Synthesis of DNA-binding proteins during the cell cycle of WI-38 cells

Synthesis of DNA-binding proteins was investigated in WI-38 human diploid fibroblast cultures after stimulation with serum containing medium. Density-inhibited confluent monolayers of young (phase II) and aging (phase III) WI-38 cells can be stimulated to synthesize DNA by replacing the medium with fresh medium containing 10% fetal calf serum. Of the phase II cells, 35–50% showed a partially synchronized burst of DNA-synthesizing activity between 15 and 24 h whereas only 4–6% of phase III cells showed DNA-synthesizing activity at 20 h, and that cell fraction was increasing even at 38 h. This suggests either an extremely prolonged G 1 in stimulated phase III cells, or a heterogeneity of the population (e.g., a mixed population of pre- and postmitotic cells) for phase III cells. At various times after the change of medium, DNA-binding protein synthesis was examined in these stimulated cultures. Protein of mol. wt 20 000–25 000 D accumulated rapidly during early G 1 and declined thereafter, whereas larger protein (40 000 and 68 000 D) accumulated during the late G 1 or G 1-S transition period indicating that accumulation of these proteins is associated with the onset of DNA synthesis in the serum-stimulated cells. In cultures where the DNA synthesis has been reduced or inhibited by an excess of thymidine, hydroxyurea or dibutyryl cAMP, the accumulation of the larger proteins (40 000 and 68 000 D) was neglible as compared with non-stimulated cultures. Hydrocortisone did not exert any effect on the DNA-binding protein synthesis in phase II cells. However, it seems to increase the cell fraction which can respond to the serum factor in phase III cells as evidenced from the pattern of DNA-binding proteins synthesis.     

Stage-dependent effects of 20-hydroxyecdysone on DNA synthesis of corpus allatum cells in the silkworm,Bombyx mori

DNA synthesis in cells of the corpus allata (CA) of the silkworm, Bombyx mori, was studied immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine (BrdU); developmental changes during the 3rd, 4th, and last larval instars and effects of 20-hydroxyecdysone treatment were examined. During both the 3rd and 4th larval instars, the number of DNA-synthesizing cells fluctuated, and relatively low levels were observed during the middle stages. On day 0 of the last larval instar, the number of DNA-synthesizing cells per gland was 9.2, which then increased on day 1 and remained at levels ranging from 12.9 and 16.9 cells per gland. A major peak level (28 BrdU-labeled cells per gland) occurred on day 8, two days after larvae entered the wandering stage. When last instar larvae were fed 20-hydroxyecdysone-supplemented mulberry leaves starting on day 0 or 1, the number of DNA-synthesizing cells dramatically decreased to very low levels and these low levels were maintained throughout the remainder of the instar. However, no effect was observed when last instar larvae were fed 20-hydroxyecdysone-supplemented mulberry leaves starting on day 3, indicating the stage-specific action of 20-hydroxyecdysone. The mechanism by which 20-hydroxyecdysone treatment inhibits DNA synthesis of CA cells was further examined by using continuous in vitro BrdU labeling for a 2-day incubation. It was found that the decrease in responsiveness of DNA synthesis of CA cells of 20-hydroxyecdysone-treated larvae to stimulation by growth factors from hemolymph may have been, at least in part, responsible for the indirect inhibitory effects of 20-hydroxyecdysone.     

DNA synthesis by partially purified replicating simian virus 40 chromosomes.

We have partially purified replicating simian virus 40 (SV40) chromosomes in a form which allows continued DNA synthesis in vitro. We first prepare a soluble DNA-synthesizing system from SV40-infected monkey cells and then sediment the components through a neutral sucrose gradient of extremely low ionic strength. Replicating SV40 chromosomes isolated from such gradients are capable of continuing DNA synthesis in vitro in the same manner as two crude subnuclear systems we have previously described (4). This indicates that the enzymes and other proteins required for in vitro DNA synthesis are bound to the replicating chromosomes.     

Temporal changes in DNA synthesis of prothoracic gland cells during larval development and their correlation with ecdysteroidogenic activity in the silkworm, Bombyx mori

DNA synthesis in prothoracic gland cells of the silkworm, Bombyx mori, was studied immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine (BrdU), and its developmental changes during the 3rd, 4th, and last larval instars were examined. During the early stages of both the 3rd and 4th larval instars, a dramatic increase in the number of DNA-synthesizing cells of the prothoracic glands was detected. However, during the latter stages of each instar, the number of DNA-synthesizing cells greatly decreased. The determination of glandular protein content showed that dramatic increases occurred during the latter stages of each larval instar. Comparison of changes in prothoracic gland cell DNA synthesis with ecdysteroidogenic activity showed that the increase in DNA synthesis precedes ecdysteroidogenesis. The cellular mechanism underlying changes in prothoracic gland cell DNA synthesis during the last two larval instars was further analyzed by determining the in vitro DNA synthesis of the glands, their responsiveness to hemolymph growth factors, and changes in the growth-promoting activity of hemolymph during development. It was found that both growth factors and the responsiveness of the prothoracic gland cells to growth factors from hemolymph may play roles in regulating DNA synthesis of gland cells.     

Effects of mitomycin C on the rate of DNA synthesis in normal and Fanconi anaemia cells

The effect of low doses mitomycin C (MMC) on DNA synthesis of fibroblast cell lines derived from normal individuals or patients with Fanconi anaemia (FA) was studied. Using low doses of MMC (12 ng/ml), little or no effect was observed on DNA synthesis of normal cells, whereas DNA synthesis of FA cells was greatly inhibited 24 and 48 h after treatment. This effect was due to a decrease in the number of DNA-synthesizing cells, while the amount of radioactivity incorporated per cell (as measured with grain counting in autoradiograms) remained the same. These findings indicate that the inhibition of semiconservative DNA synthesis induced by MMC in FA cells is not due to an inhibitory effect of unrepaired lesions on the rate of DNA synthesis but rather to a block in cell cycle progression.     

Effects of mitomycin C on the rate of DNA synthesis in normal and Faconi anaemia cells

THe effect of low doses mitomycin C (MMC) on DNA synthesis of fibroblast cell lines derived from normal individuals or patient with Fanconi anaemia (FA) was studied. Using low doses of MMC (12 ng/ml), little or no effect was observed on DNA synthesis of normal cells, whereas DNA synthesis of FA cells was greatly inhibited 24 and 48 h after treatment. This was due to a decrease in the number of DNA-synthesizing cells, while the amount of radioactivity incorporated per cell (as measured with grain counting in autoradiograms) remained the same. These findings indicate that the inhibition of semiconservative DNA synthesis induced by MMC in FA cells is not due to an inhibitory effect of unrepaired lesions on the rate of DNA synthesis but rather to a block in cell cycle progression.     

Determination of the DNA-synthesizing cell count by the immunofluorescence method

In DNA-synthesizing cells DNA is partially single-stranded. Anti-thymidine antibodies, while specifically reacting with this DNA, form a complex which may be revealed using indirect immunofluorescent technique. A comparative determination of DNA-synthesizing cell number in tumor tissue (larynx squamous cell carcinoma) was performed using immunofluorescent technique and radioautography. The former method showed the labeling index (LI) to vary from 1.2 to 9.9%, while the latter showed it to vary from 1.0 to 8.2%. The correlation ratio between the LI values obtained by the two techniques was 0.79. To eliminate a possible reaction of anti-thymidine antibodies with cellular RNA, specimens were preincubated in solutions with RNAase. No more than 6 hours were required to stain specimens using this LI estimation technique. This investigation allows to reveal DNA synthesizing cells not only in the periphery of a histological section, as does routinely radioautography, but also in its centre.     

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.     

Inhibition of mitosis in pisum root meristems by continuous gamma radiation: the influence of temperature on the synthesis of DNA, RNA and protein during inhibition

Tritiated precursors of DNA, RNA and protein were used to measure synthesis at 10 and 20C in root meristem cells of Pisum after they were mitotically arrested by continuous irradiation with gamma rays. The experiments were designed to determine if the arrested cells accumulated in a certain part of interphase, to determine the effect on DNA, RNA and protein synthesis, to find out if the effects were temperature dependent, and finally to reveal possible relationships between growth inhibition and altered synthesis. The results showed that the incorporation of DNA and RNA precursors was impaired by irradiation and that decreased temperature further increased radiation impairment of DNA synthesis. Protein synthesis on the other hand was not impaired by irradiation at either temperature. Irradiation at 20C reduced the number of DNA-synthesizing cells; at 10C this number was reduced to near zero. Although irradiated cells synthesizing RNA showed a reduction in grain counts when compared to the controls, they still retained the ability to incorporate tritiated uridine at 10C. It was hypothesized that the combination of reduced DNA and RNA synthesis and unaffected protein synthesis resulted in precocious maturation of the arrested meristem cells. Growth which occurred in the absence of cell division was attributed to meristematic cells which precociously matured and cells which were in the region of elongation.     

Kinetics of cell replication in the uterine cervix. VI. Loci of DNA-synthesizing cells and arrested mitoses in the basal-cell layer

The mode of proliferation in the basal-cell layer of the squamous cervical epithelium was investigated in C57B1 mice with the aid of 3H-thymidine and vincristine. Six hours after vincristine injection and two hours after thymidine injection, 33% of the basal cells were in DNA synthesis and 12% in mitosis. Of these, only 23% of the cells in DNA synthesis and 45% of those in mitosis were found as single cells. The remaining cells proliferated in clusters of two or more cells. As many as 59% of the cells in DNA synthesis and 30% of those in mitosis occurred in colonies of three or more consecutive cells, indicating that multicell clustering is a rather common pattern of basal cell proliferation. Multicell loci of DNA-synthesizing cells occurred contemporaneously with but independently of multicell loci of mitotic cells (the loci were nonconsecutive). Basal-cell replication in the squamous cervical epithelium thus appears to be an organized process of cell renewal.     

A study of the localization and accumulation of S-phase cells in the retina of newt Pleurodeles waltl after experimental pigment epithelial detachment

This work continues the studies of the proliferative ability of cells in the adult newt retina. The model of experimental detachment of the retina from pigment epithelium and two techniques to saturate the ocular tissues in vivo with precursors of DNA synthesis were used: (1) the method of repeated [3H]-thymidine labeling and subsequent autoradiographic analysis of semithin sections and (2) an original method for continuous labeling of thymidine analog bromodeoxyuridine and subsequent immunochemical detection. The data obtained confirm and extend our previous data on the localization of DNA-synthesizing cells in the neural retina and expose the pattern of S-phase cell accumulation after retinal detachment for each proliferation-competent cell population. In addition to cells in the growth zone of the retina, Muller glia, microglia, and minor cell population in the vitreal part of interneurons, DNA-synthesizing cells included astrocytes of the optic nerve and cells of its vascular network. Four weeks after detachment, the number of S-phase cells in the growth zone could reach 15-20%, while the above-mentioned DNA-synthesizing cells in the differentiated retina have low reproductive rate and could produce only one generation within the same period.     

DNA synthesis in the ventricular myocardium of young rats exposed to intermittent high altitude (IHA) hypoxia. An autoradiographic study.

Intermittent high altitude (IHA) hypoxia (7000 m) increased the wet weight of the right ventricular myocardium of 30-day-old rats after two 4 h/day exposures. During the same period the number of DNA-synthesizing nuclei of both muscle and non-muscle cell types increased proportionally. After 4 such exposures to hypoxia the number of 3H-thymidine-labelled nuclei in both cell types increased further. In addition, the number of labelled nuclei increased significantly in the yet un-enlarged left ventricle. While there was no difference in the number of DNA-synthesizing cells between the right and left ventricles in control animals, a significant increase in the number of cells involved in DNA synthesis in the right ventricle was found in both groups of animals exposed to IHA hypoxia. These results show that DNA synthesis in myonuclei of the ventricular myocardium can be stimulated in 30-day-old rats, i.e. at the very end of the weaning period.     

Differential lethal effects of both cytosine arabinoside and hydroxyurea on jejunal crypt cells and testosterone-stimulated accessory sex glands

DNA-synthesizing cells from mouse jejunal crypts and accessory sex glands respond differently to the DNA synthesis inhibitors cytosine arabinoside and hydroxyurea. A single injection of either agent brought about a rapid inhibition of thymidine labelling in both the tissues. Whilst both agents had a lethal effect upon the majority of S-phase cells in the crypts, only a minority of S-phase cells in the accessory sex glands showed evidence of necrosis. These differences are considered in the context of possible physiological differences between continually dividing cells and putative G0 cells. The accessory sex glands are normally quiescent proliferative tissues. They were stimulated to undergo DNA synthesis and later mitosis by testosterone propionate injections, commencing three days after castration. Cytosine arabinoside was the more effective cytocidal agent in the accessory sex glands, and when two injections were scheduled so as to affect a large number of DNA-synthesizing cells, a compensatory hyperplasia was evoked. In the coagulating gland, this compensatory response involved the proliferation of many cells which, in the absence of cytotoxic perturbation, would be non-proliferatie (Q cells).     

Autocrine activation of DNA synthesis in prothoracic gland cells of the silkworm, Bombyx mori

Autocrine activation of DNA synthesis in prothoracic gland cells in last instar larvae of the silkworm, Bombyx mori, was studied using both a long-term in vitro organ culture system and immunocytochemical labeling with 5-bromo-2'-deoxyuridine (BrdU). When prothoracic glands were incubated in a small volume of culture medium (10 microl/gland), the numbers of DNA-synthesizing cells per gland increased significantly, and DNA synthesis was stimulated less by hemolymph, as compared with glands incubated in a large volume (50 microl/gland). Moreover, glands cultured in groups (6 glands per group in a 50-microl drop) also resulted in much higher levels of DNA synthesis than those cultured individually in a 50-microl drop. The mechanism by which alternation of the volume of the incubation medium results in changes in the levels of DNA synthesis was further examined. When prothoracic glands were incubated in medium (50-microl drop per gland) that was preconditioned with glands (in a 10-microl drop individually), a dramatic increase in DNA synthesis activity was also observed, indicating that prothoracic glands may release a factor that stimulates their own DNA synthesis. The growth-promoting factor was further characterized and it was found that the factor is heat stable, and its molecular weight was estimated to be between 1,000 and 3,000 Da. Moreover, the factor also stimulated corpus allatum cell DNA synthesis in vitro. Injection of concentrated putative growth-promoting factor into day 4 last instar-ligated larvae greatly increased cell DNA synthesis of the prothoracic glands, indicating the in vivo function of the present autocrine factor.     

A study of the localization and accumulation of S-phase cells in the retina of newt <Emphasis Type="Italic">Pleurodeles waltl</Emphasis> after experimental pigment epithelial detachment

This work continues the studies of the proliferative ability of cells in the adult newt retina. The model of experimental detachment of the retina from pigment epithelium and two techniques to saturate the ocular tissues in vivo with precursors of DNA synthesis were used: (1) the method of repeated [³H]-thymidine labeling and subsequent autoradiographic analysis of semithin sections and (2) an original method for continuous labeling of thymidine analog bromodeoxyuridine and subsequent immunochemical detection. The data obtained confirm and extend our previous data on the localization of DNA-synthesizing cells in the neural retina and expose the pattern of S-phase cell accumulation after retinal detachment for each proliferation-competent cell population. In addition to cells in the growth zone of the retina, Muller glia, microglia, and minor cell population in the vitreal part of interneurons, DNA-synthesizing cells included astrocytes of the optic nerve and cells of its vascular network. Four weeks after detachment, the number of S-phase cells in the growth zone could reach 15–20%, while the above-mentioned DNA-synthesizing cells in the differentiated retina have low reproductive rate and could produce only one generation within the same period.     

DIFFERENTIAL LETHAL EFFECTS OF BOTH CYTOSINE ARABINOSIDE AND HYDROXYUREA ON JEJUNAL CRYPT CELLS AND TESTOSTERONE-STIMULATED ACCESSORY SEX GLANDS

DNA-synthesizing cells from mouse jejunal crypts and accessory sex glands respond differently to the DNA synthesis inhibitors cytosine arabinoside and hydroxyurea. A single injection of either agent brought about a rapid inhibition of thymidine labelling in both the tissues. Whilst both agents had a lethal effect upon the majority of S-phase cells in the crypts, only a minority of S-phase cells in the accessory sex glands showed evidence of necrosis. These differences are considered in the context of possible physiological differences between continually dividing cells and putative G₀ cells. The accessory sex glands are normally quiescent proliferative tissues. They were stimulated to undergo DNA synthesis and later mitosis by testosterone propionate injections, commencing three days after castration. Cytosine arabinoside was the more effective cytocidal agent in the accessory sex glands, and when two injections were scheduled so as to affect a large number of DNA-synthesizing cells, a compensatory hyperplasia was evoked. In the coagulating gland, this compensatory response involved the proliferation of many cells which, in the absence of cytotoxic perturbation, would be non-proliferative (Q cells).     

Phosphate and the regulation of DNA replication in normal and virus-transformed 3T3 cells.

3T3 cells were cultured in media with different phosphate concentrations and the effects on DNA synthesis were examined. Even a modest phosphate depletion markedly inhibited DNA synthesis and cell multiplication in proliferating cultures. Furthermore, the decrease in the proportion of DNA-synthesizing cells observed after phosphate starvation followed the same time-course as the decrease seen after serum starvation. Cells starved to quiescence in a medium with a 100-fold decrease in phosphate concentration remained viable but non-proliferating for up to 3 weeks, i.e. they had entered a state of quiescence comparable with that seen after serum starvation. Addition of phosphate to phosphate-depleted cultures restored DNA synthesis within 24h. Furthermore, the kinetics of [3H]thymidine labelling after phosphate addition were nearly identical with the labelling kinetics following addition of serum to serum-depleted cultures. In contrast, phosphate deprivation had no inhibitory effects on DNA synthesis in simian-virus-40-transformed 3T3 cells. Furthermore, the inhibitory effects on DNA synthesis in such cells caused by a complete removal of serum could not be further enhanced by decreasing the phosphate concentration in the culture medium.     

DNA synthesis in lactotrophs of primary rat anterior pituitary cell cultures. Effects of thyroliberin, bromocriptine and somatostatin

Prolactin immunostaining in combination with thymidine autoradiography was used to characterize changes in the DNA-synthesizing activity of lactotrophs in primary monolayer cultures of the rat anterior pituitary gland treated for 3 days with thyroliberin (TRH), somatostatin (SRIF) and bromocriptine (CB 154). The number of lactotrophs labelled with 3H-thymidine within the total pool of labeled pituitary cells was used to estimate DNA synthesis in prolactin-producing cells. TRH (10 ng/ml) stimulated DNA synthesis in the whole population of cultured cells but not in lactotrophs. TRH only weakly counteracted the noticeable inhibitory effect of CB 154 (0.75 microM/l) on thymidine incorporation into lactotrophs. SRIF (20 ng/ml) inhibited DNA synthesis in lactotrophs to a lesser extent than CB 154. The combination of methods used in this paper may be useful for studying the selective effects of regulators on the proliferative activity of various pituitary cell types in vivo and in culture.     

Ori-somes, nucleoprotein complexes descending from origin regions of animal chromosomal DNA replication. A micromorphological study.

Micromorphology of nucleoprotein (NP) complexes designated according to their descent and shape as Ori-somes is presented. These NP complexes of three different types harbor molecules of cytoplasmic "small" polydisperse DNA, which descend from origin regions of chromosomal DNA replication and are equipped, as shown previously, with early DNA-synthesizing activities. By negative staining the Ori-somes are visualized as particles of irregular shape, sometimes of a subunit-like structure. Micromorphological differences in size and structural compactness noted among individual Ori-somes are dependent on their type similarly as earlier shown physico-chemically and biochemically. Such differences were also confirmed by two different spreading techniques. The most unravelled structures with electron diffuse centers belong to Ori-somes of component B associated with most active DNA synthesis. In contrast, the Ori-somes of components A and C, associated with pronounced RNA synthesis, revealed large electron-dense centers. The incidence of replicative structures present in Ori-somes corresponds with the level of their DNA-synthesizing activities.