Abstract
In this paper, we show that the rate of total DNA synthesis increases twice during the Friend erythroleukemia (FL) cell culture growth cycle. The first increase takes place at the threshold of the logarithmic (early) phase of growth, while the second peak takes place in the mid-logarithmic (late) phase. This bi-phasic pattern can be reproduced in both normal and dimethylsulfoxide (DMSO)-differentiating cells, using either [3H]-thymidine or [14Cl-methyl-L-methionine as precursors for DNA labelling. In normal cells, the non-replicating strands incorporate some radioactivity originating in [3H]-thymidine, essentially in the early phase, while their labelling is insignificant in the late phase. In UV-irradiated cells, the mechanism yielding repair patches (RPs) contained by the same non-replicating strands is highly facilitated in the earlier stage in comparison with its functioning at the later stage. In contrast, the [3H]-thymidine labelling of the semiconservatively newly replicating strands is insignificant at the early stage and more substantial later, as expected. In UV-irradiated cells, the slightly decreased [3H]-thymidine labelling of replicating strands is probably explained by the concomitant small percentage of cell death. The chronology of the optimal rates of early repair and later replication is quite well confirmed by a bi-phasic regulation of the total DNA poly-merase (pol) activity: while a peak of N-ethylmaleimide (MalNEt)-resistant polß activity (accounting for base excision-repair) is seen early on, a peak of MalNEt-sensitive polα activity (accounting for replication) is found later. DMSO strikingly inhibits both the rate of the early base excision-repair (either exploiting [3H]-thymidine or [14C]-methyl-L-methionine) and the early MalNEt-resistant polß activity. It does not, however, inhibit the late replication and the late MalNEt-sensitive polα activity, when hemoglobin (Hb) synthesis is heavily induced. The time-shift of late replication in DMSO-differentiating cells is probably explained by a delay of theS-phase, as also suggested by the net decrease in the amount of DNA per nucleus during the second half of the culture growth cycle.
Riassunto
II tasso sintetico del DNA incrementa due volte durante il ciclo proliferativo delle cellule eritroleu-cemiche di Friend (FL). Il primo aumento ha luogo alla soglia della fase proliferative (precoce). Il secondo ha luogo a meta fase proliferativa (tardiva). Questo andamento bifasico si riproduce sia nelle FL di control-lo che in quelle differenziate con dimetilsulfossido (DMSO), impiegando timidina-[3H] o L-metionina-me-tile-[14C], entrambi marcatori della DNA sintesi. Nelle FL di controllo, le catene parentali incorporano radioattività proveniente dalla timidina-[3H] essenzialmente in fase précoce, mentre tale marcatura è insigni-ficante in fase tardiva. Nelle FL UV-irradiate, il meccanismo che ripara piccoli tratti (RP) di catene parentali è molto efficiente in fase precoce e poco efficiente in fase tardiva. Viceversa, come atteso, la marcatura delle catene neoreplicate con timidina-[3H] è insignificante in fase precoce e intensa in quella tardiva (nelle FL UV-irradiate, la diminuita marcatura délie catene neoreplicate con timidina-[3H] è dovuta al lieve aumento délia mortalità cellulate). La cronologia dei tassi ottimali di riparazione precoce e replica tardiva è confermata dalla regolazione bifasica dell’attività DNA polimerasica (pol): mentre un picco di attività polß, N-etilmaleimide (MalNEt)-resistente (indice di escissione-riparazione), si manifesta in fase precoce, un picco di attività pol±, MalNEt-sensibile (indice di replica), si manifesta in fase tardiva. In fase precoce il DMSO inibisce sia il tasso di escissione-riparazione (sfruttando timidina-[3H] o L-metionina-metile-[14C]) ehe l’attività polβ MalNEt-resistente. PerÒ in fase tardiva esso non inibisce né la replica né l’attività pol± MalNet-sensibile, quando peraltro c’è espressione di emoglobina (Hb). Nelle FL DMSO-differenziate, la sfasatura della replica tardiva è dovuta probabilmente al ritardo della fase S, come suggerito anche dalla diminuita quantité di DNA per nucleo nella seconda parte del ciclo proliferativo.
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Nell seduta del 13 maggio 1995.
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Eremenko, T., Tartaglini, E., Volpe, P. et al. Regulation of DNA repair and replication in proliferating FL cells. Rend. Fis. Acc. Lincei 6, 299–312 (1995). https://doi.org/10.1007/BF03001691
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DOI: https://doi.org/10.1007/BF03001691