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 immunoperoxidasestained 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 kine tics.Key terms: DNA replication, B-bromodeoxyuridine, monoclonal anti-BrdUrd antibodies, interphase nucleus There is much evidence from (3H)-thymidine (3HTdR) labeling studies that DNA synthesis in an eukaryotic nucleus consists of an ordered cascade of initiations of replicons (4), controlled by an intranuclear mechanism (36). This is particularly clear from studies on polytene chromosomes of dipteran larvae, describing various patterns of DNA synthesis, tentatively assigned to specific periods of the S-phase (1,2,28). In general, GC-rich euchromatin replicates early, whereas AT-rich heterochromatic DNA replicates late (13); these early and late DNA synthesizing sites roughly correspond to R-and G-bands on trypsinGiemsa-stained chromosomes, respectively (6). However, also in euchromatin, distinct early-and late-replicating DNA fractions have been decribed (24); Goldman et al. (1 1) have presented evidence that genes that are obligatory or potentially active in a given cell type replicate early in that cell type, whereas genes that are permanently inactive replicate late.The characteristic grain distributions observed in eukaryotic nuclei after in situ autoradiography of incorporated 3HTdR (35) may reflect both the structural organization of replicon domains and a nonrandom localization of chromosomes in the interphase nucleus (12,161. In particular, centromeric and other highly repeated nontranscribed sequence domains may be positioned on the nucleolus or nuclear membrane and act as general organizing centers for cell type-specific interphase patterns (21). Multiple adjacent replicons appear to be clustered on fixed sites of the nuclear matrix (17,271, and it has been suggested that clustered replication origins are activated simultaneously (25). Early DNA synthesis then seems to occur in replicon clusters evenly distributed over the nucleus, whereas late DNA synthesis, being primarily heterochromatic, is preferentially restricted to domains on the periphery of the nucleus and in nucleoli (35,361. However, the assignment of autoradiographic labeling patterns to spe...
Background: Incomplete tumor resections occur frequently in patients undergoing breast-conserving surgery. As the surgeon can only rely on palpation and visual inspection, real-time visualization of cancer cells during surgery is needed to increase the number of complete tumor resections. Near-infrared fluorescence (NIRF) imaging using an intra-operative camera system is a novel technique to assess the extent of disease during surgery. Advantages of NIRF light (700-900 nm) include high tissue penetration up to several centimeters deep and low autofluorescence providing sufficient signal-to-noise ratio. Currently, several NIRF dyes are available for chemical conjugation to molecules that can target tumors, e.g. antibodies. This study aimed to investigate whether monoclonal antibodies conjugated to a NIRF dye could detect primary breast carcinomas in a syngeneic rat model.Methods: The hormone-dependent syngeneic breast cancer rat model EMR86 and its derived cell line MCR86 were used. Tumors were induced by orthotopic implantation of fresh EMR86 tumor fragments of 1 mm3 into the flanks at four sites in female WAG/Rij rats (Harlan, the Netherlands). The mouse monoclonal antibody MG1 was used for tumor cell detection. MG1 (IgG2a) recognizes a membrane epitope of rat cancer cells of epithelial origin and is developed by our research group. MG1 was conjugated to the NIRF dye CW-800 (LI-COR, USA) and dye/protein ratio was determined using absorbance measurements (UltroSpec, Amersham, UK). Fluorescence imaging of cells, animals and organs was performed using IVIS Spectrum (Caliper, USA) and Odyssey (LI-COR, USA) equipment.Results: Conjugated antibodies MG1-CW800 bound specifically to cultured MCR86 cells. The signal intensity was linearly correlated with the number of cells and concentration of MG1-CW800 (R2=0.99, p<0.00001). Addition of unconjugated MG1 lowered the fluorescent signal in a competitive binding assay, showing that labeling did not influence binding properties. Intravenous injection of unconjugated MG1 (200 μg/rat) in 3 Wag/Rij rats and subsequent immunohistochemical analysis of tissue sections revealed a specific and exclusive binding of MG1 to the EMR86 tumors and no difference in staining intensity between 24 and 48hrs after injection. Intravenous injection of conjugated MG1-CW800 (200 μg/rat) demonstrated a clear fluorescent demarcation of EMR86 tumors and the surrounding tissue after spectral unmixing (paired t-test 8.17, p<0.00001). Mean fluorescence did not differ between 24 and 48hrs after injection (t=1.85, p=0.11). However, a two-fold increase of the dye/protein ratio led to a two-fold increase of the in vivo fluorescence (t=-6.40, p=0.0001).Conclusion: Intravenous injection of monoclonal antibodies MG1 conjugated to the NIRF dye CW800 enabled detection of primary breast carcinomas in the syngeneic hormone-dependent EMR86 rat model. This study demonstrates proof-of-concept for future labeling of clinically relevant antibodies (e.g. HER2/neu, EGF, VEGF). Translation of this technique to breast cancer patients can lead to intra-operative identification of optimal resection margins in order to increase the comple tumor resection rate. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5006.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.