Application of the BrdU/thymidine method to flow cytogenetics: Differential quenching/enhancement of Hoechst 33258 fluorescence of late-replicating chromosomes

Discrimination between many types of isolated mammalian chromosomes can be accomplished by dual-beam flow cytometry following DNA staining with Hoechst 33258 (HO) and Chromomycin A3 (CA3). In this report, we show that the bivariate discrimination of selected late-replicating Chinese hamster M3-1 chromosomes can be improved by appropriate treatment of the cells with 5-bromo-2-deoxyuridine (BrdU) prior to chromosome isolation and staining. Two labeling schemes are reported. In one scheme the chromosomes are collected from cells labeled with BrdU only during late S phase. The Hoechst fluorescence of the 10, 11, M2, and Y chromosomes is substantially quenched by the incorporated BrdU, thus improving their discrimination. In the other scheme, chromosomes are collected from cells labeled with thymidine (dT) during late S phase following 20 h of growth in BrdU-containing medium. The Hoechst fluorescence of the 10, 11, M2, and Y chromosomes is quenched less than the other chromosomes, again improving their discrimination. Y chromosomes from chromosome suspensions of untreated controls, of cells labeled with BrdU during late S phase, and of cells labeled with dT during late S phase following 20 h growth in BrdU were separated by dual-parameter sorting. While the purity of the sorted Y chromosome was 15% in untreated controls, it was 70–75% using the BrdU/dT labeling protocols.Work performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under contract number W-7405-ENG-48, with support from the Deutsche Forschungsgemeinschaft (Cr 60/2-2).On leave from the Institute of Human Genetics, University of Freiburg, D-7800 Freiburg i. Br., West Germany.     

Continued cell cycling ability of HL-60 cells following 3HaraC incorporation: a combination of "double-labeling" and sister chromatid analysis

This study was designed to determine if HL-60 cells could undergo one or more cycles of DNA synthesis despite containing 3H-cytosine arabinoside (3HaraC) in their genome. HL-60 cells were incubated with 3HaraC for 2 hours, washed and maintained in a medium containing bromodeoxyuridine (BrdU). At fixed time points, cells were arrested in metaphase and prepared for chromosomal analysis. Treatment of the sample by an immunofluorescent monoclonal anti-BrdU antibody allowed us to determine the differential fluorescent pattern of sister chromatids in metaphase cells that had undergone two or more rounds of DNA synthesis in the presence of BrdU. Processing the samples by autoradiography demonstrated the presence of black grains (3HaraC) overlying the chromosomes. Thus, we were able to examine each metaphase for the presence of 3HaraC as well as the number of cycles it had completed in the presence of BrdU. We showed that despite the presence of 3HaraC in their DNA, some HL-60 cells were able to undergo two or more complete rounds of DNA replication.     

Fluctuations in cellular proliferation across the light/dark cycle in the subgranular zone of the dentate gyrus in the adult male Syrian hamster

A lifelong persistent neurogenesis occurs in the dentate gyrus of the mammalian hippocampus. Research in peripheral cell tissue has shown that the timing of cellular division of these cells coincide with the light/dark cycle, however it remains unclear as to whether there is an association between the time of day and cellular proliferation in the brain. The timing of cellular division can be studied through the use of a cellular proliferation marker, such as 5-bromo-2-deoxyuridine (BrdU), which is taken up by the DNA of dividing cells during replication. The goal of this study was to determine whether the time of day affects the number of BrdU labeled cells in the subgranular zone of the dentate gyrus of adult male Syrian hamsters. Adult males received a single systemic injection of BrdU (300 mg/kg) at either the end of the light (ZT-13) or dark phase (ZT-23) of a 14:10 LD cycle and were sacrificed 24 h or 3 days later. Sections through the hippocampus were immunolabeled for BrdU. Cellular proliferation fluctuated across the light/dark cycle during the expansion phase rather than during initial cellular proliferation. A twofold increase in number was expected between 24 and 72 h following a single BrdU injection, but this increase was only seen in the population of cells injected at the end of the light phase.     

Determination of key aspects of precursor cell proliferation, cell cycle length and kinetics in the adult mouse subgranular zone

Neurogenesis studies on the adult mouse hippocampal subgranular zone (SGZ) typically report increases or decreases in proliferation. However, key information is lacking about these proliferating SGZ precursors, from the fundamental—what dose of bromodeoxyuridine (BrdU) is appropriate for labeling all S phase cells?—to the detailed—what are the kinetics of BrdU-labeled cells and their progeny? To address these questions, adult C57BL/6J mice were injected with BrdU and BrdU-immunoreactive (IR) cells were quantified. Initial experiments with a range of BrdU doses (25–500 mg/kg) suggested that 150 mg/kg labels all actively dividing precursors in the mouse SGZ. Experiments using a saturating dose of BrdU suggested BrdU bioavailability is less than 15 min, notably shorter than in the developing mouse brain. We next explored precursor division and maturation by tracking the number of BrdU-IR cells and colabeling of BrdU with other cell cycle proteins from 15 min to 30 days after BrdU. We found that BrdU and the Gap2 and mitosis (G₂/M) phase protein pHisH3 maximally colocalized 8 h after BrdU, indicating that the mouse SGZ precursor cell cycle length is 14 h. In addition, triple labeling with BrdU and proliferating cell nuclear antigen (PCNA) and Ki-67 showed that BrdU-IR precursors and/or their progeny express these endogenous cell cycle proteins up to 4 days after BrdU injection. However, the proportion of BrdU/Ki-67-IR cells declined at a greater rate than the proportion of BrdU/PCNA-IR cells. This suggests that PCNA protein is detectable long after cell cycle exit, and that reliance on PCNA may overestimate the length of time a cell remains in the cell cycle. These findings will be critical for future studies examining the regulation of SGZ precursor kinetics in adult mice, and hopefully will encourage the field to move beyond counting BrdU-IR cells to a more mechanistic analysis of adult neurogenesis.     

Different responses to mitogenic agents by Adult rat and human chromaffin cells in vitro

Adrenal medullary hyperplasia and pheochromocytomas occur frequently in laboratory rats, both in the courseof aging and in response to prolonged administration of a variety of drugs and other substances. In contrast, these lesions are rare in humans. Rat chromaffin cells proliferate throughout life, but the proliferative capacities of human chromaffin cells are unknown. To determine whether the difference in prevalence of adrenal lesions might be correlated with differences in cell proliferation, adrenal medullary cells from 3 patients undergoing radical nephrectomy were maintained in vitro for up to 2 weeks in control medium or in the presence of nerve growth factor (NGF) and/or tetradecanoyl phorbol acetate (TPA), an activator of protein kinase C. Both NGF and TPA are known mitogens for neonatal and adult rat chromaffin cells. At intervals, the cultures were pulsed for up to 36 hours with bromodeoxyuridine (BrdU) to label S-phase nuclei. They were then fixed and consecutively stained for BrdU and for tyrosine hydroxylase, to confirm that labeled cells were chromaffin cells. Cells from adult female F344 rats were similarly maintained. Human chromaffin cells labeled with BrdU were extremely rare (less than 0.1 %) under all culture conditions, and effects of NGF or TPA could not be demonstrated. Rat chromaffin cells showed little or no labeling with BrdU in control medium but, in contrast to their human counterparts studied, showed marked increases in the percentages of labeled cells in the presence of NGF (37% ± 3%), TPA (7% ± 1%), or both (31% ± 3%). The apparently lower responsiveness of human chromaffin cells to mitogenic signals, or responses to different types of signals, may contribute to the lower frequency of adrenal medullary hyperplasia and pheochromocytomas in humans compared to rats.     

Growth rate and sister chromatid exchange (SCE) incidence of bone marrow cells in Acute Myeloblastic Leukemia (AML)

The sister chromatid exchange (SCE) incidence and growth kinetics have been studied by means of an in vitro bromodeoxyuridine (BrdU) chromosome labeling method in the bone marrow cells of 17 acute myeloblastic leukemia (AML) patients with only diploid cells at diagnosis, remission, and relapse of the disease. At diagnosis, the cells tended to exhibit a low SCE frequency as compared to that during remission. An increased SCE frequency was observed after chemotherapy during remission or relapse. At diagnosis and relapse, when leukemic blast cells predominated in the marrow, they were characterized by the predominance of cells that had undergone only one cell cycle after BrdU exposure. In contrast, the marrow cells during remission tended to resemble the control pattern of growth kinetics, with a predominance of cells undergoing second and third cell cycles in the presence of BrdU. These results suggest that the growth rate of leukemic and nonleukemic cells is different, and that chemotherapy can cause an increased SCE frequency in the marrow cells of AML patients irrespective of the state of the disease.     

Polo-like激酶1反义RNA对肺癌细胞A549生长的实验研究

目的:探讨Polo-like激酶1(Plk1)基因表达下调对肺癌细胞周期分布及其生长的影响。方法:培养肺腺癌细胞株A549,构建表达Plk1反义RNA的质粒pcDNA3-Plk1,通过脂质体介导转染A549细胞,采用RT-PCR和Western blotting的方法检测Plk1基因的表达,细胞计数、BrdU脉冲标记检测细胞增殖,流式细胞仪分析细胞周期变化和凋亡,MTT法检测长春瑞宾(NVB)对各组细胞的生长抑制率。结果:A549细胞转染pcDNA3-Plk1后24h,Plk1mRNA及蛋白表达均下降;细胞变圆、漂浮、增殖减慢;S期细胞百分数(BrdU标记指数)显著低于对照组(P<0.05);转染后48hA549细胞出现G2/M期阻滞(P<0.05)并发生凋亡;等浓度化疗药物诺维本对转染pcDNA3-Plk1细胞的抑制率明显高于各对照组(P<0.05),转染pcDNA3与未转染的对照细胞差异无显著(P>0.05)。结论:pcDNA3-Plk1的转染能下调Plk1基因的表达,抑制A549细胞增殖,诱导凋亡,并能增加A549细胞对化疗药物的敏感性。     

Flow cytometric analysis of bromodeoxyuridine-induced micronuclei

The effects of DNA substitution by the thymidine analogue 5-bromodeoxyuridine(BrdU) on cell cycle progression and micronucleus inductionwere studied in different mammalian cell cultures. Simultaneousflow cytometric measurements of DNA content and side scatterof nuclei in Chinese hamster embryo (CHE) cells revealed a concentration-dependenttemporary block in the G₂/M phase of the first cell cycle. NTH3T3 cells and human amniotic fluid fibroblast-like cells, onthe contrary, did not show any cell cycle disturbances in thepresence of BrdU. Micronucleus frequency increased as soon asCHE cells started to divide and reached a plateau when all cellshave divided. The height of this plateau was almost equal for60 and 100 µM BrdU. This saturation of micronucleus inductionwas due to a saturation of BrdU incorporation into DNA alreadyat a dosis of 60 µM as shown by the BrdU/Hoechst quenchingtechnique. Indirect immunofluorescent staining of kinetochoreswith CREST antibodies revealed that nearly all BrdU-inducedmicronuclei were kinetochore-negative suggesting the presenceof acentric chromosome fragments in these micronuclei. DNA distributionsof micronuclei measured by flow cytometry showed several peaksrepresenting micronuclei which contain DNA fragments of definedsizes induced by non-random breakage of chromosomes 1 and Xas verified by flow karyotyping and C-banding.     

Polo-like激酶l反义RNA对肺癌细胞A549生长的实验研究

目的： 探讨Polo-like激酶1（Plk1）基因表达下调对肺癌细胞周期分布及其生长的影响。方法： 培养肺腺癌细胞株A549，构建表达Plk1反义RNA的质粒pcDNA3-Plk1，通过脂质体介导转染A549细胞，采用RT-PCR和Western blotting的方法检测Plk1基因的表达，细胞计数、BrdU脉冲标记检测细胞增殖，流式细胞仪分析细胞周期变化和凋亡，MTT法检测长春瑞宾（NVB）对各组细胞的生长抑制率。结果： A549细胞转染pcDNA3-Plk1后24 h，Plk1 mRNA及蛋白表达均下降；细胞变圆、漂浮、增殖减慢；S期细胞百分数（BrdU标记指数）显著低于对照组（P<0.05）；转染后48 h A549细胞出现G₂/M期阻滞（P<0.05）并发生凋亡；等浓度化疗药物诺维本对转染pcDNA3-Plk1细胞的抑制率明显高于各对照组（P<0.05），转染pcDNA3与未转染的对照细胞差异无显著（P>0.05）。结论： pcDNA3-Plk1的转染能下调Plk1基因的表达，抑制A549细胞增殖，诱导凋亡，并能增加A549细胞对化疗药物的敏感性。     

Transmission of gamma-ray-induced unstable chromosomal aberrations through successive mitotic divisions in human lymphocytes in vitro

Transmission of unstable chromosomal aberrations through successive mitotic divisions M1-M4 was analysed in 3 Gy gamma-ray-irradiated G0 human lymphocytes in vitro, harvested at 50, 72 and 96 h. Bromodeoxyuridine (BrdU) incorporation and subsequently the fluorescence plus Giemsa (FPG) method allowed the cell cycle status of each cell scored to be ascertained. Higher dicentric frequencies were observed in cells within the same post-irradiation division derived from extended culture times, indicating either a delay in cell cycle progression of aberrant cells or different lymphocyte sub-populations. The yield of complete dicentrics decreased by a factor of two in passing from M1 to M2 and showed further reductions of 26 and 44%, respectively, in moving from M2 to M3 and from M3 to M4. In the case of conventionally stained metaphases, wherein the cell cycle status does not enter the picture, the dicentric frequencies showed a reduction in yield of 39.6% at 72 h and 52.1% at 96 h compared with 50 h, because the cells analysed comprise a mixture of metaphases in different cell cycles. In the FPG stained first division metaphases, 92-100% of dicentrics analysed at 50, 72 and 96 h and in the conventionally stained metaphases, 90-94, 72-84 and 54-80% of dicentrics analysed at 50, 72 and 96 h respectively were complete dicentrics (with fragments). In the M1, M2, M3 and M4 metaphases analysed, 92-100, 50-89, 20-70 and 10-50% of dicentrics, respectively, were complete dicentrics and 55-75, 53-68, 43-57 and 36-50% excess acentrics, respectively, were seen in cells with complete dicentrics. Interindividual variation was observed in cell cycle kinetics, radiation-induced chromosomal aberrations and micronucleus frequencies. A salient feature of the delayed effects was the induction of giant cells and the mirror dicentrics observed in them. The reduction in dicentric frequencies due to either cell cycle delay or cell death in successive generations is aided by the multiplicity of aberrations. Bridge-breakage-fusion (BBF) events mediated by dicentric chromosomes may also be instrumental in the perpetuation of chromosomal instability.     

NGF／GDNF基因修饰神经干细胞植入AD模型鼠脑内的存活、分化及表达

目的 观察NGF／GDNF基因修饰神经干细胞（NGF－NSC和GDNF－NSC）在AD模型鼠脑内的存活、分化及基因产物的表达。方法 将BrdU标记的NGF－NSC和GDNF－NSC单独和联合移植入穹窿海巴伞切断的大鼠侧脑室内。移植后3周,用免疫组织化学方法对脑切片进行BrdU、Nestin、GFAP、NF、NGF及GDNF单标或双标染色。结果 脑室内见到大量BrdU阳性细胞。部分移植细胞向脑实质迁移,在切口周围、穹窿海马伞、海马、胼胝体、隔区、室管膜下区及血管壁旁均可见到BrdU阳性细胞。在皮质和切口周围可见较多的BrdU＋GFAP双标细胞;在海马内可见较多的BrdU NF双标细胞;在脑室内,两者均可见到。在脑室、皮质和海马等处均检测出BrdU＋NGF和BrdU＋GDNF免疫双标阳性细胞。结论 NGF／GDFN基因修饰NSC能在宿主脑内较好地存活,并能分化成神经元和星形胶质细胞,而且能分别表达外源基因产物NGF和GDNF。     

Evidence for the Existence of W3/13+ Monocytes in the Rat

Rat blood mononuclear cells were studied with W3/13, OX19, and W3/25 monoclonal antibodies in a dual staining procedure. Cells recognized as W3/13+ OX19- W3/25+ showed a high light scatter pattern were plastic adherent and exhibited spreading, and were identified as monocytes when stained with May-Grunwald-Giemsa. This evidence suggests that at least a subpopulation of monocytes/macrophages are W3/13+.     

Atomic force microscopy of human metaphase chromosomes after differential staining of sister chromatids

Human metaphase chromosomes, in which 5-bromo-deoxyuridine (BrdU) had been incorporated into the DNA, were treated with the fluorescent plus Giemsa (FPG) method. Use of this method distinctly stained one of the paired sister chromatids with the Giemsa solution due to the difference in content of BrdU in the two chromatids. These chromosomes with their differential staining of sister chromatids were observed by atomic force microscopy (AFM). In the air-dried specimens, one of the paired chromatids that was stained strongly with Giemsa solution was about two times higher than the counterpart that was stained faintly with Giemsa solution. In the critical point dried chromosomes, the height of the Giemsa positive chromatid roughly matched that of the Giemsa negative counterpart. These findings imply that the arrangement of the Giemsa negative chromatid after FPG staining is fragile and easily collapses due to the surface tension of water during air-drying. At higher magnifications, the surface structure differed between Giemsa positive and negative chromatids; the Giemsa positive chromatid (i.e., unifilarly BrdU-incorporated chromatid) was composed of fibrous structures while the Giemsa negative chromatid (i.e., bifilarly BrdU-incorporated chromatid) exhibited a fine granular appearance. These structural changes in the sister chromatids are thought to arise from the ultraviolet irradiation and heating of the chromosomes during FPG staining.     

Unusual SCD in cancer cells: A phenomenon related to decreased BrdU incorporation?

The simultaneous presence of chromosome segments with and without sister chromatid differentiation (SCD) in the same metaphase was observed in a human melanoma cell line after BrdU incorporation for 48-72 hr. This phenomenon was related to the time of BrdU incorporation by the cells: i.e., it was not observed after exposure to BrdU for only 17-20 hr. It is proposed, therefore, that the unusual staining pattern is caused through reduced BrdU incorporation by the cells after completing the first division.     

Simultaneous analysis of surface marker expression and cell cycle progression in human peripheral blood mononuclear cells

One method for examining cell cycle kinetics by flow cytometry uses continuous DNA labeling with bromodeoxyuridine (BrdU), a thymidine analogue. Upon incorporation into DNA, BrdU causes stoichiometric quenching of the DNA fluorochrome Hoechst 33258. After counterstaining with a secondary DNA fluorochrome (e.g., ethidium bromide), the analyst can distinguish cells in different phases of the cell cycle over a number of mitotic cycles with flow cytometry. In this report, we describe a modification of the flow cytometric BrdU-Hoechst assay that allows combined analysis of cell proliferation and immunophenotyping at the single cell level. To demonstrate an application of this method, human peripheral blood mononuclear cells were stimulated with tetanus toxoid or interleukin-2 for up to 6 days in the presence of BrdU, harvested, and immunostained for the cell surface markers CD3, CD4, CD8, CD14, CD19, and the cytokine receptor, CCR5. We used four-color flow cytometry analyses to simultaneously measure cell proliferation and surface marker expression, for the purpose of immunophenotyping and identifying specific cell subsets responding to antigen stimulation. Our successful application of this method suggests that it may be used to study immune responses at the molecular and cellular level and to identify mechanisms of immune system modulation.     

Dynamics of Gastrin-producing Cells in the Rat after Truncal Vagotomy Evaluation by Double lmmunostaining for Bromodeoxyuridine and Little Gastrin

The mechanism of hyperplasia of gastrin-producing cells (G cells) in the rat antral mucosa after truncal vagotomy was studied using double immunostaining for bromodeoxyuridine (BrdU) and little gastrin (G17). With single labeling of BrdU, a few G cells (less than 1%) showed positive immunostaining for BrdU in the nucleus throughout the experimental period in both vagotomized rats and those given a sham operation. The labeled cells in both groups demonstrated a linear increase of BrdU labeling in an identical number of cells for each experimental time-point. The labeling index of the G cells increased rapidly from day 2 to day 6 and attained a maximum level of 44.0% on day 10 in the vagotomized group after cumulative labeling. Even in this group, however, many G -cells showed no BrdU immunoreactivity throughout the experimental period. These cells did not replicate during the experimental period, but showed an intense reaction product for G17 in their cytoplasm after vagotomy. The present study indicates that the most important factor involved in G-cell hyperplasia observed after truncal vagotomy is the activation of pre existing G cells to synthesize and release hormone, together with the rapid maturation of progenitor cells to mature G cells.     

Comparison of antibodies to HBME-1 and calretinin for the detection of mesothelial cells in effusion cytology

The distinction of mesothelial cells in cytologic samples is often a diagnostic challenge. This is particularly true in potentially malignant effusions in which reactive mesothelial cells may simulate adenocarcinoma (ACA) cells, and in the differentiation of ACA vs. mesothelioma. We sought to determine the superior antibody for the positive identification of mesothelial cells in these circumstances. Cell block sections of 25 reactive and 8 malignant mesothelioma effusions were immunostained with an avidin-biotin procedure, using antibodies to HBME-1 and calretinin. No pretreatment of samples was necessary for the HBME-1-stained slides; microwave antigen retrieval was performed on all slides stained for calretinin. A negative control was performed on each sample. The staining intensity of tumor cells was scored on a scale of 0-3+, with the proportion of immunoreactive cells categorized as <25%, 25-50%, 50-75%, and >75%. The predominant staining pattern for HBME-1 was surface, with rare samples also exhibiting cytoplasmic staining as well. The calretinin-staining pattern was cytoplasmic, with peripheral condensation/prominence and accompanying nuclear staining. All samples were immunoreactive with both antibodies. Fifty-five percent (18/33) of samples showed significantly stronger immunoreactivity with calretinin than with HBME-1; 45% (15/33) of samples showed equivalent staining with the two markers. None of the samples in this study showed stronger immunoreactivity with HBME-1 than with calretinin. Sixty-one percent (20/33) of samples stained with HBME-1 at a moderate (2+) intensity. Fifty-five percent (18/33) of samples stained with calretinin at a strong (3+) intensity. While only 12% of samples showed >75% immunoreactivity for HBME-1, 58% of samples showed >75% of cells immunoreactive for calretinin. Calretinin is the preferred marker in identifying mesothelial cells in cytologic samples, showing the highest sensitivity for mesothelial cells, as evidenced by a more intense staining reaction in a higher percentage of cells than with HBME-1. Published 2001 Wiley-Liss, Inc.     

In vitro immunohistochemical localization of S-phase cells by a monoclonal antibody to bromodeoxyuridine

Bromodeoxyuridine, an analogue of thymidine, can be detected by means of monoclonal antibodies and utilized as a marker of the S-phase of the cell cycle. In vitro immunohistochemical application of the BrdU/anti-BrdU-MAb method permits a quantitative assessment of the proliferative activity of a tissue as well as the direct location of the actively replicating cells in histological sections. In this paper, a method for the detection of the labeling index of S-phase cells in normal and neoplastic tissues with in vitro BrdU labeling and standard immunohistochemical techniques using anti-BrdU-MAb and avidin-biotin peroxidase complex is described. We have employed this method in 47 human solid tumor samples, including squamous cell carcinomas of head and neck and cervix uteri, adenocarcinomas and malignant lymphomas, and also evaluated the possible application of the BrdU labeling index to estimate the cycling S-phase cells in neoplastic cell populations. In our data, the in vitro labeling index varied greatly in an individual case (3.56-29.2%) and from an area to an area within the same case. Squamous cell carcinomas of the head and neck showed higher LI than those of the cervix uteri. A case of metastatic carcinoma to the lung from ductal carcinoma of the breast had the highest LI (29.2%), in contrast to the low LI (3.6%) in the primary ductal carcinoma of breast.     

The BrdU content of DNA is decreased during reversal of inhibition of myogenesis by deoxycytidine

The mechanism by which 5-bromodeoxyuridine (BrdU) inhibits cell differentiation is unresolved. The ability of deoxycytidine to reverse the inhibition of myogenesis produced by BrdU has been cited as evidence that the inhibition is not a direct result of the incorporation of BrdU into cellular DNA. In contrast to previous work, the present study demonstrates a direct correlation between the effects of deoxycytidine on myogenic cells and a reduction in the substitution of BrdU for thymidine in the DNA. Furthermore, the reversal occurs at the same degree of BrdU substitution (20–30%) as is required to inhibit myogenesis when cells are grown in BrdU alone or with deoxycytidine in a medium that prevents the conversion of deoxycytidine to thymidine. The effects of deoxycytidine thus do not support a mechanism of action of BrdU in myogenic cells independent of its effects on DNA.