Families of replicating units in cultured hamster fibroblasts

Remington, John A.; Klevecz, Robert R.

doi:10.1016/0014-4827(73)90393-5

Cited by 25 publications

(9 citation statements)

References 52 publications

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“…They do not agree with the report of Collins (1978) that diploid WT38 cells synt,hesize DNA (and incorporate 3H-TdR) maximally in early and late S-phase. In addition, we failed t,o detect significant, oscillations in 'H-TdR incorporation throughout S-phase as previously report,ed for diploid cells (Remington andKlevecz, 1973: Klevecz andKapp, 1973). These result,s are all consist,ent with the proposal by Klevecx (1969) t.hat true diploid cell strains incorporat,e 'H-TdR in two discrete parts of S-phase with a slowing in mid-S-phase while established heteroploid cell lines show a unimodal 'H-TdR incorporation curve.…”

Section: Ui Scussioncontrasting

confidence: 67%

Rates of incorporation of radioactive molecules during the cell cycle

Gray

Pallavicini

George

et al. 1981

Journal Cellular Physiology

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We report measurements of the incorporation of radioactive molecules during short labeling periods, as a function of cell-cycle stage, using a cell-sorter-based technique that does not require cell synchronization. We have determined: (1) tritiated thymidine (3H-TdR) incorporation throughout S-phase in Lewis lung tumor cells in vitro both before and after treatment with cytosine arabinoside; (2) 3H-TdR incorporation throughout S-phase in KHT tumor cells in vitro and in vivo; (3) 3H-TdR incorporation throughout S-phase in Chinese hamster ovary cells and compared it with DNA synthesis throughout S-phase; (4) a mathematical expression describing 3H-TdR incorporation throughout S-phase in Chinese hamster M3-1 cells; and (5) the simultaneous incorporation of 3H-TdR and 35S-methionine as they are related to cell size and DNA content in S49 mouse lymphoma cells. In asynchronously growing cells in vitro and in vivo, 3H-TdR incorporation was generally low in early and late S-phase and highest in mid-S-phase. However, in Lewis lung tumor cells treated with cytosine arabinoside 3H-TdR incorporation was highest in early and late S-phase and lowest in mid-S-phase. Incorporation of 35S-methionine increased continuously with cell size and DNA content. Incorporation of 3H-TdR in CHO cells was proportional to DNA synthesis.

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Section: Ui Scussioncontrasting

confidence: 67%

Rates of incorporation of radioactive molecules during the cell cycle

Gray

Pallavicini

George

et al. 1981

Journal Cellular Physiology

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“…This explanation is offered so that other workers will come to understand that the pooling and averaging of data from several experiments must be applied judiciously (29,30) if they are not to obscure important temporal structure in the cell cycle . The fluctuations in enzyme activity are at least as great as those shown by diploid and pseudodiploid Chinese hamster fibroblasts, Don and Don C (11) and considerably greater than those in V79 (18) . Summarizing from the averaged values of Fig .…”

Section: Resultsmentioning

confidence: 78%

“…DNA content per cell was determined by reaction of 3,5-diaminobenzoic acid with the 2-deoxy sugar of DNA as described previously (18) . Fluorescence was measured using an Aminco-Bowman spectrofluorometer (American Instrument Co ., Inc., Silver Spring, Md .…”

Section: Dna Fluorescencementioning

confidence: 99%

“…However, there is a decrease in the complexity of the temporal pattern of both DNA and enzyme synthesis in cells with long culture histories . This is best exemplified by comparing diploid Don fibroblasts which display three discrete intervals of DNA synthesis within S phase (18) with established aneuploid V79 cells where only a single maximum is detectable in S (17) .…”

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confidence: 99%

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Intermittent Dna Synthesis and Periodic Expression of Enzyme Activity in the Cell Cycle of Wi-38

Klevecz

Kapp

1973

The Journal of Cell Biology

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Synchronous cultures of WI-38 were obtained using an automated system for detachment and partitioning of mitotic cells which operates without the use of inhibitors, altered medium, or lowered temperatures . The generation time in synchronous WI-38 is 19 .5 h and the duration of S phase when determined from the percentage of labeled metaphase cells or nuclei is 12 h. DNA replication in WI-38 occurs in three temporally distinct and rapid bursts separated by intervals of greatly reduced synthesis within what is nominally described as the DNA synthetic (S) period . Lactate dehydrogenase (LDH) displayed maxima in G, between 2 and 4 h and again at 10 and 16 h . Peaks in LDH activity were coordinated with DNA replication in a fashion similar to that reported for diploid Chinese hamster cells . Oscillations in LDH activity are more pronounced in normal diploid fibroblasts than in established and neoplastic lines . 564The finite life-span of diploid cells in culture has been well documented (1-4) . It has also been demonstrated that explanted fibroblasts from young individuals emigrate more rapidly from the explant and have a longer in vitro life-span than cells from older donors (2, 5) . WI-38, human fibroblasts, have been extensively studied in recent years and attempts have been made to demonstrate age-associated changes in DNA melting temperature (T.) (6), in RNA synthesis (7), in the level of glycolytic enzymes (8, 9), in lysosomal enzymes (7,8), and in the metabolism of lipids (10) .Differences in the cell cycle patterns of enzyme activity and DNA replication between heteroploid and diploid Chinese hamster fibroblasts have been reported elsewhere (11)(12)(13)(14)(15) . In heteroploid cells the pattern of enzyme and protein synthesis can be nearly linear or exponential through the cycle (13) . This may be a population effect, since some heteroploid cells (16) and cloned aneuploid derivatives of the parent heteroploid lines display periodicities in enzyme activity (15,17) . However, there is a decrease in the complexity of the temporal pattern of both DNA and enzyme synthesis in cells with long culture histories . This is best exemplified by comparing diploid Don fibroblasts which display three discrete intervals of DNA synthesis within S phase (18) with established aneuploid V79 cells where only a single maximum is detectable in S (17) .It appears that hamster fibroblasts, like rodent cells in general, are able to become established and apparently immortal cell lines without undergoing a phase III (19) crisis, presumably because the loss of proliferative capacity in diploid members of the culture is obscured by the emerging heteroploid population . Greater than 85% of Don cells in this laboratory -are diploid but maintenance of the diploid state requires careful attention to

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“…Furthermore, a number of authors have reported that the kinetics of (3H) thymidine incorporation into DNA of various synchronized cell populations is not characterized by a single peak [as would be expected if the starting time of replicon clusters was random (811, but that DNA content rather increases in a saltatory fashion (13,19,20,22,30). In some studies, the measured LI.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 1989

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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...

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Families of replicating units in cultured hamster fibroblasts

Cited by 25 publications

References 52 publications

Rates of incorporation of radioactive molecules during the cell cycle

Rates of incorporation of radioactive molecules during the cell cycle

Intermittent Dna Synthesis and Periodic Expression of Enzyme Activity in the Cell Cycle of Wi-38

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

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