A Suzuki–Miyaura method for labelling proliferating cells containing incorporated BrdU

A Suzuki–Miyaura reaction method was developed to label cellular BrdU with fluorescent boronic acid probes.

Oxygen-Independent Stabilization of the Oxygen-Labile Transcription Factor HIF-1α with Dimethyloxalyl Glycine or FG-4497 Increases Hematopoietic Stem Cell Quiescence In Vivo and Mobilization in Response to G-CSF

Abstract 2334 The endosteal region of the bone marrow (BM) is hypoxic in steady-state and most quiescent hematopoietic stem cells (HSC) reside in hypoxic, poorly perfused niches. Mobilizing doses of G-CSF renders most of the BM space hypoxic. Most cellular effects of hypoxia are mediated by O2-labile hypoxia-inducible transcription factors (HIF). At O2 concentration above 2%, HIF-α is rapidly hydroxylated on Pro residues by the prolyl hydroxylases PHD1, PHD2, and PHD3. HIF-α prolyl hydroxylation recruits the E3 ubiquitin ligase VHL, which targets HIF-α to the proteasome. In hypoxia (O2 < 2%), HIF-α proteins are stable, associate with the β subunit ARNT, and translocate to the nucleus to activate transcription. It has emerged that HIF-1α regulates HSC proliferation and is critical to maintain long-term HSC self-renewal in vivo. In this study, we investigated the effect of pharmacological stabilization of HIF-1α protein on HSC cycling and mobilization in mice using two different HIF prolyl hydroxylase (PHD) inhibitors, dimethyloxalyl glycine (DMOG) and FG-4497. We first assessed whether DMOG and FG-4497 stabilized HIF-1α protein in BM leukocytes in vivo by western-blot. Following a single injection of 400mg/kg DMOG, HIF-1α protein was stabilized for up to 6 hrs in BM leukocytes. With 20mg/kg FG-4497, HIF-1α protein persisted over 12 hours. HIF-1α protein was below detection in the BM from saline injected animals. C57BL/6 mice were injected daily with 400mg/kg DMOG to measure effect on HSC cycling and BrdU incorporation by flow cytometry. Using Hoecht33342 and FITC-conjugated anti-Ki67 antibody, a 18 day DMOG treatment increased the proportion of Lin-negative Kit+ Sca1+ CD48- (L-K+S+48-) HSC in phase G0 from 61±11% to 84±6% (p<0.001, 5 mice/group). Conversely, DMOG decreased the proportion of HSC in phase G1 from 31±5% to 14±5% (p<0.001). Similar enhancement of quiescence was observed in less primitive cells such as L-K+S+48+ cells and L-K+S- myeloid progenitors. In mice given BrdU in their drinking water for the last 3 days of the experiment, a 18 day DMOG treatment reduced 3-fold the proportion of L-K+S+48- HSC that incorporated BrdU and halved the proportion of BrdU+ L-K+S+48+ progenitors. Shorter DMOG treatments (6 days or 12 days) did not alter hematopoietic stem and progenitor cell (HSPC) cycling or BrdU incorporation. In contrast, mice treated with 20mg/kg/day FG-4497 had significantly increased proportion of HSPC in G0 phase after only 6 days of treatment, likely due to the more lasting effect of FG-4497 on HIF-1α stabilization. Proportion of HSPC that incorporated BrdU was also significantly decreased. As HIF-PHD inhibitors also increase erythropoietin (EPO) expression by stabilizing HIF in the kidney, we injected a parallel cohort of mice with EPO daily for 18 days. Despite a very strong increase in red cells and hemoglobin in the blood, EPO had no effect of HSPC cycling and BrdU incorporation. Therefore the effects of HIF-PHD inhibitors on HSPC cycling are not an indirect effect of increased EPO. Since HIF-PHD inhibitors slow HSC cycling in vivo, we tested whether they could protect HSC from sublethal irradiation. Mice were treated with 400mg/kg DMOG or saline for 22 days and then irradiated with 9.0Gy. Both cohorts were leukopenic betweens days 7 and 14 post-irradiation but DMOG treated mice had significantly higher blood leukocytes at days 22 and 30, and higher platelet numbers day 22 and all subsequent time-points suggesting that DMOG enhances HSC survival with higher blood recovery. Finally mice were injected with DMOG for 4 days and pegylated rhuG-CSF 3 and 1.5 days before harvest. DMOG doubled mobilization in response to G-CSF with 1,620±530 CFC / mL blood in response G-CSF alone, and 3,250±830 CFC / mL in the G-CSF + DMOG group (p<0.05). Without G-CSF, circulating CFC were less than 10 / mL blood. Similarly, FG-4497 pre-treatment increased the number of CFC mobilized into the spleen 4-fold compared to G-CSF alone (p<0.01). In conclusion, these data highlight the importance of the hypoxia pathway and HIF in the regulation of HPSC cycling and trafficking in vivo. Furthermore HIF-PHD inhibitors may provide therapeutic opportunities to protect HSC from deleterious effects of irradiation as well as increasing mobilization efficiency for transplantation. Disclosures: Walkinshaw: Fibrogen Inc.: Employment, Equity Ownership.

Microscopic Detection of Viable Staphylococcus epidermidis in Peri-Implant Tissue in Experimental Biomaterial-Associated Infection, Identified by Bromodeoxyuridine Incorporation

ABSTRACT Infection of biomedical devices is characterized by biofilm formation and colonization of surrounding tissue by the causative pathogens. To investigate whether bacteria detected microscopically in tissue surrounding infected devices were viable, we used bromodeoxyuridine (BrdU), a nucleotide analogue that is incorporated into bacterial DNA and can be detected with antibodies. Infected human tissue was obtained postmortem from patients with intravascular devices, and mouse biopsy specimens were obtained from mice with experimental biomaterial infection. In vitro experiments showed that Staphylococcus epidermidis incorporated BrdU, as judged from staining of the bacteria with anti-BrdU antibodies. After incubation of bacteria with BrdU and subsequent staining of microscopic sections with anti-BrdU antibodies, bacteria could be clearly visualized in the tissue surrounding intravascular devices of deceased patients. With this staining technique, relapse of infection could be visualized in mice challenged with a low dose of S. epidermidis and treated with dexamethasone between 14 and 21 days after challenge to suppress immunity. This confirms and extends our previous findings that pericatheter tissue is a reservoir for bacteria in biomaterial-associated infection. The pathogenesis of the infection and temporo-spatial distribution of viable, dividing bacteria can now be studied at the microscopic level by immunolabeling with BrdU and BrdU antibodies.

Prostaglandin E2 (PGE2) Treatment Rapidly Increases the Fraction of Non-Dividing Hematopoietic Stem Cells (HSCs)

HSCs are pluripotent cells responsible for the establishment and renewal of the entire hematopoietic system. Our group and others have established that osteoblastic cells in the bone marrow microenvironment regulate HSC cell fate decisions. Specifically, Parathyroid hormone (PTH) expands HSCs by activating osteoblasts in the HSC niche. However, the molecular mechanisms for this increase are unknown. PTH increases local production of prostaglandin E2 (PGE2) in osteoblasts by stimulating cyclo-oxygenase 2 (Cox-2). We also recently found that treatment of osteoblastic MC3T3 cells with PTH (10−7 M) rapidly induces PGE2 Synthase expression. Therefore, we hypothesized that PGE2 may act as a mediator of the PTH effect on HSCs. We have shown that in vivo PGE2 treatment caused a 2.75-fold increase in lineage− Sca-1+ c-kit+ (LSK) cells within the bone marrow compared with vehicle treated mice (p=0.0061, n=8/group). Bone marrow mononuclear cells (BMMC) from mice treated with PGE2 also demonstrated superior lymphomyeloid reconstitution in competitive repopulation analyses, suggesting that HSCs are being expanded or modulated to more efficiently reconstitute the hematopoietic system in the recipients. It is known that HSCs that reside in the G0 phase of the cell cycle have increased ability to reconstitute myeloablated recipient mice. Since PGE2 treatment resulted in superior reconstitution, we hypothesized that PGE2 may increase the percentage of HSCs residing in G0. To test this hypothesis, we treated BMMC from male C57b/6 mice with 10−6 M PGE2 or vehicle for 90 minutes. The percentage of cells in G0 vs. G1 was determined by flow-cytometric analysis using the RNA and DNA dyes, Pyronin-Y and Hoechst 33342 respectively. As we predicted, PGE2 treatment increased the percentage of wild-type LSK cells in G0 1.85 fold over vehicle-treated LSK cells (23.63% in vehicle-treated, n=4 vs. 43.7% in PGE2-treated, n=6). Since the PTH-dependent increase in HSCs is Protein Kinase A (PKA) mediated and the PGE2 receptors EP2 and EP4 signal via PKA, we assayed the effect of PGE2 on the percentage of cells in G0 in mice lacking the EP2 receptor (EP2−/− mice). Interestingly, there was no enrichment for HSC in G0 when BMMC from EP2−/− mice were treated with PGE2 (55.25% in vehicle-treated, n=4 vs. 56.06% in PGE2-treated, n=5). These findings suggest that PGE2-dependent regulation of HSC activity may involve increasing the percentage of HSCs that reside in G0 by activation of EP2, thereby augmenting their ability to reconstitute the hematopoietic system of a myeloablated recipient. 5-bromo-2-deoxyuridine (BrdU) incorporation was also used to investigate the effect of PGE2 on cell cycling of HSCs. Male 6–8 week old C57b/6 mice were injected intraperitoneally with 1 mg BrdU and PGE2 (6 mg/kg) or vehicle. After 30, 60, 90 or 120 minutes, mice were sacrificed and BMMC were subjected to flow cytometric analysis for incorporation of BrdU and DNA content in HSCs. As expected for the highly quiescent HSC population, only a small fraction of HSCs incorporated BrdU. After 30 and 60 minutes of treatment, there was no difference in the percentage of cells that incorporated BrdU between vehicle and PGE2-treated mice. However, at the 90 and 120 minute time points, there were significantly less HSCs cycling in the bone marrow from the PGE2 treated mice (12.1% vs. 5.3% at 90 min, n=2 per group; 11.1% vs. 1.8% at 120 min, n=5 per group, p=0.0060), suggesting that fewer PGE2-treated cells were synthesizing DNA. Taken together, the increase in the percentage of HSCs in G0 and the decrease in cycling HSCs after PGE2 treatment indicate that PGE2 could improve engraftment and reconstitution of the hematopoietic system by enriching for HSCs in G0. These results suggest that PGE2 may exert its beneficial effect on bone marrow reconstitution by altering cell cycle dynamics in HSCs. Identification of the molecular events mediating this novel PGE2 action on HSC could provide additional targets for HSC manipulation in clinical situations requiring rapid and efficient bone marrow reconstitution, such as recovery from iatrogenic or pathologic myeloablative injury.

Actively Growing Bacteria in the Inland Sea of Japan, Identified by Combined Bromodeoxyuridine Immunocapture and Denaturing Gradient Gel Electrophoresis

ABSTRACT A fundamental question in microbial oceanography concerns the relationship between prokaryote diversity and biogeochemical function in an ecosystem context. We combined bromodeoxyuridine (BrdU) magnetic bead immunocapture and PCR-denaturing gradient gel electrophoresis (BUMP-DGGE) to examine phylotype-specific growth in natural marine assemblages. We also examined a broad range of marine bacterial isolates to determine their abilities to incorporate BrdU in order to test the validity of the method for application to diverse marine assemblages. We found that 27 of 29 isolates belonging to different taxa could incorporate BrdU. BUMP-DGGE analysis revealed phylogenetic affiliations of DNA-synthesizing, presumably actively growing bacteria across a eutrophic to mesotrophic transect in the Inland Sea of Japan. We found that the BrdU-incorporating (growing) communities were substantially different from the total communities. The majority (34/56) of phylotypes incorporated BrdU and were presumably growing, and these phylotypes comprised 10 alphaproteobacteria, 1 betaproteobacterium, 11 gammaproteobacteria, 11 Cytophaga-Flavobacterium-Bacteroides group bacteria, and 1 unclassified bacterium. All BrdU-responsive alphaproteobacteria were members of the Rhodobacterales, suggesting that such bacteria were dominant in the growing alphaproteobacterial populations in our samples. The BrdU-responsive gammaproteobacteria belonged to the Oceanospirillales, the SAR86 cluster, the Pseudomonadales, the Alteromonadales, and the Vibrionales. Thus, contemporaneous cooccurrence of diverse actively growing bacterial taxa was a consistent pattern in our biogeochemically varied study area.

Differential Expression of Type I MAGE in New and Relapsed Multiple Myeloma: Evidence for Association with Proliferation and Progression of Disease.

The type I Melanoma Antigen GEne (MAGE) proteins belong to the Cancer-Testis family of tumor-associated antigens and are found in a broad range of solid and hematologic malignancies. We previously showed that the type I MAGE proteins CT7 (MAGE-C1) and MAGE-A3 were commonly detected in primary myeloma by both RT-PCR and immunohistochemistry (IHC). Higher levels of MAGE protein expression had a positive correlation with abnormally elevated proliferation as measured by the Plasma Cell Proliferation Index (PCPI, percentage of Ki-67+ cells in the CD138+ myeloma cell compartment). These findings suggest that MAGE may play a role in abnormal cell cycle regulation in myeloma. We explored this hypothesis by examining type I MAGE gene expression and proliferation by IHC in 46 newly-diagnosed, untreated and 35 relapsed myeloma patients, based on the clinical observation that relapsed patients exhibit lower response rates to therapy and shorter time to progression, indicative of more aggressive disease. PCPI was significantly higher in relapsed patients (19.0 ± 3.5%) compared to newly diagnosed (6.9 ± 1.3%, p<0.0002). Expression of CT7 and CT10 (MAGE-C2), a type I MAGE not previously associated with myeloma, was stable between newly-diagnosed and relapsed patients (76.0% of new samples vs. 77.1% of relapsed for CT7, 48.5% vs. 50.0% for CT10). In contrast, MAGE-A3 was detected in a significantly greater percentage of relapsed patients (77.1%) compared to newly diagnosed (35.6%, p=0.0003). The link between MAGE expression and unrestricted proliferation was further supported by in vitro studies with human myeloma cell lines. Proliferating myeloma cells were metabolically labeled with the nucleotide analog bromodeoxyuridine (BrdU) followed by intracellular staining and flow cytometry. This assay demonstrated that proliferating myeloma cells that incorporated BrdU into their genomic DNA expressed higher levels of type I MAGE protein compared to non-proliferating cells. Arresting cells at the G1-S interface by double thymidine blockade lead to the accumulation of cells expressing high levels of MAGE, which rapidly entered S phase and incorporated BrdU upon release from block. These results strongly suggest that expression of CT7 and CT10 are relatively early and stable events in the pathogenesis of myeloma, whereas activation of MAGE-A3 expression is associated with disease progression. Furthermore, MAGE expression is correlated with abnormal proliferation in vitro and in vivo, suggesting a potential functional role in the dysregulation of the cell cycle that is a hallmark of this disease. These results support the further exploration of the role of type I MAGE expression in myeloma and the development of therapeutic agents targeting them, especially tumor vaccines.

Mechanisms of granulocytosis in the absence of CD18

Genetic deficiency in CD18 leads to disease characterized by myeloid hyperplasia, including profound granulocytosis and splenomegaly. Myeloid hyperplasia could directly result from the disruption of CD18 functions essential to granulopoiesis or basal leukocyte trafficking. Alternatively, myeloid hyperplasia could be reactive in nature, due to disruption of essential roles of CD18 in leukocyte responses to microbial challenge. To distinguish between these mechanisms, the hematopoietic systems of lethally irradiated wild-type (WT) mice were reconstituted with either WT fetal liver cells or CD18-deficient fetal liver cells, or an equal mixture of both types of cells. Granulocytosis and splenomegaly developed in mice that received CD18-deficient fetal liver cells. Splenomegaly was prevented and granulocytosis was inhibited by more than 95% in mice that had received both CD18-deficient and WT fetal liver cells, suggesting that myeloid hyperplasia was largely reactive in nature. Consistent with this postulate, the circulating life spans in the blood and the fraction of neutrophils that incorporated BrdU in the bone marrow were not increased for CD18-deficient neutrophils compared with the WT. However, these animals did develop mild granulocytosis compared with mice reconstituted with WT cells alone, and a higher percentage of CD18-deficient leukocytes were neutrophils compared with the WT leukocytes. These observations suggest that the granulocytosis observed in the absence of CD18 occurs through at least 2 mechanisms: one that is dramatically improved by the presence of WT cells, likely reactive in nature, and a second that is independent of the WT hematopoietic cells, involving an alteration in the lineage distribution of blood leukocytes.

Effect of Fixation and Epitope Retrieval on BrdU Indices in Mammary Carcinomas

Studies in which 5-bromo-2′-deoxyuridine (BrdU) is used to quantify rates of cell proliferation are conducted prospectively. Therefore, the opportunity exists to select conditions that optimize detection of the BrdU epitope. The objective of this study was to quantify the extent to which the BrdU epitope was masked by formalin vs methacarn fixation in the assessment of cell proliferation. Mammary carcinomas from animals pulse-labeled with BrdU were trisected. A portion was frozen and the remaining two portions were fixed in 10% neutral buffered formalin or methacarn for 24 hr, processed, embedded in paraffin, and sections stained for incorporated BrdU using a peroxidase immunohistochemical staining technique. Antigen retrieval techniques also were applied to formalin-fixed sections. Fixation in methacarn gave the highest labeling index (16.4%), which was comparable to that observed in unfixed frozen sections (17.5%). Formalin fixation alone dramatically suppressed the labeling index (0.3%), which was only partially recovered using various antigen retrieval techniques (2.1-8.1%). Methacarn fixation is recommended for prospective studies in which BrdU detection is planned because of the quantitative recovery of epitope and the simplicity of the approach.

Long-term Oral Application of 5-Bromo-2-Deoxyuridine Does Not Reliably Label Proliferating Immune Cells in the LEW Rat

To study the lifespan of immune cell populations in the LEW rat, 5-bromo-2-deoxyuridine (BrdU) was administered in the drinking water. After 12 weeks, the epithelium of gut and skin was completely BrdU+. In contrast, thymus, bone marrow, and germinal centers of Peyer's patches contained only a few BrdU+ cells, although most should have been labeled during this time. The lack of labeling was due neither to obvious toxic effects of BrdU on these organs nor to insufficient detection of incorporated BrdU. Analysis of the kinetic pattern of the appearance of BrdU+ cells in bone marrow, blood, spleen, and lymph nodes over 12 weeks revealed that the dosage of BrdU initially was high enough to label the proliferating cells in the bone marrow, but then became too low, although the BrdU uptake of the rats was similar over the entire time. This indicates that in the LEW rat the metabolism of orally applied BrdU changes over time, leading to a reduction in the amount of BrdU available for incorporation into the DNA below a level necessary for labeling all proliferating cells. This effect appears to be species- and strain-dependent, and should be considered when the BrdU technique is used. (J Histochem Cytochem 45:393–401, 1997)