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

Abstract

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...

Detection of repair activity during the DNA damage-induced G2 delay in human cancer cells

“…Patterns of BrdU incorporation have been extensively characterized in proliferating cells (Kill et al, 1991;O'Keefe et al, 1992;Nakayasu and Berezny, 1989;Van Dierendonck et al, 1989;Fox et al, 1991). Immediately after release, cells in early-mid S phase show di use staining throughout the nucleus, while late S cells show discrete but large areas of replicating DNA.…”

Host-viral effects of chromatin assembly factor 1 interaction with HCMV IE2

“…Bromodeoxyuridine (BrdU), which can be incorporated into replicating DNA and marks DNA replication foci [9,46,47], was used to investigate whether IE2 and p150 colocalize with DNA replication foci in S-phase cells. The osteosarcoma cells U2OS expressing either HA-p150 (Supplementary information, Figure S1, a-f) or Flag-IE2 (g-l) were subjected to BrdU incorporation, followed by double immunostaining with the antibodies to BrdU (d-f and j-l) and the transfected protein (HA: a-c; Flag: g-i).…”

Nuclear organization of DNA replication in primary mammalian cells

“…Later in S-phase, a transition occurs and replication sites are found to cluster in regions of the nucleus that contain heterochromatin (Nakayasu and Berezney 1989). An important suggestion from these studies is that the nuclear organization of DNA replication is uniform in all mammalian cells (Nakamura et al 1986;Nakayasu and Berezney 1989;van Dierendonck et al 1989;O'Keefe et al 1992;Rizzoli et al 1992;Hassan and Cook 1993;Ma et al 1998;Berezney et al 2000). A further inference based on these findings is that the spatial organization of DNA replication is tightly controlled and thus unaffected by physiologic settings or by genetic changes seen in immortalization or transformation.…”