Cytochemistry for bromodeoxyuridine/DNA analysis: stoichiometry and sensitivity

This report describes an improved immunochemical procedure for staining cells in suspension for amount of incorporated bromodeoxyuridine (BrdUrd) and total DNA. In this procedure, cellular DNA is partially denatured by extracting the cells with 0.1 M HCl and then heating them to 80 degrees C in a 50% formamide solution. The cells are then immunofluorescently stained using a monoclonal antibody against BrdUrd in single-strand DNA (ssDNA) and counterstained for DNA content with propidium iodide (PI), a dye that fluoresces preferentially when bound to double-strand DNA (dsDNA). We show that the relative amounts of immunofluorescently stained BrdUrd in ssDNA and PI in dsDNA can be altered reciprocally by changing the formamide concentration, denaturation time, and denaturation temperature. We show that this new immunochemical staining procedure allows more complete DNA denaturation so that fivefold lower levels of BrdUrd incorporation can be quantified. In addition, we show that the BrdUrd-linked immunofluorescence achieved using the new denaturation procedure is more linearly related to cellular BrdUrd content than that achieved after acid DNA denaturation. However, cell loss is sufficiently severe with the thermal denaturation procedure that it may not be applicable to all cell types.     

An improved immunocytochemical procedure for high-sensitivity detection of incorporated bromodeoxyuridine

This report describes an improved immunochemical procedure to stain cells in suspension for incorporated bromodeoxyuridine (BrdUrd) and total DNA content. The procedure consists of five steps: chromatin proteins are extracted by treating with 0.1 M HCl and 0.7% Triton X-100 to facilitate DNA denaturation and to minimize nonspecific staining; cellular DNA is denatured by heating to 100 degrees C in distilled water; BrdUrd in single-stranded DNA (ssDNA) is stained using an immunochemical procedure; autofluorescence is reduced using sodium borohydride (NaBH4); and DNA is stained with the fluorescent dye propidium iodide. With this procedure, the BrdUrd incorporated by CHO cells during periods as short as a few seconds can be detected using flow cytometry. In addition, the stoichiometry of the immunofluorescent staining procedure is high.     

Detection of 5-bromodeoxyuridine (BrdUrd) incorporation by monoclonal antibodies: role of the DNA denaturation step

Immunochemical detection of cells that incorporate 5-bromodeoxyuridine (BrdUrd) requires prior denaturation of DNA in situ to make BrdUrd binding sites accessible to the antibodies. A technique is described in which the DNA denaturation step is facilitated by a) prior dissociation of histones from DNA and b) the use of low ionic strength buffer in which the cells are suspended during heating. Dissociation of histones is achieved by cell treatment with 0.08N HCl at 0 degree C, which a) increases accessibility of DNA to propidium iodide (and following the denaturation to the antibodies); b) lowers stability of DNA to thermal denaturation; c) decreases differences between various cell types due to variability in chromatin structure; and d) ensures more complete DNA denaturation. Cell heating (80-95 degrees C) at low ionic strength (1 mM Na+) eliminates the need for formamide and results in extensive and rapid DNA denaturation. The method was applied in Friend leukemia, L1210 and HL-60 cell lines, and to bone marrow, experimental animal tumor and primary human tumor cells.     

Immunohistochemical detection of bromodeoxyuridine in formalin-fixed tissues

Bromodeoxyuridine (BrdUrd) immunohistochemistry has recently been introduced for the visualization of DNA-synthesizing nuclei. In order to detect the BrdUrd incorporated into nuclear DNA in formalin-fixed, paraffin-embedded tissues, we tested several different pretreatment procedures including digestion with proteinase and hydrolysis with HCl, prior to immunoperoxidase staining. In order to determine the optimal conditions for detecting nuclear BrdUrd, mice were given BrdUrd and 3H-thymidine simultaneously, and the autoradiographic and immunohistochemical results obtained in BrdUrd-stained sections were compared. It was found that digestion with 0.05% proteinase at 37 degrees C for 20 min and hydrolysis with 1N HCl at 37 degrees C for 20 min was sufficient to detect BrdUrd immunoreactivity in 3H-thymidine-labelled nuclei, the results being virtually unaffected by the orders in which the two pretreatments were performed. Our method extends the range of application for BrdUrd immunohistochemistry in cell-kinetic studies.     

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.     

Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90 degrees and forward scatter measurements

Mitotic cells could be well discriminated from the cells in the G1-, S- and G2-phases of the cell cycle using pulse labeling of S-phase cells with bromodeoxy-uridine (BrdUrd) and staining of the cells for incorporated BrdUrd and total DNA content. Unlabeled G2- and M-phase cells could be measured as two separate peaks according to propidium iodide fluorescence. M-phase cells showed lower propidium iodide fluorescence emission compared to G2-phase cells. The fluorescence difference of M- and G2-phase cells was caused by the different thermal denaturation of their DNA. Best separation of M- and G2-phase cells was obtained after 30-50 min heat treatment at 95 degrees C. Mitotic index could be measured if no unlabeled S-phase cells were present in the cell culture. With additional measurements of 90 degree scatter and/or forward scatter signals, mitotic cells could be clearly discriminated from both unlabeled G2- and S-phase cells. The correct discrimination (about 99%) of mitotic cells from interphase cells was verified by visual analysis of the nuclear morphology after selective sorting. Unlabeled and labeled mitotic cells could be observed as pulse-labeled cells progressed through the cell cycle. We conclude that this modified BrdUrd/DNA technique using prolonged thermal denaturation and the simultaneous measurement of scatter signals may offer additional information especially in the presence of BrdUrd-unlabeled S-phase cells.     

A rapid flow cytometric method for bivariate bromodeoxyuridine/DNA analysis using simultaneous proteolytic enzyme digestion and acid denaturation

This report describes an immunocytochemical procedure for the simultaneous quantification of bromodeoxyuridine (BrdUrd) incorporated into cellular DNA and total DNA content in individual cells in suspension. Improvement of existing methods was achieved by combining acid denaturation and proteolytic enzyme digestion (0.2 mg/ml pepsin in 2N HCl for 30 min at room temperature). Acid denaturation preceded by enzyme digestion resulted in a large amount of debris and the occurrence of naked nuclei. In contrast, the simultaneous denaturation/protein digestion procedure did not damage the cellular structure, is rapid and reproducible, and has cell recoveries of more than 85%. Although experimental conditions were tested on human cultured keratinocytes, this method also appeared applicable to bone marrow cells and cells obtained from solid tissues.     

Differentiation of mitotic melanoma cells from G2 cells and their isolation by use of 5-bromo-2'-deoxyuridine and propidium iodide

This report describes a method by which mitotic cells were isolated from nonsynchronized Cloudman melanoma cells that had been pulse labeled with 5-bromo-2'-deoxyuridine (BrdUrd) and double-stained with a fluoresceinated monoclonal antibody to BrdUrd and with propidium iodide (PI). In initial experiments, melanoma cells were first pulse labeled with BrdUrd, treated with prostaglandin E1 (PGE1 10 micrograms/m1) or vehicle (0.1% ethanol) for up to 24 hours, then stained with anti-BrdUrd and PI. PGE1-treated cells monitored at 3-hour intervals were observed to migrate from S phase to G2 phase, then, enigmatically, back into the late S phase region of the distribution. In other experiments, cells treated with PGE1 were pulse labeled with BrdUrd at the end of the treatment period and harvested. In these experiments, there was a small, discrete subpopulation of cells within the late S phase region of the DNA distribution that was negative for anti-BrdUrd. This subpopulation of cells was sorted and examined by light microscopy. We observed that 95% of these BrdUrd-negative "S phase" cells were mitotic cells. Since mitotic cells and G2 cells have equivalent amounts of DNA, the reduced red fluorescence exhibited by these cells may be due to a greater sensitivity to denaturation, which has been described for DNA of mitotic cells, and would account for the phenomenon of cells appearing to move "backwards" in the cell cycle. This report indicates that although the BrdUrd/PI method can further define the cell cycle into four compartments, it can also lead to over-estimation of S phase cells in kinetic studies because of contaminating mitotic cells.     

Application of Biotin, Digoxigenin or Fluorescein Conjugated Deoxynucleotides to Label DNA Strand Breaks for Analysis of Cell Proliferation and Apoptosis Using Flow Cytometry

A flow cytometric method has recently been developed using biotinylated dUTP (b-dUTP) in a reaction catalyzed by terminal deozynucleotidyl transferase (TdT) to identify the endonuclease-induced DNA strand breaks occurring during apoptosis. Counterstaining of DNA makes it possible to relate apoptosis to cell cycle position or DNA index. In the present study, we compared this method with one using digoxigenin-conjugated dUTP (d-dUTP) to label apoptotic cells. The discrimination of apoptotic from nonapoptotic cells was similar when incorporation of d-dUTP was compared with b-dUTP. Both techniques resulted in a 20-30 fold increase in staining of apoptotic over nonapoptotic cells although somewhat less background fluorescence was observed with the d-dUTP. Direct labeling with fluo-resceinated dUTP (f-dUTP) was less sensitive in detecting DNA strand breaks, but had the advantage of simplicity. The principle of labeling DNA strand breaks using TdT was also employed to identify DNA replicating cells. To this end, the cells were incubated in the presence of BrdUrd, then exposed to UV light to selectively photolyse DNA containing the incorporated BrdUrd. DNA strand breaks resulting from the photolysis were then labeled with b-dUTP or d-dUTP. This approach is an alternative to immunocytochemical detection of BrdUrd incorporation, but unlike the latter does not require prior DNA denaturation, thus can be applied when the denaturation step must be avoided. The method was sensitive enough to recognize DNA synthesizing cells that were incubated with BrdUrd for only 5 min, the equivalent of replication of less than 1% of the cell's genome. The discrimination between apoptotic vs. BrdUrd incorporating-cells is based on different extractability of DNA following cell fixation. This method can be applied to analyze both cell proliferation (DNA replication) and death (by apoptosis) in a single measurement.     

Immunohistochemical detection of bromodeoxyuridine in formalin-fixed tissues

Summary Bromodeoxyuridine (BrdUrd) immunohisto-chemistry has recently been introduced for the visualization of DNA-synthesizing nuclei. In order to detect the BrdUrd incorporated into nuclear DNA in formalin-fixed, paraffin-embedded tissues, we tested several different pretreatment procedures including digestion with proteinase and hydrolysis with HCl, prior to immunoperoxidase staining. In order to determine the optimal conditions for detecting nuclear BrdUrd, mice were given BrdUrd and ³H-thymidine simultaneously, and the autoradiographic and immunohistochemical results obtained in BrdUrd-stained sections were compared. It was found that digestion with 0:05% proteinase at 37°C for 20 min and hydrolysis with 1 N HCl at 37°C for 20 min was sufficient to detect BrdUrd immunore-activity in ³H-thymidine-labelled nuclei, the results being virtually unaffected by the orders in which the two pretreatments were performed. Our method extends the range of application for BrdUrd, immunohistochemistry in cell-kinetic studies.     

Application of Biotin,Digoxigenin or Fluorescein Conjugated Deoxynucleotides to Label DNA Strand Breaks for Analysis of Cell Proliferation and Apoptosis Using Flow Cytometry

A flow cytometric method has recently been developed using biotinylated dUTP (b-dUTP) in a reaction catalyzed by terminal deozynucleotidyl transferase (TdT) to identify the endonuclease-induced DNA strand breaks occurring during apoptosis. Counterstaining of DNA makes it possible to relate apoptosis to cell cycle position or DNA index. In the present study, we compared this method with one using digoxigenin-conjugated dUTP (d-dUTP) to label apoptotic cells. The discrimination of apoptotic from nonapoptotic cells was similar when incorporation of d-dUTP was compared with b-dUTP. Both techniques resulted in a 20–30 fold increase in staining of apoptotic over nonapoptotic cells although somewhat less background fluorescence was observed with the d-dUTP. Direct labeling with fluo-resceinated dUTP (f-dUTP) was less sensitive in detecting DNA strand breaks, but had the advantage of simplicity. The principle of labeling DNA strand breaks using TdT was also employed to identify DNA replicating cells. To this end, the cells were incubated in the presence of BrdUrd, then exposed to UV light to selectively photolyse DNA containing the incorporated BrdUrd. DNA strand breaks resulting from the photolysis were then labeled with b-dUTP or d-dUTP. This approach is an alternative to immunocytochemical detection of BrdUrd incorporation, but unlike the latter does not require prior DNA denaturation, thus can be applied when the denaturation step must be avoided. The method was sensitive enough to recognize DNA synthesizing cells that were incubated with BrdUrd for only 5 min, the equivalent of replication of less than 1% of the cell's genome. The discrimination between apoptotic vs. BrdUrd incorporating-cells is based on different extractability of DNA following cell fixation. This method can be applied to analyze both cell proliferation (DNA replication) and death (by apoptosis) in a single measurement.     

Flow cytometric analysis of experimental parameters for the immunofluorescent labeling of BrdUrd in various tumour cell lines

This report describes the results of the comparison of three different methods and three monoclonal antibodies to stain cells in suspension for incorporated bromodeoxyuridine and total DNA content. The procedures were tested in three different experimental tumour cell lines. The sensitivity of the different procedures was expressed as the ratio of the anti-BrdUrd fluorescence intensities of the S and G1 phase cells (FS/FG1 ratio). There were remarkable differences in sensitivity between the different procedures. With the heat denaturation the most favourable FS/FG1 ratio's were obtained but substantial cell loss occurred during this procedure which is a disadvantage for clinical application. With the pepsin digestion + acid denaturation procedure cell loss was negligible. The standard acid denaturation procedure was inferior to the other two methods. Using the pepsin digestion + acid denaturation procedure we examined the variations in sensitivity for the different monoclonal antibodies and cell lines and the influence of BrdUrd concentration, labelingtime and cell concentration. The binding characteristics for the various antibodies differed considerably in our hands. Only with the IU4 antibody we obtained FS/FG1 ratio's comparable with those described in the literature. No difference was observed between the cell lines. Variation in cell concentration between 1 x 10(4) to 1 x 10(6) ml nor BrdUrd concentration appeared to influence the sensitivity of the procedure. A labelingtime of 1 h or even 30 min seems to be more than sufficient for an optimal FS/FG1 ratio. Our results indicate that using the appropriate antibody and immunofluorescence BrdUrd can be detected by flow cytometry, after incorporation into the DNA of tumour cells under a wide range of culture conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of restriction endonucleases and exonuclease III to expose halogenated pyrimidines for immunochemical staining

We describe an enzymatic procedure for exposure of single-stranded DNA (ssDNA) containing the halogenated pyrimidines (HdUrd) bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd) in single cells to antibodies that bind to HrdUrd only in ssDNA. Production of ssDNA was accomplished by digesting the DNA using either restriction endonucleases alone or endonucleases followed by exonuclease III. The enzymatic production of ssDNA was maximal when 0.1 N HCl or 0.1 M citric acid plus Triton X-100 was added to extract nuclear proteins prior to enzymatic denaturation. The restriction endonucleases Bam HI, Dde I, Eco RI, and Hind III produced significant ssDNA when used alone to allow binding of detectable amounts of the anti-HdUrd antibody IU-4 in Chinese hamster ovary cells labeled with 10 microM BrdUrd or 10 microM IdUrd. However, these treatments did not expose sufficient ssDNA to allow binding of IU-1, an anti-HdUrd antibody with lower binding affinity. IU-4 binding was most intense after treatment with Eco RI. Treatment with exonuclease III following endonuclease digestion allowed substantially more IU-4 binding.     

Immunohistological optimization of detection of bromodeoxyuridine-labeled cells in decalcified tissue

Mice were injected with a range of bromodeoxyuridine (BrdU) concentrations from 0.01 mg to 10 mg, and their jaws were fixed in buffered formalin or modified Carnoy. After EDTA or formic acid decalcification, a range of DNA denaturation schedules was assessed and immunohistological detection of BrdU-containing nuclei was performed using the Sera Lab anti-BrdU antibody MAS 250b. For Carnoy-fixed tissue, denaturation in 1 N HCl for 8 min at 60 degrees C was capable of adequately detecting an injected dose of 0.05 mg but not a dose of 0.01 mg BrdU, whereas pepsin/HCl treatment gave only weak staining after injection of 1 mg BrdU. In comparison, formalin fixation required pre-treatment with 0.2-0.4% pepsin/HCl at 37 degrees C for comparable staining intensity, but could still not adequately detect a dose of 0.1 mg BrdU. There was little detectable difference in staining between EDTA- and formic acid-decalcified tissues after injection of 10 mg BrdU.     

An improved method for the immunocytochemical detection of bromodeoxyuridine labeled nuclei using flow cytometry

A new staining protocol is described for the immunocytochemical detection of BrdUrd labeled nuclei. Pepsin treatment of ethanol fixed cells or tissue, followed by DNA denaturation at low pH, resulted in increased sensitivity of BrdUrd staining comparable to the thermal denaturation protocol, and decreased background binding. This technique is applicable to cell suspensions, including cultured cells and bone marrow cells. Furthermore, pepsin digestion of ethanol fixed tissue fragments resulted in a high recovery of nuclei in which incorporated BrdUrd could be detected. This possibility, together with the high sensitivity, make this method especially suitable for cell kinetic studies of human solid tumors in vivo.     

Bromodeoxyuridine: a diagnostic tool in biology and medicine, Part I: Historical perspectives, histochemical methods and cell kinetics

Summary Bromodeoxyuridine (BrdUrd), a thymidine analogue incorporated into DNA, can be quantified by fluorescent or chromophoric quenching of dyes bound to DNA or with antibodies to BrdUrd. These technologies have been used since the 1970s as tools for measuring DNA synthesis in isolated chromosomes and in cells and tissues. This paper is Part I of a three-part comprehensive review of the literature over the last 20 years (to the end of 1993) describing the histochemical methods for measuring BrdUrd in cells and tissues. Fixation, denaturation and staining procedures are compared for quantifying BrdUrd for microscopy and flow cytometry. Non-immunochemical methods related to the quenching of fluorescent DNA stains by BrdUrd are also described. Methods are described for the comparative assay of cell kinetic parameters by tritiated thymidine and bromodeoxyuridine. The multivariate BrdUrd/DNA assay of T_s, and T_c, and a comparison of recent methods based on the single biopsy bivariate analysis of T_pot, is presented. Recent developments in the use of double halopyrimidine label to determine kinetic parameters are also reviewed.     

Flow cytometric analysis of experimental parameters for the immunofluorescent labeling of BrdUrd in various tumour cell lines

Summary This report describes the results of the comparison of three different methods and three monoclonal antibodies to stain cells in suspension for incorporated bromodeoxyuridine and total DNA content. The procedures were tested in three different experimental tumour cell lines. The sensitivity of the different procedures was expressed as the ratio of the anti-BrdUrd fluorescence intensities of the S and G1 phase cells (FS/FG1 ratio). There were remarkable differences in sensitivity between the different procedures. With the heat denaturation the most favourable FS/FG1 ratio's were obtained but substantial cell loss occurred during this procedure which is a disadvantage for clinical application. With the pepsin digestion + acid denaturation procedure cell loss was negligible. The standard acid denaturation procedure was inferior to the other two methods. Using the pepsin digestion + acid denaturation procedure we examined the variations in sensitivity for the different monoclonal antibodies and cell lines and the influence of BrdUrd concentration, labelingtime and cell concentration. The binding characteristics for the various antibodies differed considerably in our hands. Only with the IU4 antibody we obtained FS/FG1 ratio's comparable with those desenbed in the literature. No difference was observed between the cell lines. Variation in cell concentration between 1 × 10⁴ to 1 × 10⁶ ml nor BrdUrd concentration appeared to influence the sensitivity of the procedure. A labelingtime of 1 h or even 30 min seems to be more than sufficient for an optimal FS/FG1 ratio.Our results indicate that using the appropriate antibody and immunofluorescence BrdUrd can be detected by flow cytometry, after incorporation into the DNA of tumour cells under a wide range of culture conditions.For clinical application, the pepsin digestion + acid dena uration method in combination with IU4 antibody seems to be the procedure of choice due to its good reproducibility, sensitivity and its low cell loss.     

Different sensitivity of DNA in situ in interphase and metaphase chromatin to heat denaturation

Heat denaturation of DNA in situ, in unbroken cells, was studied in relation to the cell cycle. DNA in metaphase cells denatured at lower temperatures (8 degrees-10 degrees C lower) than DNA in interphase cells. Among interphase cells, small differences between G1, S, and G2 cells were observed at temperatures above 90 degrees C. The difference between metaphase and interphase cells increased after short pretreatment with formaldehyde, decreased when cells were heated in the presence of 1 mM MgCl2, and was abolished by cell pretreatment with 0.5 N HCl. The results suggest that acid-soluble constituents of chromatin confer local stability to DNA and that the degree of stabilization is lower in metaphase chromosomes than in interphase nuclei. These in situ results remain in contrast to the published data showing no difference in DNA denaturation in chromatin isolated from interphase and metaphase cells. It is likely that factors exist which influence the stability of DNA in situ are associated with the super-structural organization of chromatin in intact nuclei and which are lost during chromatin isolation and solubilization. Since DNA denaturation is assayed after cell cooling, there is also a possibility that the extent of denatured DNA may be influenced by some factors that control strand separation and DNA reassociation. The different stainability of interphase vs. metaphase cells, based on the difference in stability of DNA, offers a method for determining mitotic indices by flow cytofluorometry, and a possible new parameter for sorting cells in metaphase.     

Induction of placental alkaline phosphatase in choriocarcinoma cells by 5-bromo-2'-deoxyuridine.

Growth of choriocarcinoma cells in the presence of 5-bromo-2'-deoxyuridine (BrdUrd) results in a 30- to 40-fold increase in alkaline phosphatase activity. The effects of BrdUrd is specific for phosphatase with an alkaline pH optimum. The induction by BrdUrd is probably not due to the production of an altered enzyme, since the induced enzyme resembles the basal enzyme in thermal denaturation and kinetic properties. Enzyme induction can be prevented by thymidine but not by deoxycytidine or deoxyuridine. The induction of alkaline phosphatase appears to require incorporation of the BrdUrd into cellular DNA. The presence of BrdUrd in the growth medium is not necessary for alkaline phosphatase induction in proliferating cells containing BrdUrd-substituted genomes. However, enzyme induction and maintenance of the induced levels of alkaline phosphatase in nonproliferating cells containing BrdUrd-substituted DNA requires the presence of the analogues in the medium. The induction of alkaline phosphatase by BrdUrd in probably an indirect process.     

Induction of placental alkaline phosphatase in choriocarcinoma cells by 5-bromo-2′-deoxyuridine

Summary Growth of choriocarcinoma cells in the presence of 5-bromo-2′-deoxyuridine (BrdUrd) results in a 30- to 40-fold increase in alkaline phosphatase activity. The effect of BrdUrd is specific for phosphatase with an alkaline pH optimum. The induction by BrdUrd is probably not due to the production of an altered enzyme, since the induced enzyme resembles the basal enzyme in thermal denaturation and kinetic properties. Enzyme induction can be prevented by thymidine but not by deoxycytidine or deoxyuridine. The induction of alkaline phosphatase appears to require incorporation of the BrdUrd into cellular DNA. The presence of BrdUrd in the growth medium is not necessary for alkaline phosphatase induction in proliferating cells containing: BrdUrd-substituted genomes. However, enzyme induction and maintenance of the induced levels of alkaline phosphatase in nonproliferating cells containing BrdUrd-substituted DNA requires the presence of the analogues in the medium. The induction of alkaline phosphatase by BrdUrd in probably an indirect process.