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

Abstract

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

Rates of incorporation of radioactive molecules during the cell cycle

“…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.…”

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

“…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.…”

Chromosome organization and chromatin modification: influence on genome function and evolution

“…Populations of some mammalian cells in culture, when synchronized, show trimodal kinetics for the rate of [ 3 H]-thymidine incorporation during S-phase. A small very early burst of DNA synthesis is followed by two larger bursts (Klevecz and Kapp, 1973;Klevecz, 1975;Holmquist et al, 1982). Between the two larger bursts, the rate of DNA synthesis slows appreciably (Brylawski et al, 2004).…”