Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VI. The correlation between UV-induced DNA lesions and chromosomal aberrations, and their modulations with inhibitors of DNA repair synthesis

The role of UV-induced DNA lesions and their repair in the formation of chromosomal aberrations in the xrs mutant cell lines xrs 5 and xrs 6 and their wild-type counterpart, CHO-K1 cells, were studied. The extent of induction of DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) due to UV irradiation in the presence or absence of 1-beta-D-arabinofuranosylcytosine (ara-C) and hydroxyurea (HU) was determined using the alkaline and neutral elution methods. Results of these experiments were compared with the frequencies of induced chromosomal aberrations in UV-irradiated G1 cells treated under similar conditions. Xrs 6 cells showed a defect in their ability to perform the incision step of nucleotide repair after UV irradiation. Accumulation of breaks 2 h after UV irradiation in xrs 6 cells in the presence of HU and ara-C remained at the level of incision breaks estimated after 20 min, which was about 35% of that found in wild-type CHO-K1 cells. In UV-irradiated CHO-K1 and xrs 5 cells, more incision breaks were present after 2 h compared with 20 min post-treatment with ara-C, a further increase was evident when HU was added to the combined treatment. The level of incision breaks induced under these conditions in xrs 5 was about 80% of that observed in CHO-K1 cells. UV irradiation itself did not induce any detectable DNA strand breaks. Accumulation of SSBs in UV-irradiated cells post-treated with ara-C and HU coincides with the increase in the frequency of chromosomal aberrations. These data suggest that accumulated SSBs when converted to DSBs in G1 give rise to chromosome-type aberrations, whereas strand breaks persisting until S-phase result in chromatid-type aberrations. Xrs 6 appeared to be the first ionizing-radiation-sensitive mutant with a partial defect in the incision step of DNA repair of UV-induced damage.     

A UV-responsive G2 checkpoint in rodent cells.

We have studied the effect of UV irradiation on the cell cycle progression of synchronized Chinese hamster ovary cells. Synchronization of cells in S or G2 phase was accomplished by the development of a novel protocol using mimosine, which blocks cell cycle progression at the G1/S boundary. After removal of mimosine, cells proceed synchronously through the S and G2 phases, allowing manipulation of cells at specific points in either phase. Synchronization of cells in G1 was achieved by release of cells after a period of serum starvation. Cells synchronized by these methods were UV irradiated at defined points in G1, S, and G2, and their subsequent progression through the cell cycle was monitored. UV irradiation of G1-synchronized cells caused a dose-dependent delay in entry into S phase. Irradiation of S-phase-synchronized cells inhibited progression through S phase and then resulted in accumulation of cells for a prolonged interval in G2. Apoptosis of a subpopulation of cells during this extended period was noted. UV irradiation of G2-synchronized cells caused a shorter G2 arrest. The arrest itself and its duration were dependent upon the timing (within G2 phase) of the irradiation and the UV dose, respectively. We have thus defined a previously undescribed (in mammalian cells) UV-responsive checkpoint in G2 phase. The implications of these findings with respect to DNA metabolism are discussed.     

Results of and prospects for methods of determination of the frequency of mutant cells for glycophorin A and T-cell receptor loci to estimate long-term genotoxic effect of ionizing radiation

The usage of two methods for assessment of somatic gene mutations for the purpose of biological dosimetry and estimation of consequences of irradiation at long time after the exposure has been discussed. The determination of cells bearing mutated glycophorin A (GPA) locus is a reliable method for biodosimetry at both short and long time after the acute irradiation according to our results and the data of other authors. For prolonged exposure, the GPA-method is less informative than in cases of acute irradiation. The determination of mutant cell frequency at T-cell receptor (TCR) locus may be used only at short time after the irradiation. Meanwhile, the TCR-method is more sensitive and informative for biodosimetry of recent irradiation than the GPA test. The both methods may be used for individual assessment of long-term health consequences after the irradiation, because persons with elevated frequencies of mutant cells may represent a group at high risk in respect to oncological diseases.     

Studies on the radiosensitivity of the function of the Golgi complex.

The uptake of 3H-glucosamine into primary human-embryo fibroblasts and into the Golgi-rich fraction isolated from liver of mice labelled in vivo was studied, after various doses of X-radiation, by autoradiography and biochemical methods. A dose of 90 rad resulted in an increased precursor uptake in interphase cells at 24 hours and in mitotic cells at 48 hours after irradiation; 226 rad had virtually no effect on the grain counts of interphase cells, but reduced the labelling of mitotic forms. The characteristic intracellular localization of the grains were not influenced by these doses. Although no immediate radiation-induced reaction could be observed in liver cells either, significant stimulation of the 3H-glucosamine incorporation was measured in isolated Golgi-rich fractions 24 hours after whole-body irradiation with 90, 450, or 905 rad. This phenomenon is discussed as a part of the somatic regeneration of membrane structures.     

Microwave irradiation in label-detection for diagnostic DNA-in situ hybridization.

Summary A DNA-in situ hybridization protocol was adapted for application to sections of routinely processed paraffin embedded material. This protocol was developed previously for detecting DNA-virus infected cells in whole cell preparations and employs biotinylated DNA as probe. Three different biotin detection methods were optimized and applied. The first uses streptavidin and a biotinylated complex of alkaline phosphatase, the second consists of an immunogold-silver staining, and the third of a peroxidase technique using a silver amplification. The alkaline phosphatase method was the most rapid, and as sensitive as the immunogold-silver staining. The peroxidase method was the most sensitive. Microwave irradiation was applied to the different incubation steps of these three detection methods. Short incubations with microwave irradiation gave very poor results when peroxidase labelled antibodies were used. Short incubation with microwave irradiation gave results comparable to those obtained with conventional incubations, when streptavidin, antibiotin, complexed alkaline phosphatase, or gold labelled goat antirabbit were used. It was thus shown that microwave irradiation creates the possibility of a very rapid label-detection for nonradioactive DNA-in situ hybridization.     

Interspecific hybridization between Nicotiana repanda Willd. and N. tabacum L. through the pollen irradiation technique and the egg cell irradiation technique

Summary Two techniques were useful in overcoming hybrid inviability between N. repanda and N. tabacum. These techniques combine gamma-ray irradiation to pollen or to egg cells (in ovules) with in vitro culture of fertilized ovules. When in vitro culture of fertilized ovules from in situ hybridization of N. repanda x N. tabacum was combined without gamma-ray irradiation to pollen or to egg cells (in ovules), all of the resulting seedlings developed chlorosis and died. Furthermore, in the case of in situ hybridization of N. repanda x N. tabacum with gamma-ray irradiated N. tabacum pollen, no viable seeds were obtained. By using both techniques, combining gamma-ray irradiation to N. tabacum pollen or to egg cells in (N. repanda ovules) with in vitro culture of fertilized ovules, we were successful in obtaining flowering hybrid plants. Thus, it appears that it may be possible to overcome hybrid inviability to a certain extent using both the pollen irradiation technique and the egg cell irradiation technique, i.e., gamma-ray irradiation to pollen or to egg cells (in ovules) before pollination and in vitro culture of fertilized ovules.The research reported in this paper is in partial fulfillment of PhD requirements for the senior author     

Indirect influences of radiation on unirradiated cells through irradiated cells.

There has been a recent upsurge of interest in radiation-induced bystander effects. Previously we reported that the accumulation of inducible nitric oxide (NO) synthase (iNOS) was induced only in human glioblastoma mutant (m) p53 cells by acute irradiation with X-rays, suggesting a suppression of iNOS induction after acute irradiation with X-rays in wtp53 cells. NO secreted from the irradiated mp53 cells induced the accumulation of p53 in unirradiated wtp53 cells. The radiosensitivity of wtp53 cells was reduced by exposure to the conditioned medium from irradiated mp53 cells, suggesting that NO is an initiator of radiation-induced bystander effects. In the present study, we found that the accumulation of iNOS in wtp53 cells was induced by chronic irradiation with gamma-rays followed by acute irradiation with X-rays, but not by each one. It is suggested that the accumulation of iNOS may be due to the depression of acute irradiation-induced p53 functions by pre-chronic irradiation. We found that chronic irradiation with gamma-rays did not inhibit the accumulation of p53 after exposure to the conditioned medium from the irradiated mp53 cells. However, the decay of accumulated p53 was stimulated by chronic irradiation with gamma-rays. At the same time, the accumulation of Hdm2 was observed; suggesting that chronic irradiation with gamma-rays may stimulate the degradation of p53 accumulated by NO-mediated bystander effects.     

Effects of electrons and neutrons on the synthesis of RNA in resistant and sensitive strains of the slime-mould Dictyostelium discoideum and the modifying effect of caffeine.

RNA synthesis was investigated after irradiation in resistant and sensitive lines of the slime-mould Dictyostelium discoideum. When 3H adenine was used as a precursor to RNA, incorporation increased after irradiation in the resistant WT line but not in the sensitive line (gammas-13). The extent of RNA synthesis after irradiation was correlated with the shoulder width on the survival curve of the resistant line. When this was reduced by irradiating with neutrons, or treatment with caffeine RNA synthesis was also reduced. No preferential synthesis of one RNA species occurred; there was increased labelling in all RNA species after irradiation. Sucrose gradient analysis of ribosomal RNA extracted from irradiated cells and free of messenger RNA revealed no apparent difference in composition from that extracted from unirradiated cells. Increased RNA synthesis after irradiation may form part of the recovery processin the resistant cells.     

Chromosome aberrations and reproductive death of mammalian cells. Quantitative relations between these effects

The data reported in the literature concerning the relationship between the yield of chromosome aberrations and the number of cells without aberrations, on the one hand, and the survival rate of mammalian cells, on the other, with a reference to different types of radiation are reviewed in this article. It is shown that the number of chromosome aberrations per one lethal damage, as to the results obtained by different authors, ranges from 0.5 to 1.5, this discrepancy is mainly due to different methods applied by different authors, and at least one chromosome aberration corresponds to a lethal damage caused by irradiation in the G1 phase.     

Ligase-deficient yeast cells exhibit defective DNA rejoining and enhanced gamma ray sensitivity.

Yeast cells deficient in DNA ligase were also deficient in their capacity to rejoin single-strand scissions in prelabeled nuclear DNA. After high-dose-rate gamma irradiation (10 and 25 krads), cdc9-9 mutant cells failed to rejoin single-strand scissions at the restrictive temperature of 37 degrees C. In contrast, parental (CDC9) cells (incubated with mutant cells both during and after irradiation) exhibited rapid medium-independent DNA rejoining after 10 min of post-irradiation incubation and slower rates of rejoining after longer incubation. Parental cells were also more resistant than mutant cells to killing by gamma irradiation. Approximately 2.5 +/- 0.07 and 5.7 +/- 0.6 single-strand breaks per 10(8) daltons were detected in DNAs from either CDC9 or cdc9-9 cells converted to spheroplasts immediately after 10 and 25 krads of irradiation, respectively. At the permissive temperature of 23 degrees C, the cdc9-9 cells contained 2 to 3 times the number of DNA single-strand breaks as parental cells after 10 min to 4 h of incubation after 10 krads of irradiation, and two- to eightfold more breaks after 10 min to 2.5 h of incubation after 25 krads of irradiation. Rejoining of single-strand scissions was faster in medium. After only 10 min in buffered growth medium and after 10 krads of irradiation, the number of DNA single-strand breaks was reduced to 0.32 +/- 0.3 (at 23 degrees C) or 0.21 +/- 0.05 (at 37 degrees C) per 10(8) daltons in parental cells, but remained at 2.1 +/- 0.06 (at 23 degrees C) or 2.3 +/- 0.07 (at 37 degrees C) per 10(8) daltons in mutant cells. After 10 or 25 krads of irradiation plus 1 h of incubation in medium at 37 degrees C, only DNA from CDC9 cells was rejoined to the size of DNA from unirradiated cells, whereas at 23 degrees C, DNAs in both strains were completely rejoined.     

Immunoperoxidase staining of cervicovaginal smears after radiotherapy.

Cervicovaginal smears from 2 women with postirradiation dysplasia, 4 women with postirradiation squamous cell carcinoma of the cervix, 30 women with irradiation atypia and 5 healthy, nonirradiated women were stained immunohistochemically with six keratin antibodies. For four of the antibodies--CK19 (BA17), EMA, PKK-1 and CAM 5.2--squamous cells showing irradiation atypia, postirradiation dysplasia or postirradiation squamous cell carcinoma were more likely to stain positively than were nonirradiated squamous cells. For three of the antibodies in which multiple squamous cells stained positively, the proportion of squamous cells showing postirradiation dysplasia or postirradiation squamous cell carcinoma staining strongly was equal to or greater than the corresponding overall proportion for squamous cells showing irradiation atypia. This was statistically significant with only one antibody, PKK-1. No statistically significant differences were seen in staining of irradiated and nonirradiated squamous cells by MAK-6 and AE1:AE3. The data show that some keratin antigens are more often expressed in the irradiated groups and that there may be differences in the degree of antigen expression between squamous cells showing postirradiation dysplasia or postirradiation squamous cell carcinoma and squamous cells showing irradiation atypia.     

Radiation effects on rat testes. V. Studies on lysosomal enzymes (acid phosphatase and acid DNAse) and their physiological significance following partial body gamma-irradiation.

Acid phosphatase is present in the nucleus and cytoplasm of cells in the seminiferous tubules and the interstitium of rat testes. The effect of irradiation on acid phosphatase is dependent on the environmental temperature and the dose of irradiation. It appears that initial rise in the enzyme at a low radiation dose and a high environmental temperature or at a high dose and low temperature is associated with a lysosomal breakdown of the germinal cells of the testes. A decrease in acid phosphatase in the advanced stages of radiation injury is a secondary radiation effect which may lead to decreased metabolic synthesis of phosphate esters owing to the unavailability of orthophosphate in the testicular tubules. The reduced acid phosphatase activity can be detected in the seminiferous tubules, suggesting that the enzyme activity is related to the state of the germ cell population. An initial increase in acid phosphatase is matched by an initial rise in acid DNAse within hours of irradiation, further suggesting that there is radiation interaction with the cells of the germinal epithelium. The enhanced activity of DNAse following a 2nd week of irradiation at 2000 R confirms the phagocytic activity of the non-germinal cells.     

Effect of salt solutions on radiosensitivity of mammalian cells. III. Treatment with hypertonic solutions.

V79 Chinese hamster cells were treated with hypertonic solutions of NaCl or KCl and irradiated rat various times before, during, or after exposure to the solution. In solutions of molarities between 0-2 and 0-5 M, the cellular radiosensitivity increases with the molarity of the bathing solution. At these molarities, the hypertonic solution need not be present during irradiation to sensitize cells. Furthermore, radiosensitivity of cells could be increased by exposing cells for longer times to the hypertonic solution before irradiation. At higher salt concentrations (at 1-5 to 1-8 M), significant radioprotection is observed. Survival curve data showed that this protection was characterized by an increase in DO and a decrease in n, while the survival curves of cells sensitized with 0-465 M NaCl or with lower concentrations exhibited mainly changes in DO. The 1-55 M NaCl solution must be present during radiation to give a protective effect. Prolonged exposure to the salt before irradiation reduced the amount of radioprotection afforded by the salt. The results are discussed in terms of the effects of ions on histones, cellular water structure and the cell-aging cycle.     

Rosette formation between rat thymocytes and guinea pig erythrocytes requires "active" fetal calf serum. II. Characterization of the receptor-bearing thymocytes.

High proportions of thymocytes from many rat strains participate in rosette formation (RF) with guinea pig erythrocytes only in the presence of non-heated fetal calf serum. Adult Lewis rat spleens, lymph nodes, peripheral blood and bone marrow contain very few cells capable of participating in RF. Adult levels of cells participating in RF are present in Lewis rat thymus from the perinatal period onward. Essentially, no cells participating in RF are found in the spleens or bone marrow of Lewis rats during the perinatal period. Thymocytes participating in RF are sensitive to high doses of cortisone and low doses of irradiation in vivo. After sublethal irradiation, thymuses are rapidly repopulated by cells participating in RF. After lethal irradiation followed by bone marrow transplantation, thymuses are repopulated by donor cells that can participate in RF. Of interest, a moderate proportion of thymocytes from many mouse strains also can participate in RF. This subpopulation of murine thymocytes are highly cortisone sensitive.     

Histological changes in spleens of radio-sensitive and radio-resistant mice exposed to low-dose X-ray irradiation

We have previously determined by using immune-assay or bio-assay methods that low-dose irradiation enhances immune and anti-oxidation functions. In this study, we examined histological changes of lymphatic follicles at 4, 24, or 48 hrs after sham, 0.25, 0.5, or 15 Gy irradiation in the spleens of BALB/c mice, which are sensitive to radiation compared with other strains, and C57BL/6J mice, which are resistant to radiation, using hematoxylin-eosin staining for lymphatic follicles or methylgreen pyronin staining for plasma cells. Results show that the lymphatic follicles in the spleens of the two mouse strains decreased at 24 or 48 hrs after 15 Gy irradiation. The number of plasma cells in the spleens of sham irradiated BALB/c mice was greater than that of sham irradiated C57BL/6J mice. At 4 hrs after 0.25 Gy irradiation, plasma cells increased in the spleens of the two mouse strains. These findings suggest, by histology, that low-dose irradiation activates the plasma cells and enhances the immune function. Although those two mouse strains have different sensitivities to radiation, the above changes were similar in both time course and degree of response. Therefore, the phenomena observed may be common in mice.     

Two types of proliferating cell nuclear antigen (PCNA) complex formation in quiescent normal and xeroderma pigmentosum group A fibroblasts following ultraviolet light (uv) irradiation.

We examined the relationship between the formation of proliferating cell nuclear antigen (PCNA) complex with DNA and nucleotide excision repair in human fibroblasts following ultraviolet light (uv) irradiation. PCNA complex formation was detected by the immunofluorescence method after methanol fixation and nucleotide excision repair activity was detected as the unscheduled DNA synthesis (UDS) by autoradiography labeled with [3H]thymidine. Quiescent normal cells showed a strong punctuated pattern of PCNA staining 5 min to 3 h and UDS 3 h after 10 J/m2 of uv irradiation, but they no longer showed PCNA staining and UDS 24 h after irradiation. In contrast, xeroderma pigmentosum group A (XP-A) cells, which lack UDS activity, did not show PCNA staining up to 30 min after irradiation; however, unexpectedly, they were stained 3 h and even 24 h after irradiation with their staining pattern being different from that in normal cells. Namely, the fluorescence spots in XP-A cells were larger in size and much smaller in number than those in normal cells. When XP-A cells were fused with normal cells with polyethylene glycol treatment, nuclei of XP-A cells showed a PCNA staining pattern similar to that of normal cells at 30 min, which was no longer detected 24 h after irradiation. These results suggest that there exist two types of PCNA complex formation, nucleotide excision repair-related and -unrelated, in human fibroblasts following uv irradiation.     

Generation of ROS in cells on exposure to CW and pulsed near-infrared laser tweezers.

We report the results of a study on generation of reactive oxygen species (ROS) and changes in the membrane potential of mitochondria of carcinoma of cervix (HeLa) and Chinese hamster ovary (CHO) cells following exposure to continuous wave (cw) or pulsed Nd: YAG laser (1064 nm). For a given laser irradiation, the generation of ROS and induced changes in the membrane potential of mitochondria were more pronounced for HeLa cells as compared to CHO cells. However, in both the cells the laser dose required to elicit a given change was much lower with pulsed laser exposure compared to that required with a cw laser exposure. This suggests involvement of photothermal effects in the laser irradiation induced changes. Mechanistic studies using quenchers for ROS suggest that laser irradiation leads to generation of hydroxyl radicals.     

Changes in the leukocyte phagocytic activity of donor blood after its UV irradiation. I. Its relation to the irradiation dose and initial level of phagocytic activity

Phagocytic activity of human mono- and granulocytes increased markedly after UV blood irradiation in the apparatus "Izolda" used in hospitals of the USSR for medical treatment. With the rise of irradiation dose the ratio of cells ingesting latex particles increased, although the average number of particles ingested per cell decreased. The integrative phagocytic index poorly depended on the irradiation dose. In patients with a low initial level of phagocytic index, after UV blood irradiation it became more pronounced than in those with the initial elevated level. The enhancement of phagocytic activity is the result of a direct UV-stimulation of cells. This stimulation not mediated by irradiated blood plasma is known to inhibit the phagocytic activity of leucocytes. A possible mechanism of phagocytic activity stimulation is discussed.     

Critical evaluation of techniques to detect and measure cell death--study in a model of UV radiation of the leukaemic cell line HL60.

The reliability of eight distinct methods (Giemsa staining, trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) double staining for fluorescence microscopy and flow cytometry, propidium iodide (PI) staining, annexin V assay, TUNEL assay and DNA ladder) for detection and quantification of cell death (apoptosis and necrosis) was evaluated and compared. Each of these methods detects different morphological or biochemical features of these two processes. The comparative analysis of the 8 techniques revealed that AO/EB (read in fluorescence microscopy) provides a reliable method to measure cells in different compartments (or pathways) of cell death though it is very time consuming. PI staining and TUNEL assay were also sensitive in detecting very early signs of apoptosis, but do not allow precise quantification of apoptotic cells. These three methods were concordant in relation to induction of apoptosis and necrosis in HL60 cells with the various UV irradiation time periods tested. Both AO/EB (read by flow cytometry) and annexin V-FITC/PI failed to detect the same number of early apoptotic cells as the other three methods. Trypan blue is valueless for this purpose. Giemsa and DNA ladder might be useful as confirmatory tests in some situations.     

Ultrastructural and morphometric alterations in bone marrow stromal tissue after 7 Gy irradiation.

To evaluate the response of marrow stroma to 7 Gy irradiation, femoral bone marrow was fixed by vascular perfusion (so as to avoid the artificial destruction of sinus endothelia), and was examined using light and electron microscopy with morphometric methods. The radiation caused a marked decrease in hematopoietic cell number (NHC) within 3 days post-irradiation, followed by total recovery of hematopoiesis, which occurred gradually over 28 days. An increased number of fat cells was seen by 7 days. During the whole course of hypoplasia and recovery, the continuity of sinus wall, three-dimensional reticular mesh work in hematopoietic parenchyma, gap junctions (GJ) between stromal cells, the adventitial cell cover of sinus wall (ACC), and the stromal cell numbers of reticular cells (RC), sinus endothelia (SE), and macrophages (MP) were maintained. The cellularity of stromal components of RC, SE, and MP seemed passively increased in contrast to a reduction in numbers of NHC. A similar tendency was observed (1) between NHC and ACC and (2) between GJ and the cellularity of fat cells, which had a statistical significant correlation (p less than 0.05; t-test). The mechanism of radio resistance in bone marrow stroma and the possible functional adaptation and cellular coordination after irradiation are discussed.