Radiation-stimulated DNA synthesis in cultured mammalian cells

A type of DNA synthesis in mammalian cells that is stimulated by ultraviolet light has been studied by means of radioautography and density gradient centrifugation. The characteristics of this synthesis are: (a) it is not semiconservative; (b) it is enhanced by the presence of 5-bromodeoxyuridine in the DNA molecule; (c) the degree of stimulation is dose dependent; (d) there is less variability in the rate of incorporation of H³-thymidine during this synthesis than during normal DNA synthesis; (e) it occurs in cells that are not in the normal DNA synthesis phase (G₁ and G₂ cells). This kind of synthesis has been found in cultured cell lines from five different species; however, in some strains, the presence of bromouracil in the DNA is required before it can be demonstrated by radioautography.     

Control of DNA synthesis in polyploid mammalian cells

To test the hypothesis that the duration of DNA synthesis is an inverse function of nuclear size or DNA content, the S phase was calculated from PLM analysis for pseudodiploid, tetraploid, and octaploid lines of Chinese hamster cells growing as a monolayer or in suspension. S phase times were found not to be significantly different between polyploid lines and the diploid lines from which they were derived, regardless of the conformation of the nucleus. There is no evidence, therefore, that would implicate the nuclear membrane, or nuclear surface area/volume relationships, in the control of DNA synthesis.     

DNA,protein, and plasma-membrane incorporation by arrested mammalian cells

Incorporation of DNA, protein, and plasma membrane during blockage by aphidicolin or by doxorubicin was studied by flow cytometry and electrorotation of three cell lines (mouse-myeloma Sp2/0-Ag14, hybridoma H73C11, and fibroblast-like L929 cells). Drug-mediated arrest at the G1-S boundary (aphidicolin) or in G2/M (doxorubicin) did not arrest synthesis of either protein or total membrane area, the increases in which outstripped growth in cell volume and apparent cell area, respectively. Measurements of membrane capacity in normal and hypo-osmotic media showed that the drugs had not changed the fundamental bilayer, but that an increase in the number or size of microvilli must have occurred. Aphidicolin-arrested cells withstood hypo-osmotic stress better than untreated cells could, indicating that the membrane excess can be utilized as a reserve during rapid cell expansion.Hypo-osmotically treated cell populations exhibited only about half the coefficient of variance (CV) in membrane properties of cells at physiological osmolality. Populations of arrested cells exhibited the same high CV as asynchronous cells, indicating that chemical arrest does not give uniformly villated cell populations. However, the lowest CV values were given by some synchronized (aphidicolin-blocked, then released) populations.Removal of aphidicolin allowed most cells to progress through S and G2, and then divide. During these processes, the membrane excess was reduced. After removal of doxorubicin, the cells did not divide: some continued protein synthesis, grew abnormally large, and further increased their membrane excess.Membrane breakdown by electric pulsing (3 X 5kV/cm, 40 sec decay time) of aphidicolin-synchronized L cells in G2/M led to a 22% loss of plasma membrane (both the area-specific and the whole-cell capacitance were reduced), presumably via endocytosislike vesiculation.This work was supported by grants of the VDI/VDE (13 MV 0305 to W.M.A. and U.Z.), of the DARA 50WB9212 (to U.Z.), and of the Deutsche Forschungsgemeinschaft (SB 176 B5 to U.Z. and W.M.A.).     

Discrepancies between precursor uptake and DNA synthesis in mammalian cells

The incorporation of tritiated thymidine and deoxycytidine into DNA of x-irradiated mammalian cells was studied. Both inhibition and stimulation were found due to pool changes rather than to effects on DNA synthesis, indicating that precursor uptake can be a misleading method to measure DNA synthesis rate.     

Stimulation of DNA synthesis in cultures of mouse embryo fibroblast-like cells

Stimulation of the DNA synthesis and mitoses in stationary cultures of mouse embryo fibroblast-like cells was induced by various agents such as ribonuclease, digitonin, fresh medium and commercial preparations of hyaluronidases. Time sequence of stimulation was similar in experiments with all these agents. Cells were activated to enter S phase from GI phase. The rise of the number of DNA-synthesizing cells was preceded by a latent period of about 8–12 hours with the maximal number of DNA-synthesizing cells being observed at 16–24 hours. Mitotic wave was observed after the wave of DNA synthesis. Stimulation of DNA synthesis and mitosis was not preceded by any significant decrease of an average cell density in the culture. The progeny of activated cells had no greater chance than other cells to be activated again when stimulation was repeated. It is concluded that similar proliferative reactions can be induced in stationary cultures by a variety of diverse agents. Possible role of cell surface changes in the induction of these reactions is discussed.     

Coupled ribonucleoside diphosphate reduction, channeling, and incorporation into DNA of mammalian cells

There is rapid and specific channeling of ribonucleoside diphosphates into DNA through reactions beginning with ribonucleotide reductase and terminating with DNA polymerase. Lysolecithin-permeabilized Chinese hamster embryo fibroblasts in culture rapidly reduced ribonucleoside diphosphates by ribonucleotide reductase action when dithiothreitol was provided as a reducing agent and incorporated these deoxynucleotides into DNA. The radioactive label provided in ribo-CDP was not diluted by added deoxyribo-CTP during its incorporation into DNA, showing that the ribo-CDP does not pass through a deoxy-CTP pool. Under the conditions that permitted rapid incorporation of ribonucleoside diphosphates, deoxynucleoside triphosphates were very poorly incorporated. Ribonucleotide reductase with the rate-limiting enzyme for the overall process. The Km values for the reductase reaction and the overall process were similar and low enough for saturation by in vivo pools. Natural feedback inhibitors dATP or dTTP inhibited incorporation of labeled ribo-CDP into deoxyribonucleotides and into DNA to the same extent. Ribonucleotide reductase behaved like other enzymes that are associated in a rapidly sedimenting form. It was concentrated in the nucleus during S phase, and most of the enzyme activity in these nuclear extracts was co-sedimented with DNA polymerase on sucrose density gradients. These data support the hypotheses that a physically associated complex of enzymes (replitase) catalyzes the production of deoxynucleotides and their incorporation into DNA in S phase cells.     

Origins of myo-inositol tetrakisphosphates in agonist-stimulated rat pancreatoma cells. Stimulation by bombesin of myo-inositol 1,3,4,5,6-pentakisphosphate breakdown to myo-inositol 3,4,5,6-tetrakisphosphate

In the rat pancreatoma cell line, AR4-2J, three inositol tetrakisphosphate isomers were identified, (1,3,4,6), (1,3,4,5), (3,4,5,6), which were increased during activation of phospholipase C by bombesin. Two other isomers were identified, (1,4,5,6) and a fifth isomer which was either (1,2,3,4) or (1,2,3,6), which have not previously been detected in any cell type. To study the metabolic interrelationships between these compounds and inositol 1,3,4,5,6-pentakisphosphate in the intact cell, their turnover was assessed under different protocols of [3H]myo-inositol labeling; the inositol phosphates were labeled to near steady state or under conditions where either rapidly or slowly turning over inositol polyphosphates were preferentially labeled. The relative specific radioactivities of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, inositol 1,3,4-trisphosphate, and inositol 1,3,4,6-tetrakisphosphate were very similar in bombesin-stimulated cells, consistent with the pathway for the conversion of inositol 1,4,5-trisphosphate to the other three inositol polyphosphates. Compared with these inositol phosphates, the turnover of inositol 1,3,4,5,6-pentakisphosphate was slow. An accumulation of radioactivity into inositol 1,3,4,5,6-pentakisphosphate was observed only under labeling conditions where its relative specific radioactivity was substantially below that of inositol 1,3,4,6-tetrakisphosphate. This indicated that the precursor for de novo synthesis of inositol 1,3,4,5,6-pentakisphosphate was inositol 1,3,4,6-tetrakisphosphate. Bombesin stimulated the net breakdown of inositol 1,3,4,5,6-pentakisphosphate and increased the level of inositol 3,4,5,6-tetrakisphosphate; the relative specific radioactivities of these two compounds were similar under all conditions. These data led to the novel proposal that inositol 3,4,5,6-tetrakisphosphate is the product of inositol 1,3,4,5,6-pentakisphosphate breakdown. This reaction was apparently stimulated by a regulated change in the enzyme(s) which interconvert inositol 1,3,4,5,6-pentakisphosphate and inositol 3,4,5,6-tetrakisphosphate.     

Effect of caffeine on DNA synthesis in irradiated and unirradiated mammalian cells

Caffeine increased the availability of replication origins, and consequently the number of growing points, in the DNA of Chinese hamster V79 and human (HeLa) cells. Caffeine also prevented the inhibition of replicon initiation normally caused by X-radiation and exposure to low doses of ultraviolet light. When caffeine was removed from the medium after irradiation, replicon initiation was inhibited. Caffeine also reversed the inhibition of replicon initiation caused by novobiocin, which is not a DNA-damaging agent. Because caffeine increases the number of growing points, it also partially reversed the inhibition of total DNA synthesis induced by hydroxyurea. It is proposed that caffeine alters the conformation of intracellular chromatin in such a way that the conformation usually induced by DNA-damaging agents is prevented.     

Separation of rapidly labeled intermediates of DNA synthesis in mammalian cells

Oxygen evolution was inhibited after reacting chloroplast membranes with four different water soluble protein modification reagents. Photosystem II photochemistry was not affected, whown by unimpaired oxidation of an alternate PSII donor, diphenyl carbazide. Concomitant with oxygen evolution inhibition by the diazonium reagent, there was a four-fold increase in covalent binding of the compound to the membranes, suggesting an electron transport dependent conformational change is involved in the effect. PSI cyclic electron flow with DCMU present did not potentiate the oxygen evolution inhibition nor the increased diazo coupling, indicating that the effect is not simply a manifestation of the same energized state driven by cyclic electron flow. Since the effects are due to non-membrane penetrating reagents, we conclude that a protein component associated with oxygen evolution is localized at the external surface of grana membranes.