Monoclonal anti-BrdUrd Antibody Research Articles

Quantitative analysis of spermatogenic DNA synthesis in the rat using a monoclonal anti-5-bromodeoxyuridine antibody.

The utility of the 5-bromodeoxyuridine (BrdUrd) labelling technique for the quantitative analysis of spermatogenic deoxyribonucleic acid (DNA) synthesis was investigated in the rat. Rat testicles were labelled by a single intraperitoneal injection of 100 mg kg-1 of BrdUrd. The testicles were removed 1 h after injection, fixed in Bouin's fluid and embedded in paraffin. BrdUrd-labelled cells were detected by immunohistochemical staining using a monoclonal anti-BrdUrd antibody. The number of BrdUrd-labelled tubules per total number of tubules (percent L.T.), the number of BrdUrd-labelled cells per total number of tubules (tubular ratio) and the number of BrdUrd-labelled cells per number of Sertoli cells (Sertoli cell ratio in BrdUrd-labelled cells) were calculated as indices of spermatogenic DNA synthesis during each stage of the seminiferous epithelial wave. BrdUrd labelling was found exclusively in the nuclei of spermatogonia and in preleptotene spermatocytes in the seminiferous epithelium. The percent L.T. was generally greater than 50%, except in stages VI, VII and XIV, and the tubular as well as Sertoli cell ratios in BrdUrd-labelled cells was greater than 2.0 and 0.15, respectively, in stages I, II-III, V, VIII, X, and XII. The tubular ratio and Sertoli cell ratio in BrdUrd-labelled cells along the seminiferous epithelial wave had two distinct peaks. The distribution of the tubular ratio using the BrdUrd-labelling technique correlated well with the distribution previously established by measuring tritiated thymidine uptake per tubule. Thus, the BrdUrd labelling technique, which is more efficient than the tritiated thymidine labelling technique, can be used to quantitatively evaluate spermatogenic DNA synthesis.

Versican/PG-M Assembles Hyaluronan into Extracellular Matrix and Inhibits CD44-mediated Signaling toward Premature Senescence in Embryonic Fibroblasts

Versican/PG-M is a large chondroitin sulfate proteoglycan of the extracellular matrix which interacts with hyaluronan at the N-terminal G1 domain, composed of A, B, and B' subdomains. Recently, we generated knock-in mice Cspg2(Delta3/Delta3), whose versican, without the A subdomain, has decreased hyaluronan (HA) binding affinity, thereby exhibiting reduced deposition of versican in the extracellular matrix. Here, we show that the Cspg2(Delta3/Delta3) fibroblasts within 20 passages proliferate more slowly and acquire senescence. Whereas the extracellular matrix of the wild type fibroblasts exhibited a network structure of hyaluronan and versican, that of the Cspg2(Delta3/Delta3) fibroblasts exhibited approximately 35 and approximately 85% deposition of versican and HA, without such a structure. The Cspg2(Delta3/Delta3) fibroblasts showed a substantial increase of ERK1/2 phosphorylation and expression of senescence markers p53, p21, and p16. Treatment of wild type fibroblasts with hyaluronidase and exogenous hyaluronan enhanced ERK1/2 phosphorylation, and treatment with an anti-CD44 antibody that blocks HA-CD44 interaction inhibited the phosphorylation. These results demonstrate that versican is essential for matrix assembly involving hyaluronan and that diminished versican deposition increases free hyaluronan fragments that interact with CD44 and increase phosphorylation of ERK1/2, leading to cellular senescence.

Leptin Increases Adult Hippocampal Neurogenesis in Vivo and in Vitro

Leptin, an adipose-derived hormone, has been implicated in several physiological processes involving the hippocampus. However, the role of leptin in adult hippocampal neurogenesis remains unknown. Here we show that leptin regulates neurogenesis in the dentate gyrus of adult mice as well as in cultured adult hippocampal progenitor cells. Chronic administration of leptin to adult mice increased cell proliferation without significant effects on the differentiation and the survival of newly proliferated cells in the dentate gyrus. The expression of the long form leptin receptor, LepRb, was detected in hippocampal progenitor cells by reverse transcription-PCR and immunohistochemistry. Leptin treatment also increased proliferation of cultured adult hippocampal progenitor cells. Analysis of signal transduction pathways revealed that leptin stimulated phosphorylation of Akt and STAT3 but not ERK1/2. Furthermore, pre-treating the cells with specific inhibitors of Akt or STAT3 attenuated leptin-induced cell proliferation in a dose-dependent manner. Taken together, our results support a role for leptin in adult hippocampal neurogenesis and suggest the involvement of the Akt and STAT3 signaling pathways in mediating the actions of leptin on neurogenesis.

Loss of Huntingtin Function Complemented by Small Molecules Acting as Repressor Element 1/Neuron Restrictive Silencer Element Silencer Modulators

Increased levels of the repressor element 1/neuron restrictive silencer element (RE1/NRSE) silencing activity promoter, and a consequent reduction in the transcription of many RE1/NRSE-bearing neuronal genes, including brain-derived neurotrophic factor (BDNF), have been demonstrated in Huntington disease (HD) and represent one possible effector of its selective neuronal vulnerability. Restoring the expression levels of neuronal genes in diseased neurons therefore seems to be an attractive therapeutic approach. To this end, we have developed a cell-based reporter assay for monitoring RE1/NRSE silencing activity and validated it by genetically inactivating the RE1/NRSE or pharmacologically stimulating global transcription. In a pilot compound screen, we identified three closely related structural analogues that up-regulate reporter expression at low nanomolar concentrations, and follow-up studies have shown that they efficaciously increase endogenous BDNF levels in HD cells. Moreover, one of the compounds increases the viability of HD cells. Our findings suggest a new avenue for the development of drugs for HD and other neurodegenerative disorders based on the pharmacological up-regulation of the production of the neuronal survival factor BDNF and of other RE1/NRSE-regulated neuronal genes.

The CB1 Cannabinoid Receptor Mediates Excitotoxicity-induced Neural Progenitor Proliferation and Neurogenesis

Endocannabinoids are lipid signaling mediators that exert an important neuromodulatory role and confer neuroprotection in several types of brain injury. Excitotoxicity and stroke can induce neural progenitor (NP) proliferation and differentiation as an attempt of neuroregeneration after damage. Here we investigated the mechanism of hippocampal progenitor cell engagement upon excitotoxicity induced by kainic acid administration and the putative involvement of the CB1 cannabinoid receptor in this process. Adult NPs express kainate receptors that mediate proliferation and neurosphere generation in vitro via CB1 cannabinoid receptors. Similarly, in vivo studies showed that excitotoxicity-induced hippocampal NPs proliferation and neurogenesis are abrogated in CB1-deficient mice and in wild-type mice administered with the selective CB1 antagonist rimonabant (N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazolecarboxamide; SR141716). Kainate stimulation increased basic fibroblast growth factor (bFGF) expression in cultured NPs in a CB1-dependent manner as this response was prevented by rimonabant and mimicked by endocannabinoids. Likewise, in vivo analyses showed that increased hippocampal expression of bFGF, as well as of brain-derived neurotrophic factor and epidermal growth factor, occurs upon excitotoxicity and that CB1 receptor ablation prevents this induction. Moreover, excitotoxicity increased the number of CB1+ bFGF+ cells, and this up-regulation preceded NP proliferation. In summary, our results show the involvement of the CB1 cannabinoid receptor in NP proliferation and neurogenesis induced by excitotoxic injury and support a role for bFGF signaling in this process.

Axin Inhibits Extracellular Signal-regulated Kinase Pathway by Ras Degradation via β-Catenin

Interactions between the Wnt/beta-catenin and the extracellular signal-regulated kinase (ERK) pathways have been posited, but the molecular mechanisms and cooperative roles of such interaction in carcinogenesis are poorly understood. In the present study, the Raf-1, MEK, and ERK activities were concomitantly decreased in fibroblasts, which inhibit morphological transformation and proliferation by Axin induction. The inhibition of the components of the ERK pathway by Axin occurred in cells retaining wild-type beta-catenin, including primary hepatocytes, but not in cells retaining non-degradable mutant beta-catenin. Axin inhibits cellular proliferation and ERK pathway activation induced by either epidermal growth factor or Ras, indicating a role of Axin in the regulation of growth induced by ERK pathway activation. ERK pathway regulation by Axin occurs at least partly via reduction of the protein level of Ras. Both wild-type and mutant Ras proteins are subjected to regulation by Axin, which occurs in cells retaining wild-type but not mutant beta-catenin gene. The role of beta-catenin in the regulation of the Ras-ERK pathway was further confirmed by Ras reduction and subsequent inhibitions of the ERK pathway components by knock down of mutated form of beta-catenin. The Ras regulation by Axin was blocked by treatment of leupeptin, an inhibitor of the lysosomal protein degradation machinery. Overall, Axin inhibits proliferation of cells at least partly by reduction of Ras protein level via beta-catenin. This study provides evidences for the role of the Ras-ERK pathway in carcinogenesis caused by mutations of the Wnt/beta-catenin pathway components.

Trans-differentiation of Alveolar Epithelial Type II Cells to Type I Cells Involves Autocrine Signaling by Transforming Growth Factor β1 through the Smad Pathway

Trans-differentiation of Alveolar Epithelial Type II Cells to Type I Cells Involves Autocrine Signaling by Transforming Growth Factor β1 through the Smad Pathway

BM88 Is a Dual Function Molecule Inducing Cell Cycle Exit and Neuronal Differentiation of Neuroblastoma Cells via Cyclin D1 Down-regulation and Retinoblastoma Protein Hypophosphorylation

Control of cell cycle progression/exit and differentiation of neuronal precursors is of paramount importance during brain development. BM88 is a neuronal protein associated with terminal neuron-generating divisions in vivo and is implicated in mechanisms underlying neuronal differentiation. Here we have used mouse neuroblastoma Neuro 2a cells as an in vitro model of neuronal differentiation to dissect the functional properties of BM88 by implementing gain- and loss-of-function approaches. We demonstrate that stably transfected cells overexpressing BM88 acquire a neuronal phenotype in the absence of external stimuli, as judged by enhanced expression of neuronal markers and neurite outgrowth-inducing signaling molecules. In addition, cell cycle measurements involving cell growth assays, BrdUrd incorporation, and fluorescence-activated cell sorting analysis revealed that the BM88-transfected cells have a prolonged G(1) phase, most probably corresponding to cell cycle exit at the G(0) restriction point, as compared with controls. BM88 overexpression also results in increased levels of the cell cycle regulatory protein p53, and accumulation of the hypophosphorylated form of the retinoblastoma protein leading to cell cycle arrest, with concomitant decreased levels and, in many cells, cytoplasmic localization of cyclin D1. Conversely, BM88 gene silencing using RNA interference experiments resulted in acceleration of cell proliferation accompanied by impairment of retinoic acid-induced neuronal differentiation of Neuro 2a cells. Taken together, our results suggest that BM88 plays an essential role in regulating cell cycle exit and differentiation of Neuro 2a cells toward a neuronal phenotype and further support its involvement in the proliferation/differentiation transition of neural stem/progenitor cells during embryonic development.

Ribosomal Stress Couples the Unfolded Protein Response to p53-dependent Cell Cycle Arrest

Protein misfolding in the endoplasmic reticulum (ER) triggers a signaling pathway termed the unfolded protein response path-way (UPR). UPR signaling is transduced through the transmembrane ER effectors PKR-like ER kinase (PERK), inositol requiring kinase-1 (IRE-1), and activating transcription factor 6 (ATF6). PERK activation triggers phosphorylation of eIF2alpha leading to repression of protein synthesis, thereby relieving ER protein load and directly inhibiting cyclin D1 translation thereby contributing to cell cycle arrest. However, PERK(-/-) murine embryonic fibroblasts have an attenuated G(1)/S arrest that is not attributable to cyclin D1 loss, suggesting a cyclin D1-independent mechanism. Here we show that the UPR triggers p53 accumulation and activation. UPR induction promotes enhanced interaction between the ribosome proteins (rpL5, rpL11, and rpL23) and Hdm2 in a PERK-dependent manner. Interaction with ribosomal proteins results in inhibition of Hdm2-mediated ubiquitination and degradation of p53. Our data demonstrate that ribosomal subunit:Hdm2 association couples the unfolded protein response to p53-dependent cell cycle arrest.

The Transcriptional ETS2 Repressor Factor Associates with Active and Inactive Erks through Distinct FXF Motifs

The transcriptional ETS2 repressor factor (ERF) is phosphorylated by Erks both in vivo and in vitro. This phosphorylation determines the subcellular localization and biological function of ERF. Here, we show that active and inactive Erk2 proteins bind ERF with high affinity through a hydrophobic pocket formed by the alphaF and alphaG helices and the activation loop of Erk2. We have identified two FXF motifs on ERF that mediate the specific interaction with Erks. One of these motifs is utilized only by active Erks, whereas the other mediates the association with inactive Erks but also contributes to interaction with active Erks. Mutation of the phenylalanines of these motifs to alanines resulted in decreased association and phosphorylation of ERF by Erks both in cells and in vitro. ERF proteins carrying these mutations exhibited increased nuclear accumulation and increased inhibition of cellular proliferation. Expression of ERF regions harboring these motifs could inhibit Erk activity in cells. Our data suggest that, in the proper context, FXF motifs can mediate a strong and specific interaction not only with active but also inactive Erks and that these interactions determine protein function in vivo.

Increased Susceptibility of Mice Lacking Clara Cell 10-kDa Protein to Lung Tumorigenesis by 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone, a Potent Carcinogen in Cigarette Smoke

Ninety percent of all human lung cancers are related to cigarette smoking. Both tobacco smoke and lung tumorigenesis are associated with drastically reduced levels of Clara cell 10-kDa protein (CC10), a multifunctional secreted protein, naturally produced by the airway epithelia of virtually all mammals. We previously reported that the expression of CC10 is markedly reduced in animals exposed to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK, a potent carcinogen in tobacco smoke. Furthermore, it has been reported that CC10 expression, induced in certain tumor cells, reverses the transformed phenotype. We demonstrate here that NNK exposure of CC10-knock-out (CC10-KO) mice causes a significantly higher incidence of airway epithelial hyperplasia and lung adenomas compared with wild type (WT) littermates (30% CC10-KO versus 5% WT, p = 0.041). We also found that compared with NNK-treated WT mice, CC10-KO mice manifest increased frequency of K-ras mutation, elevated level of Fas ligand (FasL) expression, and increased MAPK/Erk phosphorylation, all of which are considered predisposing events in NNK-induced lung tumorigenesis. We propose that CC10 has a protective role against NNK-induced lung tumorigenesis mediated via down-regulation of the above-mentioned predisposing events.

Nodal and ALK7 Inhibit Proliferation and Induce Apoptosis in Human Trophoblast Cells

Nodal, a member of the transforming growth factor-beta superfamily, is known to play critical roles in early vertebrate development, but its functions in extraembryonic tissues are unclear. ALK7 is a type I receptor for Nodal. Recently, we demonstrated that Nodal mRNA and several ALK7 transcripts are expressed in human placenta throughout pregnancy (Roberts, H. J., Hu, S., Qiu, Q., Leung, P. C. K., Cannigia, I., Gruslin, A., Tsang, B., and Peng, C. (2003) Biol. Reprod. 68, 1719-1726). In this study, we determined the role of Nodal and ALK7 in trophoblast cell proliferation and apoptosis. Overexpression of Nodal in normal trophoblast cells (HTR8/SVneo) and several choriocarcinoma cell lines resulted in a significant decrease in the number of metabolically active cells. The effect of Nodal could be mimicked by constitutively active ALK7 (ALK7-ca), but was blocked by kinase-deficient ALK7. The growth inhibitory effect of Nodal was also blocked by dominant-negative Smad2/3. Overexpression of Nodal and ALK7-ca induced apoptosis in trophoblast cells as determined by Hoechst staining, flow cytometry, and caspase-3 Western blotting. In addition, Nodal and ALK7-ca decreased the number of proliferating cells as measured by bromodeoxyuridine assays. Furthermore, overexpression of Nodal or ALK7-ca increased p27 expression, but reduced the levels of Cdk2 and cyclin D(1). Taken together, this study demonstrates for the first time that Nodal, acting through ALK7 and Smad2/3, inhibits proliferation and induces apoptosis in human trophoblast cells. Our findings also suggest that the Nodal-ALK7 pathway inhibits cell proliferation by inducing G(1) cell cycle arrest and that this effect is mediated in part by the p27-cyclin E/Cdk2 pathway.

Contribution of the Helix-Loop-Helix Factor Id2 to Regulation of Vascular Smooth Muscle Cell Proliferation

Smooth muscle cell (SMC) proliferation plays a key role in vascular proliferative disorders. The molecular mechanisms that control cell cycle entry of SMCs in response to vascular injury are not well understood. Id2 (inhibitor of DNA binding) is a member of the helix-loop-helix (HLH) family of transcription regulators that are known to promote cell cycle progression. Thus, we investigated the role of Id2 in SMC growth and cell cycle regulation. The results demonstrated that overexpression of Id2 resulted in a significant enhancement of SMC growth via increased S-phase entry. A possible mechanism of Id2-enhanced SMC growth is via regulation of p21 expression, as overexpression of Id2-inhibited transcriptional activity of a 2.3-kb p21 promoter/luciferase reporter construct as well as p21 protein levels. Id2 enhancement of SMC growth and inhibition of p21 expression were dependent on phosphorylation of Id2 by cyclin E/cdk2, as an Id2 cDNA containing a mutation in the cdk2 phosphorylation site (serine 5) failed to regulate SMC cell cycle progression or p21 promoter activity. The mechanism of cyclin E/cdk2 control of the Id2 effect may in part involve regulation of nuclear transport; unlike wild-type Id2, the Id2 mutant was not transported to the nucleus. Finally, in a rat carotid model of arterial injury, Id2 was expressed in a temporal pattern that parallels the kinetics of cellular proliferation. In summary, these results provide evidence that the Id2 protein is integrated into the cell cycle regulatory cascade that results in SMC proliferation following vascular injury and suggest that this effect is at least in part via a cdk2-dependent inhibition of p21 gene expression.

Morphometric analysis of bromodeoxyuridine distribution and cell density in the rat Dunning prostate tumor R3327-AT1 following treatment with radiation and/or hyperthermia.

To monitor cellular response to single doses of radiation (RT) and/or local tumor hyperthermia (LTH) proliferation kinetics were determined in the anaplastic prostate adenocarcinoma R3327-AT1 grown in Copenhagen rats. Tumor-bearing animals were injected i.v. with a bolus of bromodeoxyuridine (BrdUrd), and at defined times after treatment the tumors were surgically removed, fixed and embedded in paraffin. BrdUrd incorporated into the DNA of S-Phase nuclei was detected on 4-6 microns-thick tissue sections using a monoclonal anti-BrdUrd antibody followed by streptavidin-biotin and alkaline phosphatase as a reporter system. Cell nuclei were stained with the fluorescence dye DAPI (Diaminophenylindole). Morphometric analysis was performed using a computer-assisted Leitz-TAS/plus system. Depending on tumor size, up to 18,000 nuclei were routinely analyzed. Untreated tumors of standardized size (8-10 mm) exhibited a BrdUrd-labeling index (LI) of (6.9 +/- 1.6)%. In general, the LI was higher in the periphery than in the center, being more pronounced in larger tumors. After 6 Gy gamma-rays, the mean LI decreased to 1.8% (24 h) and rose afterwards to 5.4% by 168 h. Following LTH (43.5 degrees C, 35 min water bath), the mean LI rapidly decreased to 2% (8 h), rose to 9.8% (48 h), and plateaued at 6% after 168 h. A combined treatment consisting of irradiation (6 Gy) followed by LTH yielded smallest LI (2.4 +/- 0.18%) and lowest cell density (111 +/- 0.6 nuclei per field) by 168 h. The morphometric procedure was reliable and reproducible and can be used to characterize and compare the effects of different therapies on cell kinetics. Of particular value is that these analyses are done on an intact tissue architecture and hence enable a better interpretation of flow cytometric results of treatment-induced alterations within different topohistological regions in solid tumors. Moreover, the technique provides the basis for 3D reconstruction of the cellular activity and heterogeneity of experimental neoplasms.

Cca1 is required for formation of growth-arrested confluent monolayer of rat 3Y1 cells.

A novel cDNA fragment, named cca1 (confluent 3Y1 cell-associated 1), was previously isolated on the basis of preferential accumulation of the corresponding mRNA in growth-arrested confluent but not in growing subconfluent rat 3Y1 cells. The cca1 cDNA was found to consist of 5022 nucleotides with an open reading frame of 1905 nucleotides, encoding a protein of 635 amino acids. Unlike the 3Y1 cell case, cca1 mRNA was not detected in confluent 3Y1 BU, 3Y1 BU/pTK, 3Y1-16E6, or F2408 cells, whose growth patterns monitored by phalloidin staining and bromodeoxyuridine incorporation were different from those of the confluent 3Y1 cells. A restoration of the confluent 3Y1-type growth pattern was observed in the cca1 cDNA-introduced 3Y1 BU and 3Y1 BU/pTK cells after reaching confluence but not in the cDNA-introduced 3Y1-16E6 or F2408 cells. The results allow us to conclude that cca1 is required but not sufficient for formation of growth-arrested confluent monolayer of 3Y1 cells.

The characterization of the monoclonal antibody Th‐10a, specific for a nuclear protein appearing in the S phase of the cell cycle in normal thymocytes and its unregulated expression in lymphoma cell lines

A monoclonal antibody (Th-10a) specific for the nuclear protein appearing in the S phase of the cell cycle in normal mouse thymocytes was derived by immunizing Wistar rats with a murine thymic lymphoma (TIGN), and its isotype was rat IgG2a and had kappa light chain. Immunohistochemical staining of frozen sections of B10.Thy1.1 newborn thymus and embryonic intestine revealed that this monoclonal antibody reacted strongly with the nuclear proteins of subcortical thymocytes and the basal layer of the mucosa, where many cells were dividing, but not with that of the thymic medullary area. To evaluate the expression of the nuclear proteins during the cell cycle in detail, the results of an immunofluorescence analysis of the thymocytes from hydroxyurea-treated B10 mice using Th-10a monoclonal antibody were compared with those of DNA synthesis of these cells with the use of the FITC-conjugated anti-BrdUrd monoclonal antibody. The results indicated that the nuclear protein detected by Th-10a monoclonal antibody was highly expressed in the S phase of normal thymocytes, while the cells in G1, G2 and M phases exhibited a low level of the expression. Moreover, the variations in expression of the nuclear proteins in the thymocytes at different times after hydroxyurea treatment were observed to correspond with the frequency of DNA synthesizing cells. In contrast, the high level and unregulated expression of the nuclear protein detected by Th-10a monoclonal antibody was observed throughout the cell cycle of the mouse lymphoma cell lines examined. Since Th-10a monoclonal antibody does not react with the nuclear proteins derived from human, hamster or rat proliferating cells, this antibody may recognize a murine specific epitope of the nuclear protein. To further characterize the nuclear proteins, we extracted them from normal thymocytes or thymic lymphomas, and analysed them by immunoblotting or immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis. The nuclear protein(s) detected by Th-10a monoclonal antibody was mostly 95 kDa and also 83 kDa polypeptide. The results also indicated that the 95 kDa nuclear protein was phosphorylated in vivo.

Immunocytochemical detection of active DNA synthesis by in vivo labelling with anti-bromodeoxyuridine antibodies in glomeruli of rats with nephrotoxic serum nephritis.

We studied DNA synthesis in rats with nephrotoxic serum nephritis (NTSN), a model of a glomerular disease, using in vivo labelling with 5-bromo-2'-deoxyuridine (BrdUrd). NTSN was induced by intravenous injection of subnephritogenic doses of rabbit anti-rat GBM antiserum into male Sprague-Dawley rats. Each rat received a single injection of the DNA precursor, 3H-thymidine analog (BrdUrd), ten min before the tissues were removed. For immunocytochemical detection of DNA synthesis, semithin sections were prepared at various intervals (4 h up to 84 days) after pulse labelling. Using a monoclonal anti-BrdUrd antibody, BrdUrd-incorporated DNA-synthesizing cells were noted in the proliferative zone of the gastric mucosa at all times. In NTSN, BrdUd incorporated DNA-synthesizing cells were detected in the glomeruli from 4h through 28 days after inoculation, with the peak occurring at days 2 to 4. On those days, up to half of the glomeruli showed BrdUrd-incorporated cells, with 8 cells per glomerulus as a maximum. From days 7 to 28, few glomerular cells incorporated BrdUrd, and none did so after day 28. The majority of the BrdUrd-incorporated cells were endothelial. These results suggest that active DNA synthesis by glomerular endothelial cells occurs during a short period of the heterologous phase in this model, and that the lack of mesangial cell proliferation might explain the self-limiting nature of this model. By using in vivo labelling with BrdUrd, we were also able to easily and accurately detect active DNA synthesis without consideration of the normal cell renewal.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of 5-bromo-2-deoxyuridine (BrdUrd) incorporation with monoclonal anti-BrdUrd antibody after deoxyribonuclease treatment.

We studied the effects of deoxyribonucleases on the detection of 5-bromo-2-deoxyuridine (BrdUrd) by anti-BrdUrd monoclonal antibodies (mAbs). After DNase I treatment, BrdUrd was detected in cells fixed on slides with the anti-BrdUrd mAbs, B44 and BMC9318. The level of detection related to the degree of DNA digestion. DNA digestion of 25-75% resulted in levels of staining comparable to control preparations in which DNA was denatured by heating with formamide. Staining with the mAbs of DNase I-treated cells was abolished with S1 nuclease, a single-stranded DNA-specific nuclease. When exonuclease III was used after DNase I treatment, the staining intensity of cells fixed on slides increased, and BrdUrd could be detected in suspended cells by flow cytometry. Since this enzymatic method leading to the detection of BrdUrd does not involve cell loss, or destruction of either cellular morphology or epitope reactivity, as occurs with traditional DNA denaturation procedures, it is useful for kinetic studies of phenotypically mixed populations. Furthermore, staining with anti-BrdUrd mAb of cells treated with exonuclease III offers a simple approach to quantitation of apoptotic cells, in which an endogenous endonuclease is activated.

Bromodeoxyuridine labelling of transitional cell carcinoma of the bladder--an index of recurrence?

Nineteen patients with newly diagnosed transitional cell carcinoma of the bladder consented to receive an intravenous bolus of bromodeoxyuridine (BrdUrd) 3 to 6 h prior to transurethral resection of bladder tumour. Routine paraffin embedded sections were stained with anti-BrdUrd monoclonal antibody in order to determine the S phase fraction of the bladder tumours and to correlate this with tumour grade, category and the development of recurrence during follow-up. BrdUrd labelling of nuclei was reliably detected in 100% of the tumours. The mean labelling index of Ta tumours (n = 8) was 3.14%; in T1 tumours (n = 7) it was 8.6% and in T2-3 tumours (n = 4) it was 16.4%. A similar correlation was found in association with tumour grade. The patients were followed up for a mean of 23 months (range 18-30). Seven of the 15 superficial tumours recurred during the period of follow-up. These tumours had a mean labelling index (at diagnosis) of 8.4%, whereas the tumours that did not recur had a mean labelling index of 3.4%. The in vivo determination of BrdUrd uptake in transitional cell carcinoma of the bladder may be useful in predicting which superficial tumours are likely to recur following resection and so rationalise both the time to follow-up and intravesical chemoprophylaxis.

DNA double labelling with IdUrd and CldUrd for spatial and temporal analysis of cell proliferation and DNA replication.

A procedure was developed that very effectively distinguishes between IdUrd and CldUrd incorporated in the DNA of cell nuclei and chromosomes. For double staining we used the rat anti-BrdUrd monoclonal antibody from Sera-lab that binds specifically to CldUrd and BrdUrd but not to IdUrd, in combination with the mouse anti-BrdUrd monoclonal antibody from Becton Dickinson. This antibody binds to all three halogenated deoxyuridines, but when the nuclei are washed in TRIS buffer with a high salt concentration the antibodies linked to CldUrd-labelled DNA are removed. When analysing the effect of the deoxyuridines on the cell cycle we found that the growth kinetics of Chinese hamster cells were not changed by adding IdUrd or CldUrd for 30 min at a concentration of 10 microM, whereas adequate double labelling required only 2 min pulses. The effectiveness of the technique was demonstrated in two model experiments. The first test concerned the assessment of cell recruitment in the central areas of slow-growing clones, after addition of fresh medium. The second experiment focussed on the spatial resolution of the method. Double-labelled metaphase chromosomes showed interspersed green and red replication bands with a spacing corresponding with medium resolution Giemsa banding patterns.