or three rounds of replication. The preparations were treated with 20% PCA at 55 degrees C for 20-30 min, or 5N HCl at 55 degrees C for 15-20 min. After staining with Giemsa, unifilarly BrdU-substituted chromatids stained faintly and bifilarly substituted chromatids stained darkly. Such a pattern of sister chromatid differential staining was confirmed by the examination of metaphase cells grown with BrdU for three rounds of replication.     

Insights into the mechanisms of sister chromatid exchange formation

The DNA lesions responsible for the formation of sister chromatid exchanges (SCEs) have been the object of research for a long time. SCEs can be visualized by growing cells for either two rounds of replication in the presence of 5-bromo-2'-deoxyuridine (BrdU) or for one round with BrdU and the next without. If BrdU is added after cells were treated with a DNA-damaging agent, the effect on SCEs can only be analyzed in the second post-treatment mitosis. If one wishes to analyze the first post-treatment mitosis, cells unifilarily labeled with BrdU must be treated. Due to the highly reactive bromine atom, BrdU interacts with such agents like ionizing and UV radiation enhancing the frequency of SCEs. However, its precise role in this process was difficult to assess for a long time, because no alternative technique existed that allowed differential staining of chromatids. We have recently developed a method to differentially label sister chromatids with biotin-16-2'-deoxyuridine-5'-triphosphate (biotin-dUTP) circumventing the disadvantage of BrdU. This technique was applied to study the SCEs induced by ionizing and UV radiation as well as by mitomycin C, DNaseI and AluI. This article is a review of the results and conclusions of our previous studies.     

Three-Way Differentiation of Chinese Hamster Ovary Chromosomes by Immunoperoxidase Technique Using a Monoclonal Anti-Bromodeoxyuridine Antibody

A new permanent staining procedure for sister chromatid differentiation (SCD) in cultured Chinese hamster ovary cells has been established by combining the three-way differentiation in third mitosis (M3) chromosomes and the immunoperoxidase reaction developed with 3,3'-diaminobenzidine using a monoclonal anti-bromodeoxyuridine (BrdU) antibody. This procedure allows SCD at very low BrdU concentrations, and the evaluation of the sister chromatid exchange frequencies on a per cell-cycle basis.     

The role of chromosomal proteins in the induction of a differential staining of sister chromatids by light

Summary Fixed chromosomes of human lymphocytes, cultured in the presence of bromodeoxyuridine (BrdU) during two cell cycles, were exposed to near-ultraviolet irradiation, stained with Giemsa, and after destaining, were subjected to either Coomassie Blue or Feulgen-Schiff staining. A differential reaction of sister chromatids was first revealed by Coomassie Blue staining. Differential staining with Giemsa required a longer irradiation time. This appeared to be reduced after the addition of dithiodipyridine to the cells during their last few hours of culture. The differential pattern obtained after Coomassie Blue staining was the inverse of that obtained after Giemsa staining. From these findings we concluded that the induction of sister chromatid differentiation by light in BrdU-substituted DNA containing chromosomes occurs primarily via chromosomal proteins, presumably by differential breakage of their disulphide bonds. The results of the Feulgen-Schiff staining indicated that differential depurination of BrdU-containing DNA could occur, although only after very prolonged irradiation. A faint though distinctly differential Feulgen-Schiff pattern of sister chromated staining, resulting from differential removal of DNA, was observed after photosensitization by specific DNA-binding dyes. Thus, DNA seems to be affected only under more extreme conditions.     

Lymphocyte proliferation in Down's syndrome measured by sister chromatid differential staining

Summary Lymphocyte proliferation in PHA stimulated cultures from Down's syndrome patients and normals was measured by the BrdU/Giemsa method for demonstrating sister chromatid differential staining. Both Down's syndrome patients and normals had 1st, 2nd and 3rd divisions present. However, Down's syndrome patients had more 3rd division metaphases and fewer 2nd division metaphases than the normals. There was no difference in the mitotic index between the two groups.