Sensitivity to exposure to postradiation repair inhibitors and to repeat irradiation in the progeny of irradiated cells

In experiments on HeLa cells a study was made of the response of near and distant descendants of irradiated cells to repeated irradiation and to the effect of inhibitors of repair and replicative synthesis of DNA, that is, arabinosyl cytosine (Ara-C) and hydroxyurea (HU). Throughout 12-15 postirradiation generations the descendants of preirradiated cells were more sensitive to ionizing radiation and to the effect of Ara-C and HU: the dose-response curves had no shoulder and the Do value decreased by 1.7 times. In generations 18 to 24, the sensitivity to the damaging effect of the agents under study was normalized and the resistance somewhat increased. The data obtained indicate that some DNA lesions persist in many generations of the exposed cells.     

Long-term DNA damage and mammalian cell survival

On the basis of our own data and those reported in the literature we have made an attempt to follow the fate of the DNA lesions which remain unrepaired during a long period of time, and their possible role in the fate of irradiated cells. The presence of long-lived ("residual") damages is determined by the changes in survival of exposed cells treated, at different times after irradiation, with a mixture of arabinoside cytosine and hydroxyurea. It is shown that "residual" damages can probably exist in the exposed generation and be retained in that following the irradiated one, i.e. after the first mitosis. The nearest descendants of exposed cells (the 3d-5th generations) exhibit a 50% decrease in the rate of DNA synthesis and fall of their proliferative activity, as well as a decrease in the rate of reproduction of their remote descendants. The comparison of the results obtained with those reported by other authors enable us to assume that "residual" DNA lesions play an important role in the fate of exposed cells, that is, in reproductive death, radiation mutagenesis, and malignant transformations.     

Damaging agitation intensities increase DNA synthesis rate and alter cell-cycle phase distributions of CHO cells

The effects of fluid-mechanical force (agitation) on the cell cycle kinetics of Chinese hamster ovary (CHO) cells cultured in suspension in 2-L bioreactors has been examined. A two-color flow cytometry method was used to determine the fraction rate of DNA synthesis. With increased agitation intensity, cell viability decreased as a result of increased cell death. However, increased agitation induced the viable cells of the culture to a higher proliferative state relative to a control culture. The fraction of viable cells of the high-agitation culture (250 rpm) in S phase was higher (up to 45%) and in G1 phase was lower (up to 50%) compared with the viable cells of the control culture (80 rpm). The DNA synthesis rate per viable S-phase cell of the high-agitation culture was confirmed by recovery experiments, which were conducted to measure the apparent specific growth rate and the cell cycle kinetics of the high-agitation culture upon reduction in the agitation rate from 250 rpm back to 80 rpm. The apparent specific growth rate of the test culture, calculated for the first 12 h of the recovery period, was greater than the apparent specific growth rate of the control culture. Furthermore, the proliferative state of the viable cells of the test culture, which had become higher relative to the control culture during the high agitation period, gradually approached the level of the control culture during recovery. Results also show that the magnitude of the agitation intensity; the culture agitated at 250 rpm attained a greater proliferative state than a parallel culture agitated at 235 rpm. The 250-rpm culture had a higher fraction of S-phase and a lower fraction of G1-phase cells than the 235-rpm culture. The DNA sunthesis rate per viable S-phase cell of the 250-rpm culture was greater than of the 235-rpm culture. (c) 1992 John Wiley & Sons, Inc.     

Age-dependent changes in cell proliferation and cell death in the periodontal tissue and the submandibular gland in mice: a comparison with other tissues and organs

This study investigated the age-dependent changes in the number of BrdU- and TUNEL-positive cells in murine gingival tissue and submandibular gland, and compared the findings with those in other tissues and organs. The cell proliferative activity was decreased after 20 weeks of age in epithelial cells of the gingiva, tongue, buccal mucosa and skin. A decreased cell proliferative activity was also associated with aging in the liver and kidney parenchymal cells. Meanwhile, cell death showed peculiar changes in gingival subepithelial tissue, and mucous and serous acini of the submandibular gland. An increase of TUNEL-positive cells was demonstrated in gingival subepithelial tissue after 20-week-old of age. A significant increase of TUNEL-positive cells was also found in the mucous acinar cells in the 20-week-old mice and in the serous acini after 20 weeks. The fluctuation in the number of TUNEL-positive cells in the subepithelial tissue of the skin, and BrdU- and TUNEL-positive staining ratios in the liver was smaller than that in other tissue and organs throughout life. This study may provide useful information for better understanding the influence of aging on the functional alteration that occurs in the gingival tissue and submandibular gland of the elderly.     

Inhibition of DNA synthesis in isolated human endometrial cells by a levonorgestrel-releasing intrauterine device

OBJECTIVE: To estimate the effect of an intrauterine device (IUD) releasing 20 micrograms levonorgestrel (LNG) per 24 hours on DNA synthesis in human endometrial cells before and after 12 months of use. STUDY DESIGN: Endometrial specimens were collected from the anterior or posterior wall of the miduterus from 6 females on cycle day 10-12 before insertion of the IUD and after 12 months of use. RESULTS: Previous results from our group did not reveal any influence on endometrial DNA cell content when a levonorgestrel IUD releasing 2 micrograms/24 h was used for 12 months in a group of fertile females. In this study, the IUD release rate, 20 micrograms LNG/24 h, was statistically significantly different from the results in the previous studies. The effect of the levonorgestrel IUD on endometrial proliferation was dose dependent, and a significant correlation could be found between continuous exposure to LNG and inhibition of DNA synthesis in endometrial cells. CONCLUSION: Inhibition of proliferative activity in endometrial cells seems to be reflected by a decrease in DNA synthesis per cell nucleus and contributes to the clinical performance of the LNG-releasing IUD.     

Cell cycle related [3H]thymidine uptake and its significance for the incorporation into DNA

Cellular uptake of [3H]thymidine [( 3H]TdR) and incorporation into DNA of Ehrlich ascites tumour cells were studied in relation to the cell cycle by measuring the activity in the acid-soluble and insoluble parts of the cell material. Cells were synchronized at various stages of the cell cycle using centrifugal elutriation. The degree of synchrony of the various cell fractions was measured by flow-cytofluorometric DNA analysis. From the cellular uptake, the TdR triphosphate (dTTP) concentration of a mean cell in an unseparated cell population was calculated to be 20 X 10(-18) mol/cell. The pool activity of G1 cells was unmeasurable but rose to maximum values at the border of the G1-S phase. It decreased again during G2. The [3H]TdR incorporation into DNA was low during early S phase, reached a maximum value at two-thirds of the S phase and decreased again during late S phase. These changes in DNA synthesis were not due to changes in the dTTP pool being a limiting factor. During maximum DNA synthesis, 10% X min-1 of the dTTP pool was utilized, at which time the pool size also decreased by about 30%. Changes in pool size during the cell cycle have to be taken into account when the results of incorporation of radioactive TdR into DNA are discussed.     

Expression of the Viral Thymidine Kinase Gene in Herpes Simplex Virus-Transformed L Cells

In these studies, the expression of thymidine kinase (TK) in normal and herpes simplex virus (HSV)-transformed L cells has been compared. In asynchronously dividing cultures of L cells, the TK activity rose and declined rapidly and coordinately with DNA synthesis. When net cell increase stopped, TK activity was at a minimum. In contrast, TK activity of HSV-transformed cells remained at a minimum during rapid DNA synthesis and gradually increased as the rate of DNA synthesis decreased. When net cell increase stopped, TK activity was at a maximum. In synchronous cultures of L cells, TK activity rose and fell coordinately with the rate of DNA synthesis. In synchronous cultures of HSV-transformed cells, no increase in TK activity was observed during the period of rapid DNA synthesis, i.e., the S phase. These findings indicated that the viral TK gene in HSV-transformed cells was not placed under the control of the cellular mechanisms which normally modulate the host cell TK gene. Lytic infection of HSV-transformed cells with a TK(-) mutant of HSV-1 induced a four-to fivefold increase in viral TK. The TK of HSV-1 was induced in the HSV-1-transformed cells and HSV-2 in the HSV-2-transformed cells by this TK(-) mutant. The same infection of normal L cells decreased the cellular TK activity by 80%. This stimulation, rather than inhibition, suggest that the viral gene in HSV-transformed cells retain some of its original viral characteristics.     

5-fluorodeoxyuridine induces radioresistant synthesis of DNA and sensitizes HeLa cells to gamma radiation

It was shown that preincubation of HeLa cells with 5-fluorodeoxyuridine (10(-6) M) induced DNA synthesis resistant to gamma-radiation (6 Gy). At the same time, the death rate of exposed cells increased and nucleoid relaxation decreased. The role of DNA synthesis inhibitors in the reproductive death of exposed cells is discussed.     

Inhibitory effects of S-nitrosoglutathione on cell proliferation and DNA synthesis: possible role of glyoxalase I inactivation

We investigated the inhibitory effects of S-nitrosoglutathione (GSNO) on cell proliferation, DNA synthesis and several enzymatic activities using spontaneously immortalized human endothelial cells (ECV304). Proliferation of ECV304 was inhibited by GSNO in a dose-dependent manner (125-1000 microM). DNA synthesis was decreased 2 h after addition of GSNO to cells and was markedly repressed from 20 h after the addition. The activity of ribonucleotide reductase, a rate-limiting enzyme for DNA synthesis, was unchanged in GSNO-treated cells. GSNO inhibited less than 40% of mitochondrial respiration activity, and the membrane potential and cellular levels of ATP were not significantly decreased by GSNO. GSNO had no inhibitory effect on activities of glutathione peroxidase, glutathione S-transferase and glutathione reductase. However, glyoxalase I (Glo I) activity was decreased to 20% of the control level within 60 min, and was consistently repressed during exposure to GSNO for 20 h. A membrane-permeable Glo I inhibitor, S-bromobenzylglutathione diethylester, inhibited proliferation of ECV304 cells, while methylglyoxal (MG), a toxic metabolite generated during glycolysis and a substrate for Glo I, failed to inhibit the cell growth even at 100 microM. Glo I in several mammalian cell lines was inactivated by GSNO with a pI shift. Although we failed to detect accumulation of MG under conditions of Glo I inactivation, these results suggest that the inhibitory effects of GSNO on cell proliferation and DNA synthesis might be at least partly due to inactivation of Glo I.     

DNA polymerase alpha and the regulation of entry into S phase in heterokaryons

We have previously reported that the DNA polymerase alpha activity/unit cellular protein is decreased in late-passage (senescent) human diploid fibroblast-like (HDFL) cultures due to the cellular enlargement associated with in vitro aging. In the studies described here, we have used cell fusion technology to investigate the formal kinetic relationship between the concentration of DNA polymerase alpha and the rate of reinitiation of DNA synthesis in nuclei from senescent cells. Heterokaryons were derived from the fusion of senescent cells to a series of actively dividing cell types with inherently different DNA polymerase alpha activities per cell. A kinetic analysis revealed a first-order relationship between the entry into S phase of senescent nuclei and the concentration of DNA polymerase alpha activity calculated to be in heterokaryons. This result suggests that increases in cell volume may be related to the decline in proliferative activity of late-passage HDFL cells, via "dilution" of factors essential for cellular replication.     

Association of poly(ADP-rib) synthesis with cessation of DNA synthesis and DNA fragmentation

CHO cells and cs-4-D3 cells were used to investigate the association between poly(ADP-rib) synthesis and the cessation of DNA synthesis and DNA fragmentation. The cs4-D3 cells are cold-sensitive DNA synthesis arrest mutants of CHO cells. Upon incubation at 33 degrees C, DNA synthesis in the cs4-D3 cells stops and the cells enter a prolonged G1 or G0 phase. The events that occurred when cs4 cells were incubated at 33 degrees C were similar to those that occurred when wild-type CHO cells grew to high density. (1) In both cases, DNA synthesis and cell growth stopped. (2) The NAD+ concentration/cell was 20-25% lower in growth-arrested cells than in logarithmically growing cells. (3) Poly(ADP-rib) synthesis was 3-4 fold higher in growth-arrested cells than in logarithmically growing cells. (4) The growth-inhibited cells developed DNA strand breaks which resulted in large percentages of their DNA appearing in the low molecular weight range of alkaline sucrose gradients. (5) Both the increased rate of poly(ADP-rib) synthesis and the development of DNA strand breaks appears to be characteristic of the G1 phase of the cell cycle. (6) When growth-inhibited cells were restored to conditions favorable for DNA synthesis and cell growth, the DNA strand breaks were repaired. (7) Prolonged incubation under growth-restrictive conditions resulted in the accumulation of more DNA strand breaks than the cells could repair. This was followed by cell death when the cells were restored to conditions favorable for cell growth.     

Correlation between nuclear morphology and rate of deoxyribonucleic acid synthesis in a normal cell line.

Chinese hamster fibroblasts were investigated for the existence of correlations between proliferative activity and nuclear morphology. As a proliferative parameter, the rate of DNA synthesis of individual cells was determined by quantitative 14C-autoradiography. In a second step the images of the Feulgen-stained nuclei were digitized for extraction of features of morphology and texture. These features were correlated with the corresponding DNA synthesis rate values. The following relationships were found: Round nuclei have higher rates of DNA synthesis than flat ones. The more chromatin is packed at the nuclear rim, possibly representing heterochromatin, the lower the rate of DNA synthesis. The DNA synthesis rate also correlates with the graininess of chromatin. Larger areas of condensed chromatin are associated with lower rate values. A fine and irregular network of chromatin, as is typical of immature cell types, is associated with a high rate of DNA synthesis. Although these results are presently confined to the cell line investigated, parallels seem to exist to other cell types, such as erythropoietic cells, which await further investigation.     

The diverse effects of 5'-bromodeoxyuridine on enzyme activities in cultured H35 hepatoma cells.

Reuber (H35) hepatoma cells were grown in medium containing 10(-5)M bromodeoxyuridine (BrdU), which was incorporated into their DNA. Cell growth rate was unaffected by BrdU for the first two generations, after which it was reduced by about 50%. The effect of BrdU incorporation on the activities of several enzymes with rapid turnover rates was examined to test the hypothesis that the synthesis of such enzymes will be preferentially inhibited by BrdU. Tyrosine amino-transferase (TAT) activity decreased by 70% within two generations whereas thymidine kinase activity remained at control values. PEP carboxykinase activity was unchanged during the first generation in BrdU-containing medium but, during the second, its activity increased by at least 30%. Ornithine decarboxylase levels decreased by about 50% only after two generations in the presence of BrdU. There appeared to be no simple relationship between turnover rates and the effect of BrdU on enzyme activity. Incorporation of BrdU was found to inhibit the induction of both TAT and PEP carboxykinase by dexamethasone and to enhance the inhibition of cell growth by this steroid. These results are discussed with respect to possible mechanisms of gene expression and development in both normal and neoplastic cells.     

Mechanisms and kinetics of monoclonal antibody synthesis and secretion in synchronous and asynchronous hybridoma cell cultures.

The kinetics of monoclonal antibody synthesis and secretion have been studied in synchronous and asynchronous mouse hybridoma cell cultures. Pulse-labelling of IgG followed by immunoprecipitation and quantitation of synthesized and secreted IgG in synchronous cultures show maximum production during G1/S phases. Secretion takes place through exocytotic release of vesicle contents. Pulse-chase experiments show that 71% of the synthesized IgG is secreted within 8 h of the pulsing period and only a further 4% is secreted by 22 h. Higher specific antibody production (QA) is obtained if (a) cells are arrested and then maintained in G1/S phases, (b) viability is decreased during the death phase of batch culture, (c) the dilution rate is decreased in continuous culture or (d) cells are subjected to hydrodynamically induced stress. The increase in QA in all these cases is mainly due to the passive release of the accumulated intracellular antibody. DNA and protein synthetic activity peak during the early exponential phase and decline rapidly during mid and late exponential and death phases. Metabolic activity however peaks up to 20 h after the peak in DNA synthesis, and declines similarly during the death phase. The data are consistent with the idea that slow growth and higher death rates increase QA and that Ig secretion is probably subject to complex intracellular control.     

Starch Synthesis and Programmed Cell Death during Endosperm Development in Triticale(× Triticosecale Wittmack)

Triticale(× Triticosecale Wittmack) grains synthesize and accumulate starch as their main energy source.Starch accumulation rate and synthesis activities of ADP-glucose pyrophosphorylase,soluble starch synthases,granule-bound starch synthase and starch-branching enzyme showed similar pattern of unimodal curves during endosperm development.There was no significant difference in activity of the starch granule-bound protein isolated from total and separated starch granules at different developmental stages after anthesis in triticale.Evans Blue staining and analysis of DNA fragmentation indicated that cells of triticale endosperm undergo programmed cell death during its development.Dead cells within the endosperm were detected at 6 d post anthesis(DPA),and evidence of DNA fragmentation was first observed at 21 DPA.The period between initial detection of PCD to its rapid increase overlapped with the key stages of rapid starch accumulation during endosperm development.Cell death occurred stochastically throughout the whole endosperm,meanwhile,the activities of starch biosynthetic enzymes and the starch accumulation rate decreased in the late stages of grain filling.These results suggested that the timing and progression of PCD in triticale endosperm may interfere with starch synthesis and accumulation.     

Regulatory role of endogenous regucalcin in the enhancement of nuclear deoxyribonuleic acid synthesis with proliferation of cloned rat hepatoma cells (H4-II-E)

The role of endogenous regucalcin in the regulation of deoxyribonuleic acid (DNA) synthesis in the nuclei of the cloned rat hepatoma cells (H4-II-E) with proliferative cells was investigated. Cells were cultured for 6-96 h in a alpha-minimum essential medium (alpha-MEM) containing fetal bovine serum (FBS; 1 or 10%). Cell number was significantly increased between 24 and 96 h after culture with 10% FBS; cell proliferation was markedly stimulated by culture with 10% FBS as compared with that of 1% FBS. In vitro DNA synthesis activity in the nuclei of cells was significantly elevated 6 h after culture with 10% FBS and its elevation was remarkable at 12 and 24 h after the culture. Nuclear DNA synthesis activity was significantly reduced in the presence of various protein kinase inhibitors (PD98059, staurosprine, or trifluoperazine) in the reaction mixture containing the nuclei of cells cultured for 12 and 24 h with FBS (1 and 10%). The addition of regucalcin (10(-7) and 10(-6)M) in the reaction mixture caused a significant inhibition of nuclear DNA synthesis activity. The presence of anti-regucalcin monoclonal antibody (25-100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS resulted in a significant increase in nuclear DNA synthesis activity. This increase was completely blocked by the addition of regucalcin (10(-6) M). The effect of anti-regucalcin antibody (100 ng/ml) in increasing nuclear DNA synthesis activity was significantly inhibited in the presence of various protein kinase inhibitors. DNA synthesis activity was significantly enhanced in the presence of anti-regucalcin antibody (100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS in the presence of Bay K 8644 (2.5 x 10(-6) M). Culture with Bay K 8644 did not cause a significant increase in DNA synthesis activity in the absence of anti-regucalcin antibody. The present study demonstrates that endogenous regucalcin plays a suppressive role in the enhancement of nuclear DNA synthesis with proliferative cells.     

An autoradiographic study of cellular proliferaton,DNA synthesis and cell cycle variability in the rat liver caused by phenobarbital-induced oxidative stress: The protective role of melatonin

The protective effect of melatonin against phenobarbital-induced oxidative stress in the rat liver was measured based on lipid peroxidation levels (malondialedyde and 4-hydroxyalkenals). Cellular proliferation, DNA synthesis and cell cycle duration were quantitated by the incorporation of ³H-thymidine, detected by autoradiography, into newly synthesized DNA. Two experiments were carried out in this study, each on four equal-sized groups of male rats (control, melatonin [10 mg/kg], phenobabital [20 mg/kg] and phenobarbital plus melatonin). Experiment I was designed to study the proliferative activity and rate of DNA synthesis, and measure the levels of lipid peroxidation, while experiment II was for cell cycle time determination. Relative to the controls, the phenobarbital-treated rats showed a significant increase (P < 0.01) in the lipid peroxidation levels (30.7%), labelling index (69.4%) and rate of DNA synthesis (37.8%), and a decrease in the cell cycle time. Administering melatonin to the phenobarbital-treated rats significantly reduced (P < 0.01) the lipid peroxidation levels (23.5%), labelling index (38.2%) and rate of DNA synthesis (29.0%), and increased the cell cycle time. These results seem to indicate that the stimulatory effect of phenobarbital on the oxidized lipids, proliferative activity, kinetics of DNA synthesis and cell cycle time alteration in the liver may be one of the mechanisms by which the non-genotoxic mitogen induces its carcinogenic action. Furthermore, melatonin displayed powerful protection against the toxic effect of phenobarbital.     

Radiation induction of delayed recombination in Schizosaccharomyces pombe

Ionizing radiation is known to induce delayed chromosome and gene mutations in the descendants of the irradiated tissue culture cells. Molecular mechanisms of such delayed mutations are yet to be elucidated, since high genomic complexity of mammalian cells makes it difficult to analyze. We now tested radiation induction of delayed recombination in the fission yeast Schizosaccharomyces pombe by monitoring the frequency of homologous recombination after X-irradiation. A reporter with 200 bp tandem repeats went through spontaneous recombination at a frequency of 1.0 x 10(-4), and the frequency increased dose-dependently to around 10 x 10(-4) at 500 Gy of X-irradiation. Although the repair of initial DNA damage was thought to be completed before the restart of cell division cycle, the elevation of the recombination frequency persisted for 8-10 cell generations after irradiation (delayed recombination). The delayed recombination suggests that descendants of the irradiated cells keep a memory of the initial DNA damage which upregulates recombination machinery for 8-10 generations even in the absence of DNA double-strand breaks (DSBs). Since radical scavengers were ineffective in inhibiting the delayed recombination, a memory by continuous production of DNA damaging agents such as reactive oxygen species (ROS) was excluded. Recombination was induced in trans in a reporter on chromosome III by a DNA DSB at a site on chromosome I, suggesting the untargeted nature of delayed recombination. Interestingly, Rad22 foci persisted in the X-irradiated population in parallel with the elevation of the recombination frequency. These results suggest that the epigenetic damage memory induced by DNA DSB upregulates untargeted and delayed recombination in S. pombe.     

Results of experimental radiobiological studies made within the 10-kilometer zone of the Chernobyl AES accident]

A study was made of the effect of high radioactive contamination on the animal organism (C57BL/6 mice) and HeLa cell culture within the ten-kilometer zone of the Chernobyl A.P.S. accident. The total radiation dose, as calculated by a gamma-component, was 0.09 to 2 Gy. A long-term exposure of mice within the zone (cumulative dose of 1.8 to 2 Gy) caused a significant decrease in bone marrow stem potencies and changes in the brain vascular system; subsequent acute exposure of animals increased interferon titres in the serum to a much greater extent than a single acute exposure did. As to HeLa cells, irradiation there of with doses of 0.09 to 0.4 Gy during 15-20 postirradiation generations caused a decrease in the proliferative activity, an emergence of cells with micronuclei and of giant cells, and remote cell death.