DNA replication in mammalian cells after the action of physical, chemical or biological factors. VI. The recovery of DNA synthesis in a culture of irradiated cells

The kinetics of DNA synthesis restoration in cultured HeLa cells and in L-929 mouse fibroblasts irradiated by gamma-rays of 60Co with a dose of 10 Gy was studied. Early after irradiation the rate of DNA synthesis in HeLa cells measured with 3H-thymidine incorporation was seen to decrease. Two hours later the incorporation starts to increase to reach the control level 4 hours after irradiation and then becomes even higher than this level. The distribution of cells among phases of the cell cycle measured with flow cytometry undergoes changes. 4-6 hours after irradiation part of S-phase cells increased contributing presumably to the elevating of 3H-thymidine incorporation observed at this time. The restoration of the incorporation was suppressed by inhibitors of protein and RNA synthesis--cycloheximide and actinomycin D. It is suggested that the processes of restoration of DNA synthesis in irradiated cells can be of inducible nature. In irradiated HeLa and L-929 cells the restoration of DNA synthesis is resistant to novobiocin, an inhibitor of DNA replication.     

Disruption of DNA replication in non-irradiated cells in basal cell nevus syndrome and the effect of ionizing radiation

Analysis of DNA fiber autoradiograms from basal cell nevus syndrome (BCNS) skin fibroblasts has revealed for the first time a new defect in DNA replication earlier unknown in other chromosomal instability syndromes, that involves a significantly decreased rate of DNA-chain growth in unirradiated cells. Here we present evidence that the defect may be due to a marked reduction in number of simultaneously operating groups of replicons compared to that in normal cells, the rate of fork movement and the fusion of neighbouring units in the group remaining unchanged. Radioresistant DNA synthesis was observed in the BCNS cells. The exposure of cells derived from normal donor to gamma-rays at a dose of 5 Gy reduces the number of simultaneously operating groups of replicons to the level occurring in unirradiated BCNS cells, the rate of folk movement being unchanged in both cell types. However, the incidence of fusion between neighbouring units within the group is lower in the cells exposed to gamma-rays, due perhaps to a radiation-induced lesion in the group. Thus, ionizing radiation reduces the rate of DNA synthesis to the same level, however from different initial levels. Our data suggest that the phenomenon of radioresistant DNA synthesis may be explained by the presence of the initial defect in DNA replication in BCNS or any other chromosomal instability disorders.     

Mechanisms of disruption of DNA in human cells. Mutagenesis of a virus in xeroderma pigmentosum cells as an indicator of a defect in DNA repair

Lymphocytes of two patients with Xeroderma pigmentosum, their mothers and cell lines isolated from the third patient were examined. The reaction of vaccinia virus treated with mutagens, the index of virus-induced mutagenesis and DNA repair synthesis were the criteria of estimation of cell repair activity. Significant inhibition of DNA repair synthesis was detected in cells of all the patients observed (primary and established lines) in the experiments with UV-irradiation and 4-nitroquinoline-4-oxide. These indexes correlated with decreased virus reactivation and increased level of virus-induced mutagenesis. Inhibition of DNA repair synthesis was also revealed in lymphocytes of both mothers. These data claim a possibility of discovering heterozygotes, the carriers of mutant genes. The data on virus-induced mutagenesis serve as a simple test for detection of DNA repair disorders.     

DNA synthesis and repair in permeable cells of Micrococcus radiodurans.

Cells permeable to deoxyribonucleoside triphosphate were prepared from Micrococcus radiodurans, and DNA synthesis and rejoining of strand scissions induced by gamma-rays were investigated. DNA synthesis was stimulated by ATP at an optimal concentration of 1mM. This reaction requires four deoxyribonucleoside triphosphates and MgCl2. NAD inhibited the reaction, but no rejoining of primer DNA was observed. Even in the presence of NAD, DNA which was synthesized in the unirradiated permeable cells had a peak molecular weight of only 1.3 - 10(6). DNA synthesis was stimulated by irradiation of the permeable cells with gamma-rays, but this stimulatory effect was eliminated by the addition of NAD. Both primer and synthesized DNA in the irradiated permeable cells were rejoined in vitro in the presence of NAD and deoxyribonucleoside triphosphates, while those in the unirradiated permeable cells were not rejoined.     

Aromatase activity and the effect of estradiol and testosterone on DNA synthesis in endometrial carcinoma cell lines

Human endometrial and breast carcinoma cell lines were examined for aromatase activity and the effects of sex steroids (estradiol and testosterone) on DNA synthesis. Aromatase activity was high (greater than 500 fmol/10⁷ cells/24 h) in the cell lines MCF-7 and OMC-2, moderate (100–499 fmol/10⁷ cells/24 h) in the cell lines HEC-59 and Ishikawa, and low (less than 100 fmol/10⁷ cells/24 h) in the HHUA cell line. A substantial stimulation of DNA synthesis by estradiol (10⁻⁹M) was observed in cell lines HEC-59, OMC-2, and MCF-7, with an increase in [³H]thymidine uptake of over 250%. The Ishikawa cell line was stimulated moderately (115–249%). No estradiol-induced increase in DNA synthesis was observed in HHUA. Responsiveness of DNA synthesis to testosterone was observed in cell lines that showed the greatest response to estradiol, namely HEC-59, OMC-2, and MCF-7. Otherwise, estrogen-responsiveness did not always correlate with a significant aromatase activity. These data suggest that some but not all endometrial carcinomas may possess an aromatase-dependent growth stimulating system.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. V. The correlation of DNA strand breaks and base damage to chromosomal aberrations and sister-chromatid exchanges induced by X-irradiation

The X-ray-sensitive Chinese hamster ovary (CHO) mutant cell lines xrs 5 and xrs 6 were used to study the relation between X-ray-induced DNA lesions and biological effects. The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCE) were determined in wild-type CHO-K1 as well as mutants xrs 5 and xrs 6 cells following X-irradiation under aerobic and anaerobic conditions. Furthermore, we used a newly developed immunochemical method (based on the binding of a monoclonal antibody to single-stranded DNA) to assay DNA single-strand breaks (SSBs) induced by gamma-rays in these CHO cells, after a repair time of up to 4 h. For all cell lines tested the frequency of X-ray-induced chromosomal aberrations was strongly increased after irradiation in air compared with hypoxic conditions. When compared to the wild-type line, the xrs mutants known to have a defect in repair of DNA double-strand breaks (DSBs) exhibited a markedly enhanced sensitivity to aerobic irradiation, and a high OER (oxygen enhancement ratio) of 2.8-3.5, compared with 1.8-2 in CHO-K1 cells. The induction of SCE by X-rays was relatively little affected in CHO-K1 irradiated in air compared with hypoxic conditions (OER = 0.8), and in xrs 5 (OER = 0.7). A dose-dependent increase in the frequency of SCEs was obtained in xrs 6 cells treated with X-rays in air, and a further increase by a factor of 2 was evident under hypoxic conditions (OER = 0.4). With the immunochemical assay of SSB following gamma-irradiation, no difference was found between wild-type and mutant strains in the number of SSBs induced. The observed rate of rejoining of SSBs was also the same for all cell lines studied.     

Degradation and repair of rat hepatoma cell DNA following exposure to gamma rays and methylnitrosourea]

Single-strand breaks induced in DNA of ascitic hepatoma cells by gamma-rays and N-methyl-N-nitrosourea (MNU), resp., may be effectively repaired. Double-strand breaks of DNA from MNU-treated hepatoma cells are also effectively repairable in vivo. Only a small part of double-strand breaks induced by gamma-rays in DNA of these cells is repaired in the postradiation period. The combined action of gamma-rays and MNU on the hepatoma cells causes a complete inhibition of repair of DNA and its further degradation. Under these conditions, inhibition of the repair of DNA synthesis and repression of DNA polymerase I activity is observed.     

Radioresistant DNA synthesis in the fibroblasts of a Down's syndrome patient

DNA synthesis after gamma-irradiation was analysed either by direct assay of the amount of 3H-Td incorporated into DNA of fibroblasts derived from normal donor and from a patient with Down's syndrome, or by analysis of the steady-state distribution of 3H-DNA in alkaline sucrose gradients. Doses of gamma-radiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition in fibroblasts of the patient with Down's syndrome. The radioresistant DNA synthesis in cells of this patient was mainly due to a much less inhibition of replicon initiation than that in normal cells. Thus, in the case of Down's syndrome, the cells fail to go through the delays during which DNA lesions can be repaired, unlike the situation being the case in normal cells.     

Tn10 transposition does not respond to environmental stress

The rate of DNA synthesis after gamma-irradiation was studied either by analysis of the steady-state distribution of daughter [3H]DNA in alkaline sucrose gradients or by direct assay of the amount of [3H]thymidine incorporated into DNA of fibroblasts derived from a normal donor (LCH882) and from Down's syndrome (LCH944), Werner's syndrome (WS1LE) and xeroderma pigmentosum (XP2LE) patients with chromosomal sensitivity to ionizing radiation. Doses of gamma-irradiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition of DNA synthesis in the cells from the affected individuals. The radioresistant DNA synthesis in Down's syndrome cells was mainly due to a much lower inhibition of replicon initiation than that in normal cells; these cells were also more resistant to damage that inhibited replicon elongation. Our data suggest that radioresistant DNA synthesis may be an intrinsic feature of all genetic disorders showing increased radiosensitivity in terms of chromosome aberrations.     

Establishment of cell lines derived from ataxia telangiectasia and xeroderma pigmentosum patients with high radiation sensitivity

Four human fibroblast cell lines, three of which were derived from a patient with ataxia telangiectasia and the other from a patient with xeroderma pigmentosum, were established after transfection with cloned SV40 DNA. These 4 cell lines showed some phenotypes characteristic of neoplastically transformed cells, and had a human karyotype with heteromorphisms identical to those of the parental fibroblasts. Their sensitivity to the cytotoxic effects of gamma-rays or ultraviolet irradiation was as high as those of their parental fibroblasts.     

Induction of apoptosis in bone marrow cells after treatment of mice with WR-2721 and gamma-rays: relationship to the cell cycle

Apoptosis and cell proliferation are accepted to be responsible for the maintenance of homeostasis in the hematopoietic system. Understanding of the mechanisms of action of the aminothiols and ionizing radiation on normal hematopoietic cells requires determination of the correlation between apoptotic cell death and cell cycle distribution. The effects of WR-2721 ((S)-2-/3-aminopropylamino/ethylphosphorothioic acid; Amifostine) and ⁶⁰Co gamma-rays on apoptosis and cell cycle progression in the mouse bone marrow were determined. Adult male Swiss mice were exposed to 6 Gy gamma-rays only, or pretreated with WR-2721, at a dose of 400 mg/kg body weight, 30 min before gamma-irradiation. The laser scanning cytometry APO-BRDU^TM assay based on simultaneous analysis of cellular DNA content and the in situ detection of DNA strand breaks was used to identify apoptotic cells and to reveal the cell cycle position of apoptotic and nonapoptotic cells. Temporary changes in the frequency of apoptotic cells with fluorescein isothiocyanate (FITC) labeling of DNA strand breaks, and all bone marrow cells including apoptotic and nonapoptotic ones, whose DNA stained with propidium iodide, were observed in the particular phases of the cell cycle throughout the 96-h period after WR-2721 application and gamma-irradiation. The cell cycle phase specificity of WR-2721 and ⁶⁰Co gamma-irradiation was shown in terms of induction of apoptosis in bone marrow cells. The patterns of alterations in the frequency of apoptotic cells and all bone marrow cells with respect to their cell cycle position were dependent on the agent(s) applied and the time interval after treatment of mice with WR-2721 and/or gamma-rays. A modulatory, suppressive action of WR-2721 on apoptosis induction and the cell cycle perturbation caused in normal cells of the mouse bone marrow by gamma-rays was found.     

The action of N-methyl-N-nitrosourea on non-established human cell lines in vitro. I. Cell cycle inhibition and aberration induction in diploid and Down's fibroblasts.

The effect of N-methyl-N-nitrosourea (MNU) on the cell cycle, DNA synthesis and chromosomal sensitivity of cultivated diploid fibroblasts and fibroblasts with trisomy 21 was investigated in vitro. With the exception of the inhibition of G2, Down's cells proved to be more sensitive than diploid cells with respect to the decrease of the mitotic and labelling index, the inhibition of the progression of cells through the early and middle S and the frequency of induced chromosomal aberrations. The chromosomal sensitivity was dependent on the position of cells in the cell cycle during treatment with MNU. If treated during late S no differences concerning the S block and aberration frequencies were found between diploid and Down's cells. However, if MNU treatment took place in the middle and early S, Down's cells were more sensitive. The higher aberration frequencies in Down's cells resulted from elevated levels of chromatid breaks, multiple fragmentations and chromatid translocations. Possible reasons for the increased sensitivity of Down's cells are discussed.     

Immunofluorescence study of the adenovirus type 2 single-stranded DNA binding protein in infected and transformed cells.

High-titer monospecific antiserum against highly purified adenovirus 2 (Ad2) single-stranded DNA binding protein (DBP) was used to study, by indirect immunofluorescence (IF), the synthesis of DBP in Ad2-infected human cells and adenovirus-transformed rat, hamster, and human cell lines. In infected cells the synthesis of DBP was first detected in the cytoplasm at 2 to 4 h postinfection and reached a maximum intensity at 6 h postinfection. At this time DBP began to accumulate in the nucleus, where it reached maximum intensity at about 14 h postinfection. The cytoplasmic IF was diffuse, whereas nuclear IF appeared as dots that coalesced into large globules as infection progressed. In cells treated with 1-beta-d-arabinofuranosylcytosine to inhibit viral DNA synthesis, strong nuclear IF was observed in the form of dots, but the large fluorescent globules were not observed. The Ad2 (oncogenic group C) anti-DBP serum reacted very strongly by IF with Ad5 (group C)-infected, to a lesser extent with Ad7 and Ad11 (group B)-infected, and weakly with Ad12 and Ad18 (group A)-infected KB cells (treated with 1-beta-d-arabinofuranosylcytosine). These results may indicate that Ad2 DBP is closely related immunologically to DBPs induced early after infection by adenovirus serotypes in oncogenic group C, moderately related to DBPs of serotypes in oncogenic group B, and perhaps distantly related to DBPs of serotypes in oncogenic group A. The following adenovirus-transformed cell lines were examined for DBP synthesis by IF with the Ad2 anti-DBP serum: six rat cell lines (T2C4, F17, 8662, 8638, 8617, and F161) transformed by Ad2 virus, three hamster cell lines transformed by Ad2 virus (Ad2HT1) and Ad2-simian virus 40 hybrid virus (ND1HK1 and ND4HK4), and one rat (5RK) and one human (293-31) cell line transformed by transfection with Ad5 DNA. T2C4 and 8662 appeared weakly positive, whereas Ad2HT1 and ND4HK1 were strongly positive. The other transformed cell lines did not produce DBP detectable by IF. Thus, some but not all transformed cell lines produce DBP, which indicates that DBP is not required for maintenance of cell transformation and that transformed cells can express "nontransforming" viral genes as protein.     

Effect of ionizing radiation on DNA synthesis in ataxia telangiectasia cells.

The effect of ionizing radiation on DNA synthesis in control and ataxia telangiectasia (AT) lymphoblastoid cell lines was determined. A dose dependent decrease in DNA synthesis was observed in control cells, and the rate and extent of thi decrease in synthesis increased with time after irradiation. No decrease in DNA synthesis was obtained in AT cells, immediately following irradiation, at doses up to 400 rads. At longer times postirradiation, inhibition of synthesis increased but the extent of inhibition was less in AT cell than controls at all doses used. An immediate depression of DNA synthesis was evident in control cells after a radiation dose of 200 rads reaching a maximum at 90 min postirradiation. Little or no decrease in DNA synthesis was evident in AT cells up to 60 min after the same radiation dose, but a decrease occurred between 60 and 90 min after irradiation. The rate of recovery of DNA synthesis to normal levels was more rapid in AT cells than in controls.     

Ataxia telangiectasia: an anomaly in DNA replication after irradiation.

The effect of increasing dose of gamma-radiation on DNA synthesis in an ataxia telangiectasia lymphoblastoid cell line and a number of control lymphoblastoid cell lines was investigated. No significant inhibition of low molecular weight DNA synthesis was observed in the AT cell line at doses which resulted in considerable inhibition in the control cell lines. At higher doses, 600 to 800 rad, low molecular weight DNA synthesis and chain elongation were enhanced in the AT cell line. At time course study of DNA synthesis after 200 rads of gamma-radiation, revealed no appreciable inhibition of low and high molecular weight DNA synthesis up to 60 minutes postirradiation. However, in control cell lines, overall DNA synthesis was depressed to a level 50% of that shown by the unirradiated cells.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells: III. Comparison of Simian Virus 40 Lytic Infection in Three Different Monkey Kidney Cell Lines

A comparative study of simian virus 40 (SV40) lytic infection in three different monkey cell lines is described. The results demonstrate that viral deoxyribonucleic acid (DNA) synthesis and infectious virus production begin some 10 to 20 hr earlier in CV-1 cells and primary African green monkey kidney (AGMK) cells than in BSC-1 cells. Induction of cellular DNA synthesis by SV40 was observed in CV-1 and AGMK cells but not with BSC-1 cells. Excision of large molecular weight cellular DNA to smaller fragments was easily detectable late in infection of AGMK cells. Little or no excision was observed at comparable times after infection of CV-1 and BSC-1 cells. The different kinds of responses of these three monkey cell lines during SV40 lytic infection suggest the involvement of cellular functions in the virus-directed induction of cellular DNA synthesis and the excision of this DNA from the genome.     

Damage by gamma rays and heavy ions to superhelical structures of nuclear DNA

Chinese hamster cells (V79-4), human lymphocytes and mouse ascites cell were exposed to gamma-rays and heavy ions (4He and 12C). Sedimentation of complexes containing DNA was studied after cell lysis by centrifugation in a neutral sucrose gradient. The distinctions noted after irradiation with gamma-rays and heavy ions are consistent with the idea of the superhelical organization of DNA into discrete and membrane-bound compact units. According to the estimates made the diameter of these complexes was approximately 0.2 micron and DNA content, about 2 X 10(9) dalton.     

Faster rates of DNA unwinding under alkaline conditions in xrs-5 cells may reflect chromatin structure alterations.

The Chinese hamster ovary (CHO) cell line xrs-5 is a radiation-sensitive mutant isolated from CHO-K1 cells. The radiation sensitivity is associated with a defect in DNA double-strand break rejoining. The DNA alkaline unwinding technique was used to measure the DNA single-strand breakage caused by gamma-rays in xrs-5 and CHO-K1 cells. Greater rates of DNA unwinding were found in xrs-5 cells as compared to CHO-K1. Independent measurement of DNA strand breakage by DNA filter elution or pulsed-field gel electrophoresis failed to show any difference between the two cell lines. The greater rate of unwinding in xrs-5 cells may reflect an alteration in chromosome structure.     

Deoxyribonucleoside triphosphate pools and differential thymidine sensitivities of cultured mouse lymphoma and myeloma cells

The effects of various concentrations of thymidine on DNA synthesis and deoxyribonucleoside triphosphate contents of a highly thymidine-sensitive cultured mouse lymphoma cell line (WEHI-7) and a relatively resistant mouse myeloma cell line (HPC-108) have been studied by 32P-labelling techniques. DNA synthesis in the myeloma cells was inhibited by thymidine at concentrations of 10(-3) M or greater, while DNA synthesis in the lymphoma cells was inhibited by concentrations 30-fold lower, consistent with the 25-fold difference between the two cell lines in sensitivity to growth inhibition by thymidine. Thymidine caused marked elevation of the dTTP and dGTP pools, slight elevation or no change in the dATP pool and a marked decrease in the dCTP pool in cells of both lines. The greater resistance of HPC-108 cells to thymidine inhibition was related to the finding that they normally contained a much higher concentration of dCTP than did the WEHI-7 cells. Pool size measurements on thymidine-treated (10(-4) M) cells of an additional seven sensitive lymphoma and six relatively resistant myeloma cell lines indicated that in all 15 lines studied, with one exception, a critical concentration of dCTP of about 32 nmol per ml of cell volume was required for the maintenance of normal rates of DNA synthesis. The dCTP content found normally in the lymphoma cells was only a little above this concentration. Amongst the myeloma lines, three contained similarly low levels of dCTP, but were more resistant to thymidine inhibition probably because of their inefficient production of dTTP from thymidine. Cells of the other four myeloma lines (including HPC-108) normally contained much higher dCTP concentrations. The mechanism of thymidine action was explained by reference to the known allosteric properties of ribonucleotide reductase.