Double labeling and in vitro versus in vivo incorporation of bromodeoxyuridine in patients with acute nonlymphocytic leukemia

A monoclonal antibody against bromodeoxyuridine (BrdUrd) was produced, and a rapid slide technique (RPMB technique) was developed for the estimation of S-phase cells in a population using this antibody. Bone marrow cells from patients with acute nonlymphocytic leukemia (ANLL) were studied by both the RPMB technique and tritiated thymidine (3HdThd) labeling index studies. The percentage of S-phase cells obtained by each method was compared in 50 samples, and the correlation coefficient was r = 0.89. A "double label" method is also described in which cells were simultaneously incubated with either BrdUrd and 3HdThd or BrdUrd and tritiated cytosine arabinoside (3HAra-C). The samples were first processed by the RPMB technique and then by autoradiography. Results showed only black grains overlying the nuclei of fluorescent cells in each group. An automated microphotometer was used to quantitate grains and fluorescence from each cell. This demonstrated an almost direct relationship between grains and fluorescence from BrdUrd + 3HdThd slides, whereas different patterns of relationship were noted from BrdU + 3HAra-C slides of leukemic patients. Their implications are discussed in the text. Finally, intravenous infusions of BrdUrd was given to five leukemic patients. S-phase cells were recognized distinctly within 5 min of starting the infusion. The percentage of S-phase cells was almost identical from in vivo and in vitro samples. Various possibilities of studying the biological behavior of acute leukemias and analyzing cell cycle characteristics are discussed.     

Mapping of the pattern of DNA replication in polytene chromosome from Chironomus thummi using monoclonal anti-bromodeoxyuridine antibodies

We present results from a nonautoradiographic study of DNA replication in polytene chromosomes from dipteran larvae. Monoclonal antibodies with specificity for 5-bromodeoxyuridine (BrdUrd) were used to localize by indirect immunofluorescence the sites of BrdUrd incorporation and to follow the dynamics of DNA synthesis in salivary gland cells of 4th instar Chironomus thummi larvae. This technique presents numerous advantages over autoradiographic procedures and allows mapping of DNA synthesis patterns at the level of resolution of one chromosomal band. Several replication patterns were observed, classified according to characteristic features, and tentatively assigned to specific periods of the S-phase. In early S-phase, DNA synthesis is first detectable in puffs and interbands, later in bands. Most chromosomal bands appear to initiate DNA synthesis synchronously; however, in bands within centromeric and heterochromatic regions the start of synthesis is delayed. At mid S-phase, all the bands show uniform staining. Subsequent staining patterns are increasingly differential with the bands displaying characteristic fluorescence intensities. As replication progresses through the late S-phase period, the chromosomes show a decreasing number of fluorescent bands. The last bands to terminate replication are located in centromeric and heterochromatic DNA-rich regions and a few bands of low DNA content in region IIAa-c.     

A quantitative method for evaluating bivariate flow cytometric data obtained using monoclonal antibodies to bromodeoxyuridine.

A method is presented for analyzing data from bivariate analysis of cell populations exposed to bromodeoxyuridine and subsequently examined both for the presence of BrdUrd and for the cellular DNA content. It is shown that certain features may be defined in the bivariate data which are constant independent both of cell type and, within limits, experimental variability. These landmark features include the ratio of red, DNA, fluorescence of G2 + M cells to G1 cells, the ratio of green fluorescence corresponding to the non-specific binding of unlabeled G2 + M cells to unlabeled G1 cells, and the distribution of green fluorescence in unlabeled cells. The landmarks make it possible to standardize rules for establishing the separation line between-labeled and unlabeled cells as required in these experiments to obtain estimates of cytokinetic parameters. Values obtained for the DNA synthesis time and the potential doubling time which result from different decision rules for distinguishing labeled from unlabeled are compared in two murine tumor lines. The potential doubling time, but not the DNA synthesis time is shown to depend sensitively on the separation line. Suggestions are presented for analyzing clinical data with this procedure.     

A monoclonal antibody reactive with 5-bromo-2-deoxyuridine that does not require DNA denaturation

We describe a mouse monoclonal antibody (BU-1) reactive with 5-bromo-2-deoxyuridine (BrdUrd). The antibody is different from previously described BrdUrd monoclonal antibodies in that BU-1 does not require pretreatment of cells with strong DNA denaturants in order for the antibody to react with BrdUrd incorporated in the DNA. The antibody can be used in immunocytochemical and indirect immunofluorescent assays and can be used to identify cells that have incorporated BrdUrd. Double staining with BU-1 antibody and propidium iodide has been used to confirm S-phase measurements with the BU-1 antibody. Immunocytochemical stains using the BU-1 antibody do not destroy cell morphology and allow cell identification to be performed simultaneously with S-phase measurements. Flow cytometer two-color fluorescence analysis allows the simultaneous identification of cell surface or cytoplasmic markers and S-phase quantitation. The BU-1 antibody should broaden the application of cell kinetic measurements to individual elements of cell populations that are heterogeneous with respect to morphology, surface marker, and other biological features.     

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.     

SCE induction and harlequin staining in mycoplasma-contaminated Chinese hamster cells

Chinese hamster V79 and CHO cells infected with Mycoplasma hyorhinis show elevated sister-chromatid exchange (SCE) levels but normal cell proliferation and levels of chromosomal aberrations when compared with uninfected cells. Harlequin staining patterns differ from those seen with uninfected cells at similar levels of bromodeoxyuridine (BrdUrd), indicating that BrdUrd is rapidly depleted from the medium by the mycoplasmal uridine phosphorylase and therefore becomes unavailable over the two cell cycles necessary for harlequin staining. Continuous treatment with the antibiotic minocycline restores the SCE level and harlequin staining to that seen in uncontaminated cells. The results suggest that mycoplasma infection should be suspected if harlequin staining patterns indicate a sudden decrease in incorporation of BrdUrd in cells grown in normal levels of BrdUrd.     

Subdivision of S-phase by analysis of nuclear 5-bromodeoxyuridine staining patterns

After pulse labeling of mammalian cells in vivo or in vitro with 5-bromodeoxyuridine (BrdUrd), followed by immunostaining with a monoclonal antibody to DNA-incorporated BrdUrd, various intranuclear staining patterns are observed. These results are obtained in various labeling, fixation, denaturation, and staining conditions. We defined five different patterns in immunoperoxidase-stained monolayers of human and rodent cancer cells and compared mean nuclear areas as measured by computerized planimetry. Furthermore, we evaluated frequency distributions of these patterns in partly synchronized cell populations and correlated these results with flow cytometric DNA histograms. The results indicate that the observed patterns reflect the spatial and temporal organization of DNA synthesis, which seems to be characterized by at least three successive stages of replication. Evaluation of these patterns may have various applications in studies on cell cycle kinetics.     

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.     

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.     

BrdUrd incorporation studies for evaluation of spermatogenesis in the blue fox.

The utility of BrdUrd incorporation techniques for studies of spermatogenesis was investigated in the blue fox. BrdUrd was injected intraperitoneally followed by collection of testicular tissue by castration/hemicastration at intervals up to 35 days after pulse labelling. Fluorescent tagged monoclonal antibodies against BrdUrd allowed detection of cells with incorporated tracer in histological sections by fluorescent light microscopy as well as in isolated testicular cells by bivariate BrdUrd/DNA flow cytometry. The duration of the spermatogenic cycle was estimated by following the labelled cohort of preleptotene spermatocytes by immunofluorescence in sections through the various stages of maturation to the late spermatid stage. These data were confirmed by bivariate BrdUrd/DNA flow cytometry of testicular cells in suspensions. Furthermore, estimations of the S phase durations and length of the spermatogonial cell cycle were possible. A consistent and satisfactory fluorescence intensity of incorporated label throughout the study shows that degradation of the incorporated label is no practical problem for this type of study, and suggests that the method is an excellent tool for studying aspects of proliferation and maturation during normal as well as perturbed spermatogenesis. Advantages of the described method include avoidance of potential radiation influence on spermatogenesis from commonly used radiolabelled tracers, e.g., 3H-TdR, and that both large and small animals can be investigated at modest cost since the unlabelled BrdUrd is considerably less expensive than labelled tracers.     

Fluorescence enhancement of DNA-bound TO-PRO-3 by incorporation of bromodeoxyuridine to monitor cell cycle kinetics.

BACKGROUND:The detection of DNA-incorporated bromodeoxyuridine (BrdUrd) in mammalian cells is a well-known and important technique to study cell cycle. The use of TO-PRO-3 for detection of BrdUrd substitution of DNA by dual-laser flow cytometry has been investigated. METHODS:Fluorescence enhancement of TO-PRO-3 in BrdUrd-labeled cells is registered in combination with the fluorescence emission of the intercalating dye propidium iodide (PI) as a total DNA stain to give bivariate DNA/BrdUrd histograms. By the low concentration of only 0.3 mircoM TO-PRO-3, BrdUrd detection is optimized, and undisturbed total DNA content by PI can be detected as well. TO-PRO-3 is excited by a red HeNe laser and PI by an argon ion laser. RESULTS:In order to understand the binding of TO-PRO-3, energy transfer from PI to TO-PRO-3 has been measured as well as the influence of an external DNA binding dye such as Hoechst 33258 with Adenine-Thymine (AT) binding specificity. Cell cycle studies of human SCL-2 keratinocytes and mouse 3T3 cells prove the method to be as generally applicable as the classical BrdUrd/Hoechst quenching technique, but without need for expensive ultraviolet laser excitation. No BrdUrd sensitivity could be found for the similar dyes TO-PRO-1 and YO-PRO-3, whereas TO-PRO-5 and YOYO-3 showed only very little sensitivity to BrdUrd labeling as compared with TO-PRO-3. CONCLUSIONS:Cell cycle studies of mammalian cells can be done by dual-laser flow cytometry without the need for ultraviolet lasers by using the BrdUrd-dependent fluorescence enhancement of TO-PRO-3. Total DNA content can be measured simultaneously using PI.     

Extensive cochleotopic mapping of human auditory cortical fields obtained with phase-encoding FMRI

The primary sensory cortices are characterized by a topographical mapping of basic sensory features which is considered to deteriorate in higher-order areas in favor of complex sensory features. Recently, however, retinotopic maps were also discovered in the higher-order visual, parietal and prefrontal cortices. The discovery of these maps enabled the distinction between visual regions, clarified their function and hierarchical processing. Could such extension of topographical mapping to high-order processing regions apply to the auditory modality as well? This question has been studied previously in animal models but only sporadically in humans, whose anatomical and functional organization may differ from that of animals (e.g. unique verbal functions and Heschl''s gyrus curvature). Here we applied fMRI spectral analysis to investigate the cochleotopic organization of the human cerebral cortex. We found multiple mirror-symmetric novel cochleotopic maps covering most of the core and high-order human auditory cortex, including regions considered non-cochleotopic, stretching all the way to the superior temporal sulcus. These maps suggest that topographical mapping persists well beyond the auditory core and belt, and that the mirror-symmetry of topographical preferences may be a fundamental principle across sensory modalities.     

Mechanism of formation of chromatid exchanges

Chinese hamster cells with different patterns of distribution of 5-bromodeoxyuridine (BrdUrd) between chromosome subunits were subjected, during the G2 stage, to UV irradiation, which only produced breaks in BrdUrd substituted DNA. The frequency of chromatid and subchromatid interchanges as well as isochromatid aberrations was estimated. It was found that only BrdUrd containing chromatids were involved into aberrations; this result challenges the so called "molecular theory" for aberration production proposed by Leenhouts and Chadwick. A very small increase of the aberration yield in chromosomes without BrdUrd may be connected with the action of UV on the frequency of recombination. The observed frequency of interchanges was not proportional to the BrdUrd content in chromosomes and depended on the time of its incorporation: more exchanges were induced in the chromatids incorporating BrdUrd during the last round of replication. These regularities may be connected with some molecular peculiarities of chromosome structure and function.     

Granule-containing cells in the human superior cervical ganglion.

The fine structure of granule-containing cells in the human superior cervical ganglion is described. These cells are larger than the typical SIF cells in mammals and exhibit green-yellow fluorescence. They are characterized by numerous granular vesicles (80-140 nm in diameter) in the cytoplasm, but have many features in common with ordinary ganglion cells. They emit several long processes which form bundles together with ordinary nerve fibers. No synapses are found where the cells are presynaptic, although a few synapses are observed there where nerves are prosynaptic on the perikarya and processes of the cells. No close topographical relations are seen between the cells and blood vessels. It is suggested that the granule-containing cells are a special type of postganglionic aminergic neurons.     

In vitro transformation by bromodeoxyuridine and X irradiation in C3H 10T1/2 cells

The effect of 10(-5) M bromodeoxyuridine (BrdUrd) substitution in C3H 10T1/2 cells was evaluated. Cellular toxicity increased rapidly for BrdUrd exposure times that were longer than the population doubling time. Radiosensitization by BrdUrd exposure was almost complete after one cell doubling time and was characterized by a decrease in D0 and the survival curve shoulder. Exposure to BrdUrd for one cell doubling time produced only very low transformation levels, but for prolonged BrdUrd exposure times, the transformation frequency per viable cell increased significantly. BrdUrd incorporation also enhanced radiation induction of transformation above the transformation levels resulting from the independent action of X rays or BrdUrd treatment. These results show that BrdUrd is a transforming agent in C3H 10T1/2 cells and thus may be a carcinogen and that BrdUrd can enhance radiation-induced transformation.     

A method to measure the duration of DNA synthesis and the potential doubling time from a single sample

A method is described whereby the DNA synthesis time, Ts, can be calculated using data of a single sample of cells taken several hours after labelling with bromodeoxyuridine (BrdUrd). The method involves a simple calculation using flow cytometry data of BrdUrd incorporation (green fluorescence, FITC-labelled anti-BrdUrd-DNA antibody) and total DNA content (red fluorescence, propidium iodide). The movement of BrdUrd-labelled cells through the S phase can be quantified by measuring their mean red fluorescence relative to that of G1 and G2 cells. Assuming the movement of the labelled cells toward G2 is linear with time, Ts can be calculated by measuring their relative movement at any one time. The method was tested on cells in vitro and on bone marrow and tumor cells in vivo. Reasonable agreement was seen with published estimates of Ts for these tissues.     

Validation of bromodeoxyuridine immunohistochemistry for localization of S-phase cells in decalcified tissues. A comparative study with tritiated thymidine autoradiography

Summary Immunohistochemical detection of the thymidine analogue 5-bromo-2-deoxyuridine (BrdUrd), which is incorporated by S-phase cells, offers a convenient way of studying the proliferation kinetics of cells in normal skeletal tissues and in bone containing/derived tumours. To assess the validity of using this approach on decalcified, paraffin embedded tissues, the BrdUrd method was compared with tritiated thymidine (³H-TdR) autoradiography, using rat tibiae labelled with both³H-TdR and BrdUrd, fixed in Carnoy's fluid and decalcified in EDTA, prior to routine paraffin embedding. The distribution of BrdUrd-labelled cells correlated with the sites of cell proliferation in the growing rat tibia.Independent studies with each method on paired serial sections of double-labelled tissue, showed a highly significant correlation (r=0.81, p<0.0003) in the numbers of labelled cells seen in autoradiographs and immunostained sections from the proximal tibial growth plate. Combined BrdUrd immunohistochemistry and³H-TdR autoradiography showed that the majority of labelled cells in cartilage, bone marrow, and fibrous perichondrium and periosteum had incorporated both labels. These results show that BrdUrd immunohistochemistry is a valid technique for the study of dividing cells in mineralized tissues after decalcification.     

Effect of 5-fluoro-2'-deoxyuridine (FdUrd) on 5-bromo-2'-deoxyuridine (BrdUrd) incorporation into DNA measured with a monoclonal BrdUrd antibody and by the BrdUrd/Hoechst quenching effect

The purpose of this study was to improve the application of bromodeoxyuridine (BrdUrd) for the flow cytometric analysis of cell kinetics. In order to obtain a quantitative measure of the DNA synthesis rate (or the number of divided cells), BrdUrd should replace thymidine (dThd) completely in the newly synthesized DNA strands. The de novo synthesis of dThd monophosphate competing with BrdUrd incorporation was stopped by fluorodeoxyuridine (FdUrd). Cells of a human leukemic cell line (REH) were exposed to BrdUrd for either 20 min, 8 h, or 24 h. Bromodeoxyuridine incorporation was determined by a monoclonal antibody as well as by the BrdUrd/Hoechst (H) technique. Counterstaining of the DNA was performed with propidium iodide or ethidium bromide. DNA fluorescence was measured in both techniques with a two-parameter flow cytometer, the histograms being analyzed by computer. It was found that FdUrd is required in the BrdUrd/H technique for replacement of dThd at low BrdUrd concentrations and long incubation times. With short incubation periods, as used for detection by the monoclonal anti-BrdUrd antibody, FdUrd increases the incorporated BrdUrd amount when BrdUrd concentrations of 10 microM or less are applied.     

Differential giemsa staining of sister chromatids and the study of sister chromatid exchanges without autoradiography

Chinese hamster ovary cells grown for two rounds of DNA replication in the presence of BrdUrd contain sister chromatids that fluoresce differentially when stained with Hoechst 33258. If such fluorescent treatments are followed by incubation in 2 X SSC or water at 62° C and staining in 3% Giemsa, the chromosomes now contain one dark (unifilarly substituted) chromatid and one light (bifilarly substituted) chromatid, i.e. are harlequinized. These preparations do not fade and can be studied without resorting to fluorescence microscopy. Sister chromatid exchanges (SCE's) are seen with great clarity and resolution; and all the chromosomes in a cell can be scored, which is contrary to the usual experience with autoradiography. It was found that a) the yield of SCE's is dependent upon the concentration of BrdUrd in which the cells are grown and that the maximum number of SCE's that can occur spontaneously is 0.15 per chromosome per division cycle, b) the yield of SCE's doubles if the cells are exposed to visible light that can cause the photolysis of BrdUrd-containing DNA, and c) chromosomes that appear isolabelled in autoradiographic preparations come from observable multiple exchanges and are not the result of the segregation of DNA from a binemic chromosome. Furthermore, the staining patterns obtained in endoreduplicated cells clearly confirm that the polynucleotide strands of the DNA segregate into sister chromatids as though the newly synthesized strands were laid on the outside of the replicating double helix.