A correlation between radiation sensitivity and initial chromatid breaks in cancer cell lines revealed by Calyculin A-induced premature condensation

Three human malignancy cell lines were irradiated with ⁶⁰Co γ-rays. Initial chromatid breaks were measured by using the chemically induced premature chromosome condensation technique. Survival curves of cells exposed to gamma rays was linear-quadratic while the efficiency of Calyculin A in inducing PCC of G₂ PCC was about five times more than G₁ PCC. A dose-dependent increase in radiation-induced chromatid/isochromatid breaks was observed in G₁ and G₂ phase PCC and a nearly positive linear correlation was found between cell survival and chromatin breaks. This study implies that low LET radiation-induced chromatid/isochromatid breaks can potentially be used to predict the radiosensitivity of tumor cells either in in vitro experimentation or in in vivo clinical radiotherapy.     

Chromatid breaks induced in Chinese hamster cells by low doses (12–100 rad) ofπ −-mesons

Summary Monolayer cultures of the fibroblast-like Chinese hamster cell-line 19/1 were irradiated in the G₂-phase of the cell cycle by ^–-mesons (6 rad/min peak-pion dose rate). Frequencies of induced single- and isochromatid breaks, acentric fragments and interchanges were compared with data obtained from 140 kV X-rays.The RBE-values were for the pion dose peak between 0.8–1.2 and for the pion dose plateau 0.5–0.9. Whereas for single chromatid breaks there was no significant difference between X-rays and peak pions for identical physical doses, the isochromatid breaks alone showed a significantly higher frequency for 100 rad peak pions.     

The effect of G2-treatments with 2′-deoxyadenosine on the frequency of chromatid aberrations in human lymphocytes depends on the type of culture

The effect of G₂-treatments with 2-deoxyadenosine (dAdo) on the frequency of chromatid aberrations in X-irradiated and unirradiated human lymphocytes depends on the method of culture. In whole-blood cultures dAdo alone produced very few if any aberrations, but in the presence of inhibitors of adenosine deaminase (ADA), such as EHNA or coformycin, a high frequency of chromatid gaps, chromatid breaks, and isochromatid breaks were produced. In cultures of purified lymphocytes, dAdo produced aberrations even in the absence of an ADA inhibitor. Apparently the lymphocytes are protected against the chromosome-damaging effect of dAdo by the ADA activity of the erythrocytes. — When given as a post-treatment, dAdo also enhances the frequency of chromatid aberrations induced by X-rays in G₂. In whole-blood cultures this effect is obtained even in the absence of an ADA inhibitor, although the concentration required to produce enhancement is about twenty times higher than in the presence of the inhibitor.     

Determining the repair rate for radiation-induced chromatid deletions during the first 24 hours after irradiation

The number of chromatid plus isochromatid deletions present in the testes and bone marrow of the Chinese hamster was measured as a function of time following acute exposure to cobalt-60 gamma irradiation. The number of breaks remaining scorable at any time after irradiation exposure could be represented by a simple exponential equation (N = Noe-kt). The values of No and k, calculated after 100 roentgens of cobalt-60 gamma irradiation, were 1.15 breaks/cell and 0.16/hour, respectively, for the testes, and 0.53 break/cell and 0.14/hour, respectively, for the bone marrow. The average length of time that the breaks remain scorable, 1/k, was found to be 6.3 hours for the testes and 7.1 hours for the bone marrow. By suitable formulation, it was possible to predict the number of breaks present by using varied radiation exposure rates and times after exposure. This formulation could be used for both single and multiple radiation exposures.     

Absence of Chromosome Damage in the bone marrow of rats fed detergent actives for 90 days

Four detergent actives, sodium lauryl sulphate, DOBS 055, Dobanol 25 sulphate LCU and Dobanol 25 sulphate HCB, were fed to rats in the diet for 90 days at the maximum tolerated dose, 1.13 percent active ingredient in each case. Sodium lauryl sulphate and DOBS 055 were also fed at half this concentration. Chromosome preparations were made from the bone marrow and scored for the presence of rearrangements, chromatid gaps and breaks and isochromatid gaps and breaks. The four detergent actives were found to have no effect on the chromosomes of rat bone marrow cells.     

Mechanisms of chromosomal aberration production II. Aberrations induced by 5-bromodeoxyuridine and visible light

We have allowed synchronized V79B Chinese hamster tissue culture cells to incorporate 5-bromodeoxyuridine (BUdR) during one DNA synthetic (S) period of the cell cycle and then determined chromosomal aberration yields induced by illumination of the cells with visible light during the succeeding pre- and post-DNA-synthetic (G₁and G₂) phases of the cell cycle. At the level used, BUdR by itself induces no aberrations. Illumination during the G₁ phase following incorporation induces aberrations of the chromatid type, but none of the chromosome type. All types of chromatid aberrations are induced, including isochromatid deletions and exchange types. In contrast, when cells are illuminated during the immediately following G₂ phase, large numbers of achromatic lesions and chromatic deletions are seen at the first post-illumination mitosis, but no isochromatid deletions and few exchange-type aberrations occur. When G₂-illuminated cells are examined in their second mitosis, however, chromatid aberrations of all types are again seen.

These results are interpreted within the “repair” model of chromosomal aberration production by UV light presented earlier³. The model assumes that the vertebrate chromosome is mononeme, consisting of but a single DNA double helix during the prereplication G₁ phase. The initial lesions induced by illumination of BUdR-containing DNA are believed to be single-chain breaks, and the observation that G₁ illumination produces only chromatid-type aberrations is taken as additional evidence for the mononeme chromosome. Conversion of single-chain breaks into double chain breaks through the action of a single-strand nuclease is postulated to account for the production of chromatid deletions at the first mitosis of G₂-illuminated cells. The action of this enzyme, plus a recombinational or post-replication repair mechanism, are postulated to account for the production of isochromatid deletions in G₁-illuminated cells. A rapid decline in achromatic lesion frequency with increasing time between G₂ illumination and fixation of the cells is considered evidence for rapid rejoining of most of the initial chain breaks.     

High yields of isochromatid breaks and successive formation of chromosome exchanges may lead to reproductive cell death following high-LET irradiation

To clarify the relationship between cell death and chromosomal aberrations following exposure to heavy-charged ion particles beams, exponentially growing Human Salivary Gland Tumor cells (HSG cells) were irradiated with various kinds of high energy heavy ions; 13 keV/μm carbon ions as a low-LET charged particle radiation source, 120 keV/μm carbon ions and 440 keV/μm iron ions as high-LET charged particle radiation sources. X-rays (200 kVp) were used as a reference. Reproductive cell death was evaluated by clonogenic assays, and the chromatid aberrations in G2/M phase and their repairing kinetics were analyzed by the calyculin A induced premature chromosome condensation (PCC) method. High-LET heavy-ion beams introduced much more severe and un-repairable chromatid breaks and isochromatid breaks in HSG cells than low-LET irradiation. In addition, the continuous increase of exchange aberrations after irradiation occurred in the high-LET irradiated cells. The cell death, initial production of isochromatid breaks and subsequent formation of chromosome exchange seemed to be depend similarly on LET with a maximum RBE peak around 100–200 keV/μm of LET value. Conversely, un-rejoined isochromatid breaks or chromatid breaks/gaps seemed to be less effective in reproductive cell death. These results suggest that the continuous yield of chromosome exchange aberrations induced by high-LET ionizing particles is a possible reason for the high RBE for cell death following high-LET irradiation, alongside other chromosomal aberrations additively or synergistically.     

Folic acid and chromosome breakage. III. Types and frequencies of spontaneous chromosome aberrations in proliferating lymphocytes

The types and frequencies of spontaneous chromosome aberrations were studied in human lymphocytes cultured for 96 h in minimal essential medium (MEM) or MEM without folic acid (MEM-FA). In both media, the most frequent aberrations were chromatid gaps, isochromatid gaps and chromatid breaks. Chromosome (isochromatid) breaks and dicentrics were seen less frequently. Neither of these less frequent aberrations was seen in 4000 cells from MEM, but both were seen in 4000 cells from MEM-FA.     

Enhancement of chromosome aberrations by the combination of DNA substitution with halogenated deoxyuridine and streptonigrin treatments

We treated CHO cells with streptonigrin (SN) alone, in combination with BrdUrd or IdUrd substitution, and with or without the addition of caffeine. The cells assessed for chromosome damage by SN were in the G₂ period and the magnitude of the damage was expressed as monosubstituted chromatid breaks, bisubstituted chromatid breaks and boundary regions breaks (boundary regions indicate the point of exchange of mono- and bisubstituted chromatids). We found that the combination of BrdUrd or IdUrd substitution with SN treatments produced a remarkable increase in the frequency of breaks over the frequencies observed with the halogenated compound only. The effect was more evident with IdUrd than with BrdUrd, and more dramatic in bisubstituted than in monosubstituted chromatids. The frequency of boundary breaks in cells treated with BrdUrd plus SN was similar to the frequency of breaks in monosubstituted chromatids treated similarly. Conversely, the damage in boundary regions was almost similar to that in bisubstituted chromatids in cells challenged with IdUrd plus SN. The addition of caffeine to BrdUrd-substituted chromosomes gave rise to a marked enhancement of breakages with a gradient of chromatid damage that was: bisubstituted > monosubstituted > boundary regions. A further increase of chromatin breaks maintaining the gradient indicated above was obtained when the cells were treated with BrdUrd plus SN plus caffeine. We propose that BrdUrd and IdUrd substitution alone or in combination with caffeine treatments and with SN in its capacity to bind DNA, give rise to different chromatin structures capable of modulating the DNA damage induced along the chromatin fibril by the active oxygen species liberated by SN-DNA complexes.     

Types and frequencies of chromosomal aberrations induced by irradiation of different parts of interphase in Crepis capillaris

Quantitative and qualitative estimates of chromosomal damage in roots of Crepis capillaris were made in metaphase cells at many time intervals after irradiation with 200 or 400 rad of ⁶⁰Co gamma-rays. The results have confirmed the general pattern described for cells of other organisms, and have revealed in addition the following new facts. (1) The formation of aberrations of chromosome and chromatid type is not determined by the time of chromosome duplication alone. (2) The relative frequencies of different types of discontinuity form peaks with the following time succession: single gaps, chromatid breaks, isolocus breaks. (3) The location of peaks does not depend on the radiation dose, and shows no correlation which the time of synthesis. (4) Irradiation of G₂ induces a significant number of chromosome-type exchanges in Crepis. (5) Higher doses of radiation in G₂ favour the formation of chromatid over chromosome exchanges and of isochromatid breaks over chromosome breaks. A new interpretation of the production of certain types of aberration is discussed.     

Folic acid and chromosome breakage III. Types and frequencies of spontaneous chromosome aberrations in proliferating lymphocytes

The types and frequencies of spontaneous chromosome aberrations were studied in human lymphocytes cultured for 96 h in minimal essential medium (MEM) or MEM without folic acid (MEM-FA). In both media, the most frequent aberrations were chromatid gaps, isochromatid gaps and chromatid breaks. Chromosome (isochromatid) breaks and dicentrics were seen less frequently. Neither of these less frequent aberrations was seen in 4000 cells from MEM, but both were seen in 4000 cells from MEM-FA.     

Characteristics of γ-ray-induced chromosomal aberrations in mutagen-sensitive mutants of L5178Y cells

We have examined the chromosomal radiosensitivities of an ionizing-radiation- and MMS-sensitive mutant (M10), and a UV- and 4NQO-sensitive mutant (Q31), isolated from mouse lymphoma L5178Y cells, with regard to killing effects. In the first mitoses after 100 R γ-irradiations, it was found that M10 cells were highly radiosensitive in terms of chromosomal aberrations accompanying longer mitotic delay (3 h); the frequencies of both chromatid-type and chromosome-type aberrations were, respectively, about 7 and 4 times higher than that of wild-type L5178Y cells. Furthermore, chromatid exchanges, particularly triradials, isochromatid breaks with sister union, and chromatid gaps and breaks were markedly enhanced at G₁ phase of M10 cells. In contrast, the chromosomal radiosensitivity of Q31 cells after 100 R irradiation was similar to that of L5178Y cells. On the other hand, spontaneous aberration frequencies (overall breaks per cell) of M10 and Q31 cells were, respectively, 5.1 and 2.2 times higher than that of wild-type L5178Y cells. The chromosomal hypersensitivity to γ-rays in M10 cells is discussed in the light of knowledge obtained from ataxia telangiectasia cells.     

Localisation of 7-12-Dimethylbenz(a)anthracene induced chromatid breaks and sister chromatid exchanges in chromosomes 1 and 2 of bone marrow cells of rat in vivo

The frequency of chromatid breaks associated with sister chromatid exchanges at the break point was determined in rat bone marrow cells treated in vivo with 7–12 DMBA, during the late S phase of the cell cycle. The chromosomal aberrations and SCEs were scored in the same cells. Under the experimental conditions employed, more than 40% of the chromatid breaks were found to be associated with an SCE, a frequency expected according to Revell's hypothesis for the formation of chromatid breaks.     

Mentha piperita (Linn.) leaf extract provides protection against radiation induced chromosomal damage in bone marrow of mice

Oral administration of M. piperita (1 g/kg body weight/day) before exposure to gamma radiation was found to be effective in protecting against the chromosomal damage in bone marrow of Swiss albino mice. Animals exposed to 8 Gy gamma radiation showed chromosomal aberrations in the form of chromatid breaks, chromosome breaks, centric rings, dicentrics, exchanges and acentric fragments. There was a significant increase in the frequency of aberrant cells at 6 hr after irradiation. Maximum aberrant cells were observed at 12 hr post-irradiation autopsy time. Further, the frequency of aberrant cells showed decline at late post-irradiation autopsy time. However, in the animals pretreated with Mentha extract, there was a significant decrease in the frequency of aberrant cells as compared to the irradiated control. Also significant increase in percentage of chromatid breaks, chromosome breaks, centric rings, dicentrics, exchanges, acentric fragments, total aberrations and aberrations/damaged cell was observed at 12 hr post-irradiation autopsy time in control animals, whereas Mentha pretreated irradiated animals showed a significant decrease in percentage of such aberrations. A significant decrease in GSH content and increase in LPO level was observed in control animals, whereas Mentha pretreated irradiated animals exhibited a significant increase in GSH content and decrease in LPO level but the values remained below the normal. The radioprotective effect of Mentha was also demonstrated by determining the LD(50/30) values (DRF = 1.78). The results from the present study suggest that Mentha pretreatment provides protection against radiation induced chromosomal damage in bone marrow of Swiss albino mice.     

Molecular mechanisms involved in the production of cromosomal aberrations II. Utilization of neurospora endonuclease for the study of aberration production by X-rays in G1 and G2 stages of the cell cycle

Chinese hamster ovary cells (CHO) were X-irradiated in G₁ and G₂ stages of the cell cycle and subsequently Neurospora endonuclease (NE) (E.C.3.1.4), an enzyme which is specific in cleaving single-stranded DNA, was introduced into the cells, after making the cells permeable by treatment with inactivated Sendai virus. With this treatment all classes of X-ray-induced chromatid aberrations increased in G₂ cells, whereas in G₁ cells an increase in cromosome type of aberrations was found, associated with a profound induction of chromatid type of aberrations as well. Duration of the availability of single-strand gaps for the action of NE has been studied in G₂ cells following X-irradiation and the influence of different parts of the G₂ stage on the type and frequencies of chromatid aberrations was discerned. While the increase in chromosome type of aberrations by NE in X-irradiated G₁ cells has been interpreted as due to the conversion of DNA single-strand breaks or gaps to double-strand breaks by NE, the induction of chromatid aberrations in G₁ has been assumed to be due to conversion of some of the damaged bases strand breaks by NE. Biochemical evidence is presented for the conversion by NE of DNA single-strand breaks induced by X-rays into double-strand breaks using neutral sucrose gradient centrifugation.     

Persistence of chromatid aberrations in the cells of solid mouse tissues exposed to 137Cs gamma radiation

Primary mouse ear and kidney cultures were established for determination of cytogenetic aberrations at short (3 days to 1 month) and long (12-23 months) times after exposure of their right sides to 7.5 Gy of (137)Cs gamma radiation. In every case, higher levels of aberrations were observed in primary cultures established from the irradiated tissues than in those established from the contralateral tissues. The most common aberrations in the contralateral tissues and those from nonirradiated mice were chromatid and isochromatid breaks and small chromatid fragments. Primary cells from irradiated tissues removed from animals within a month of exposure displayed a variety of unstable chromosome-type aberrations characteristic of recent exposure to ionizing radiation including rings, dicentrics, double minutes, and large acentric fragments. The percentages of cells exhibiting chromatid breaks and small chromatid fragments were also markedly elevated. Although the levels of chromosome-type aberrations found in primary cells from irradiated tissues dropped to near background levels a year or more after exposure, chromatid-type aberrations remained elevated. These results are consistent with long-term persistence of damage in the genomes of ionizing radiation-exposed cells in solid tissues and the induction of genomic instability in vivo.     

G2 chromatid damage and repair kinetics in normal human fibroblast cells exposed to low- or high-LET radiation

Radiation-induced chromosome damage can be measured in interphase using the Premature Chromosome Condensation (PCC) technique. With the introduction of a new PCC technique using the potent phosphatase inhibitor calyculin-A, chromosomes can be condensed within five minutes, and it is now possible to examine the early damage induced by radiation. Using this method, it has been shown that high-LET radiation induces a higher frequency of chromatid breaks and a much higher frequency of isochromatid breaks than low-LET radiation. The kinetics of chromatid break rejoining consists of two exponential components representing a rapid and a slow time constant, which appears to be similar for low- and high-LET radiations. However, after high-LET radiation exposures, the rejoining process for isochromatid breaks influences the repair kinetics of chromatid-type breaks, and this plays an important role in the assessment of chromatid break rejoining in the G2 phase of the cell cycle.     

Effects of G2 treatments with inhibitors of DNA synthesis and repair on chromosome damage induced by X-rays and chemical clastogens in root tips of Vicia faba. Comparison with corresponding effects in cultured human lymphocytes

The frequencies of chromatid aberrations produced in roots of Vicia faba by clastogenic (chromosome-damaging) agents were strongly enhanced by exposing the root-tip cells to inhibitors of DNA synthesis during the G2 phase. Chromosome damage produced by both S-dependent (maleic hydrazide, methyl methanesulfonate, thio-TEPA) and S-independent (X-rays, streptonigrin) mechanisms was enhanced by the inhibitor treatments. The types of aberrations affected by the inhibitors were mainly chromatid gaps and breaks and isochromatid breaks of the non-union type. Most effective among the inhibitors tested were hydroxyurea (HU) and 5-fluorodeoxyuridine (FdUrd). Post-treatments with caffeine were effective in enhancing clastogen-induced chromosome damage when given during the S phase. All types of aberrations, exchanges as well as breaks, were enhanced by the post-treatments. When given during the G2 phase, caffeine enhanced only the frequency of chromatid aberrations produced by X-rays. The enhancement was slight and obtained only when the cells were irradiated in the G2 phase and immediately post-treated with caffeine. Clastogen-treated cultures of human lymphocytes responded to post-treatments with inhibitors of DNA synthesis in very much the same way as clastogen-treated root-tip cells of Vicia faba. Thus, the frequencies of chromatid gaps and breaks and isochromatid breaks of the non-union type were strongly enhanced by exposing clastogen-treated lymphocytes to inhibitors of DNA synthesis during the G2 phase. The efficiency of the inhibitors, however, varied considerably in the two materials. On the whole, the number of inhibitors capable of enhancing induced chromosome damage was much larger in lymphocytes than in bean root tips. Only HU was equally effective in both materials. The most striking difference between the two materials was found when caffeine was given as a post-treatment. Thus, in human lymphocytes the frequencies of chromatid aberrations induced by most clastogenic agents were strongly enhanced when caffeine was given during the G2 phase, but little affected by post-treatments with caffeine during the S phase.     

Chromosome damage induced by decay of 3H and 125I incorporated into DNA of Chinese hamster cells

The present study was undertaken to compare the frequency of chromatid-type aberrations in Chinese hamster cells with previous results on accumulation of unrepaired DNA-strand breaks after incorporation of 3H-TdR or 125IUdR into DNA. A linear-quadratic function was fitted by the weighted-least-square method to the data on yield of chromatid aberrations at different dpm values. Based on a significant linear response at low doses, RBE for 125I in relation to 3H was calculated for (i) chromatid breaks (17 +/- 6), (ii) the sum of isochromatid breaks and chromatid exchanges (21 +/- 9), and (iii) the total number of chromatid aberrations (18 +/- 5). Analogously, the RBE for accumulation of DNA-strand breaks was determined (13 +/- 6). Our results are consistent with the assumption that chromosomal aberrations mainly originate from unrepaired DNA-strand breaks.     

Inhibition of erythroid cell growth by allogeneic murine lymphocytes. Evidence for a synergism between lymph node cells and thymocytes

Murine lymphoid cells from thymus and lymph nodes were tested for synergistic response in a graft-vs-host test. The test is based on the principle that allogeneic lymphocytes inhibit erythroid cell proliferation in the spleens of irradiated mice infused with syngeneic bone marrow cells.I was observed that mixtures of thymocytes and lymph node cells from the same parental strain yielded graft-vs-host responses in irradiated F₁-hybrids higher than expected by summing the responses of the two cell populations tested separately. A similar synergistic response was obtained using mixtures of thymocytes and lymph node cells obtained from the two parental strains of the hybrid, whereas such an effect was not detected using mixtures of lymph node cells or mixtures of thymocytes from the two parental strains. Nor could synergy be demonstrated between parental strain lymph node cells and thymocytes syngeneic with the bone marrow target cells. Thymocytes obtained from one parental strain which were injected into its irradiated F₁-hybrid transformed into a population of sensitized cells in the spleens of the recipients. This transformation was suppressed by the simultaneous injection of lymph node cells from the second parental strain. Since there is a synergistic immune response by such cell mixtures it is concluded that thymocytes may enhance the graft-vs-host response of lymph node cells. Parental strain thymocytes and lymph node cells, the latter being specifically immunologically tolerant to the bone marrow target cells, failed to give a synergistic response indicating that thymocytes do not transform unresponsive lymphocytes into responsive, but rather enhance the reactivity of existing, specifically responsive cells.The results thus show that thymocytes may enhance the response of lymph node cells in this specific graft-vs-host assay.