Low doses of bromo- and iododeoxyuridine produce near-saturation labeling of adult proliferative populations in the dentate gyrus.

Cell proliferation can be detected by the incorporation of tritiated thymidine (3H-dT) or halopyrimidines during DNA synthesis in progenitor cells. Administration of two thymidine analogues at different times can further determine the cell-cycle kinetics of proliferating cells. Traditionally, this was done by combining bromodeoxyuridine (BrdU) immunocytochemistry and 3H-dT autoradiography, or by BrdU and iododeoxyuridine (IdU) double-labeling using two mouse antibodies. However, these methods either require lengthy exposure time or involve complicated histological procedures for differentiating between two antibodies of the same species. Here we report a simple and reliable method of distinguishing BrdU- and IdU-labeled cells by immunofluorescence. This method uses a mouse monoclonal antibody that recognizes both BrdU and IdU and a rat anti-BrdU antibody that has no cross-reactivity with IdU. When combined with species-specific secondary antibodies that are conjugated to different fluorophores, this method identifies BrdU- and IdU-incorporation as doubly and singly labeled cells, respectively. This method has broad applications. First, we demonstrate that this method can distinguish mouse cortical neurons generated on different embryonic days. Second, by administering IdU and BrdU at varying intervals, we used this method to calculate that the length of S-phase of neural progenitor cells in the adult mouse dentate gyrus is approximately 6 h. Finally, we show that a six-fold higher concentration of IdU detects only 10% more cells than the standard dose of BrdU (50 mg/kg) using the double-labeling method. These results suggest that the standard dose of BrdU is sufficient to label the majority of proliferative populations in the S-phase in pulse labeling experiments.

Site-specific and temporally controlled initiation of DNA replication in a human cell-free system.

We have recently established a cell-free system from human cells that initiates semi-conservative DNA replication in nuclei isolated from cells which are synchronised in late G1 phase of the cell division cycle. We now investigate origin specificity of initiation using this system. New DNA replication foci are established upon incubation of late G1 phase nuclei in a cytosolic extract from proliferating human cells. The intranuclear sites of replication foci initiated in vitro coincide with the sites of earliest replicating DNA sequences, where DNA replication had been initiated in these nuclei in vivo upon entry into S phase of the previous cell cycle. In contrast, intranuclear sites that replicate later in S phase in vivo do not initiate in vitro. DNA replication initiates in this cell-free system site-specifically at the lamin B2 DNA replication origin, which is also activated in vivo upon release of mimosine-arrested late G1 phase cells into early S phase. In contrast, in the later replicating ribosomal DNA locus (rDNA) we neither detected replicating rDNA in the human in vitro initiation system nor upon entry of intact mimosine-arrested cells into S phase in vivo. As a control, replicating rDNA was detected in vivo after progression into mid S phase. These data indicate that early origin activity is faithfully recapitulated in the in vitro system and that late origins are not activated under these conditions, suggesting that early and late origins may be subject to different mechanisms of control.

Novel formula for cell kinetics in xenograft model of hepatocellular carcinoma using histologically calculable parameters.

The growth rate of tumors should be assessed in terms of both tumor cell proliferation and death. The former is considered to be determined by growth fraction and cell-cycle time, whereas the latter is mainly determined by apoptosis, especially in tumors with a low level of necrosis. While most hepatocellular carcinomas (HCCs) in a relatively early stage contain only a small amount of necrosis, the growth rate supposedly depends mainly on growth fraction, cell-cycle time, and apoptosis. However, their quantitative relationship remains unknown. We have derived a novel theoretical formula for determining this relationship in nonnecrotic HCC, using Ki-67-positive index, apoptotic score, and a correction factor, all calculable by histological assessment without injecting labeling agents. Furthermore, we confirmed the reliability of this formula, using a xenograft model of human HCC with less than 15% necrosis. In this model the values of cell-cycle time calculated from the formula were very close to those estimated by a conventional double-labeling method and showed high correlations. Since our novel formula can clarify the cell kinetics without cumbersome labeling procedures, it is expected to be clinically applicable to HCC with a small portion of necrosis, using the radiographically measured growth rate and the histologically assessed cell kinetic parameters.

A new immunocytochemical technique for ultrastructural analysis of DNA replication in proliferating cells after application of two halogenated deoxyuridines.

We describe a colloidal gold immunolabeling technique for electron microscopy which allows one to differentially visualize portions of DNA replicated during different periods of S-phase. This was performed by incorporating two halogenated deoxyuridines (IdUrd and CldUrd) into Chinese hamster cells and, after cell processing, by detecting them with selected antibodies. This technique, using in particular appropriate blocking solutions and also Tris buffer with a high salt concentration and 1% Tween-20, prevents nonspecific background and crossreaction of both antibodies. Controls such as digestion with DNase and specific staining of DNA with osmium ammine show that labeling corresponds well to replicated DNA. Different patterns of labeling distribution, reflecting different periods of DNA replication during S-phase, were characterized. Cells in early S-phase display a diffuse pattern of labeling with many spots, whereas cells in late S-phase show labeling confined to larger domains, often at the periphery of the nucleus or associated with the nucleolus. The good correlation between our observations and previous double labeling results in immunofluorescence also proved the technique to be reliable.

Bromodeoxyuridine and iododeoxyuridine double immunostaining for epoxy resin sections.

To establish bromodeoxyuridine (BrdUrd)/iododeoxyuridine (IdUrd) double immunostaining for thick sections of epoxy resin-embedded tissues, young hamsters received intra-peritoneal injections of IdUrd and BrdUrd 3 hr and 1 hr before sacrifice, respectively. The intestines were fixed with phosphate-buffered 4% paraformaldehyde and embedded in an Epon-Araldite mixture. The epoxy resin was removed completely by a sodium methoxide/benzene/methanol solution. This epoxy resin removal method was effective for BrdUrd/IdUrd immunostaining using a mono-specific antibody for BrdUrd (Br-3) and a bi-specific antibody for BrdUrd (Br-3) and a bi-specific antibody for BrdUrd and IdUrd (IU-4), followed by the ABC complex method. Epoxy sections stained with these antibodies showed clear localization of nuclei incorporating the two thymidine analogues with precise morphology of labeled cells. Furthermore, ultrastructural observation of thin sections adjacent to thick sections immunostained for BrdUrd/IdUrd confirmed the cell type and ultrastructural features of cells labeled with these thymidine analogues.

Simultaneous immunohistochemical detection of IUdR and BrdU infused intravenously to cancer patients.

Cell cycle kinetics of solid tumors in the past have been restricted to an in vitro labeling index (LI) measurement. Two thymidine analogues, bromodeoxyuridine (BrdU) and iododeoxyuridine (IUdR), can be used to label S-phase cells in vivo because they can be detected in situ by use of monoclonal antibodies (MAb) against BrdU (Br-3) or IUdR (3D9). Patients with a variety of solid tumors (lymphoma, brain, colon cancers) received sequential intravenous IUdR and BrdU. Tumor tissue removed at the end of infusion was embedded in plastic and treated with MAb Br-3 and 3D9 sequentially, using a modification of a previously described method. Clearly single and double labeled cells were visible, which enabled us to determine the duration of S-phase (Ts) and the total cell cycle time (Tc), in addition to the LI in these tumors. Detailed control experiments using tissue culture cell lines as well as bone marrow cells from leukemic patients are described, including the comparison of this double label technique with our previously described BrdU-tritiated thymidine technique. We conclude that the two methods are comparable and that the IUdR/BrdU method permits rapid and reliable cell cycle measurements in solid tumors.

Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication.

Monoclonal antibodies specific for 5-bromodeoxyuridine have been produced and applied in detecting low levels of DNA replication on a cell-by-cell basis in vitro. The immunoglobulin-producing hybridomas were derived from spleen cells of mice immunized with a conjugate of iodouridine and ovalbumin. The cells were fused with the plasmacytoma line SP2/0Ag14. The antibodies produced are highly specific for bromodeoxyuridine and iododeoxyuridine and do not cross-react with thymidine. DNA synthesis in cultured cells exposed to bromodeoxyuridine for as short a time as 6 minutes can be detected easily and rapidly by an immunofluorescent staining method and quantitated by flow cytometry.

Deoxyribonucleic acid replication in single cells and chromosomes by immunologic techniques.

Antibodies to 5-bromodeoxyuridine (BrdU) or iododeoxyuridine may be used to identify cells or regions of chromosomes in which de novo deoxyribonucleic acid synthesis has occurred. The antibodies to BrdU were produced in rabbits by injection of the antigen, a conjugate between bovine serum albumin and bromouridine (BrU), or iodouridine. Specific antibodies were produced by affinity chromatography on AH-Sepharose 4B to which had been coupled BrU. Anti-BrU cross-reacts with iodeodeoxyuridine. Indirect antibody techniques have been used to monitor deoxyribonucleic acid synthesis in nuclei; anti-BrdU treatment was followed by goat anti-rabbit immunoglobulin G labeled with either fluorescein or horseradish peroxidase. By use of these techniques, labeling indices were determined in cell cultures which had been pulsed with 3H-BrdU. The immunologic technique compared favorably with the autoradiographic methods performed concurrently on the same cultures. Metaphase chromosomes from synchronous CHO cell which had been pulse labled with BrdU at different time intervals during S phase were subjected to these immunologic procedures. Chromosome banding was observed with both the fluoresence and peroxidase methods. Chromosomes from cells not containing BrdU did not exhibit banding.

Allergic contact dermatitis caused by idoxuridine. Patterns of cross reactivity with other pyrimidine analogues.

Idoxuridine has been used for many years in the treatment of herpex simplex infections of the eye. Use of the drug for herpes simplex infection of the skin is increasing. Ophthalmologists have noted occasional conjunctival and corneal irritant reactions, but no true delayed cutaneous hypersensitivity has been verified. We report four cases of allergic contact dermatitis from idoxuridine, sensitized by both eye and skin applications. Cross reactivity to brominated and chlorinated, but not fluorinated, pyrimidine analogues is noted. Extensive patch testing indicates the general relationship between the structure of pyrimidine compounds and their antigenic cross reactivity.