Cell survival and radiation induced chromosome aberrations

Summary Existing mathematical formulations to predict the frequency of radiation induced chromosome aberrations in 2nd post-irradiation division are based on the Poisson distribution [3, 4]. Meanwhile several studies have shown that intercellular distributions exist, deviating from Poisson. In the present study a modified model was developed which permits the application of empirical distributions. Transmission and survival parameters of aberrations can be iteratively computed. A general formula was derived for the calculation of cell survival from 1st to 2nd division.     

PHOTOCONTROL OF THE ORIENTATION OF CELL DIVISION IN ADIANTUM. I. EFFECTS OF THE DARK AND RED PERIODS IN THE APICAL CELL OF GAMETOPHYTES

When protonemata of Adiantum capillus-veneris L. which had been grown filamentously under continuous red light were transferred to continuous white light, the apical cell divided transversely twice, but the 3rd division was longitudinal. An intervening period of darkness lasting from 0 to 90 hr either between the 1st and the 2nd cell division or between the 2nd and the 3rd one did not affect the number of protonemata in which the 3rd cell division was longitudinal. The insertion of red light instead of darkness greatly decreased the percentage of 1st longitudinal divisions occurring at the 3rd division, and increased the number of transverse divisions. Fifty percent reduction of induction of 1st longitudinal division was caused by ca. 50 hr exposure to red light between 1st and 2nd division and by ca. 20 hr between 2nd and 3rd division, and total loss was induced by an exposure of ca. 100 hr or longer to red light in the former and by ca. 40 hr longer in the latter. Thus, by using an appropriate intervening dark period or exposure to red light, the orientation and timing of cell division could be controlled in apical cell of the fern protonemata.     

Effects of Radiation,Antibiotics and Alkylating Agents on Cell Division and Growth in the Ciliate Spathidium spathula*

SYNOPSIS. Spathidium spathula is very sensitive to division inhibition after X-irradiation. Five kr delivered to animals 1 hr into the cell cycle prolong the period until the next division to about 2 times the normal length. The next 2 cell cycles, however, are shorter than normal, and by the 4th division irradiated cells have recovered the normal division rate. During this division delay, scanning interference microscopy shows that growth in dry mass continues; at the 1st post-irradiation division the cells average 3 times the normal dry mass. After the 2nd post-irradiation division, dry mass is 1.5 times the normal amount. Dry mass measurements were not made beyond the 2nd division. Giant cells produced by X-rays have enlarged macronuclei, indicating that DNA synthesis is not inhibited by a dose of X-rays that blocks division. Mitomycin C and triethylene melamine, agents which attack or damage DNA, also produce division blockage and giantism in Spathidium. This suggests that damage to DNA in either the macronucleus, the micronucleus or other organelles may be much more effective in delaying cell division than cell growth.     

Postnatal changes in the DNA content of mouse cerebral hemispheres.

Changes in the wet weight of the cerebral hemispheres and in their DNA content and concentration were studied in CBA mice (non-SPF, Velaz, Prague) aged from 1 to 270 days. It was found that hemisphere wet weight rose by 350% between the 1st and 14th day and by a further 11% between the 14th and 180th day. In the next three months it remained stable. The total DNA content rose by 30% between the 1st and 2nd day and by 45% between the 1st and 10th day; changes between the 10th and 180th day were non-significant, but a decrease of 16% occurred by the 270th day. Between the 1st and 2nd day the DNA concentration did not alter, or rose non-significantly (+20%). Towards the end of the 2nd postnatal week it fell exponentially (-75%). Changes in the DNA content and concentration indicate that the rate of cell proliferation in mouse cerebral hemispheres is highest on the first two days after birth, while the general chemical composition of the hemisphere develops fastest between the 2nd and 14th day. The constancy of the DNA content between the 10th and 180th day implies that cell division in the hemispheres of adolescent and adult mice primarily reflects renewal of the non-neuronal cell population.     

Regulation of Cortical Proteins in Euplotes*

SYNOPSIS. A method of isolating cortical organelles from single specimens of Euplotes eurystomus, involving lysis in an induced electric current, is described. The isolation technic was coupled with radioautography to study the patterns of incorporation and conservation of labeled proteins in the membranellar band (MB). Isolated single cells of known age were followed thru one or more divisions. A method of distinguishing between daughter cells (proters and opisthes) at division was utilized in some experiments. The old MB, which is apparently morphostatic, incorporates significant amounts of labeled proteins. The pattern of incorporation in total cell proteins at the 1st and 2nd divisions after pulse-chase indicates that the levels of incorporation among daughter cells is equivalent. However, at the 1st division after pulse-chase, the new (opisthe) MB is generally more heavily labeled than the old (proter) MB, and at the 2nd and 3rd divisions new MBs incorporate less label as their development is farther removed in time from the beginning of the chase. The higher level of incorporation in the MB of the 1st division opisthe is maintained thru subsequent divisions, indicating that in Euplotes proteins of the MB are relatively stable.     

Gamma Irradiation of Stentor polymorphus

SYNOPSIS. Stentor polymorphus was irradiated with ⁶⁰Co gamma rays at 525 rads/minute to examine the effect on survival, cell division, oral membranellar frequency and oxygen uptake. Both survival and cell division were studied on single cells. The LD₅₀ is 285 kilorads but cell division is inhibited below this dose; 110 kilorads doubles the 1st post-irradiation division interval, and a delay of 240 hours occurs at the maximum tolerated dose of 310 kilorads. Conjugating cells are more sensitive, with an LD₅₀ of approximately 40 kilorads.
The frequency of cilia in the membranellar band, measured stroboscopically, is reduced by 30% after 14.5 kilorads, and stopped by higher doses. Recovery has a similar time scale to recovery of fission. A Clarke electrode was used to measure changes in oxygen uptake after irradiation. Depressions of up to 50% were found. Recovery followed a similar pattern to that of ciliary activity and cell division.     

Chromosomal aberrations and sister-chromatid exchanges in tool and die workers

Because tool and die workers are exposed to a number of potentially genotoxic agents, including mutagenic metals, polyaromatic hydrocarbons, and nitrosamines, and may be at increased cancer risk, the present study was undertaken to test whether chromosomal damage in peripheral blood cells is associated with work in the tool and die industry. Lymphocyte cultures were established from 27 tool and die fabrication workers from one manufacturing plant who had worked in the trade for more than 15 years. 15 of these workers also had some form of malignancy at the time of the study, but had not been treated with radiation or chemotherapies that could themselves induce chromosomal damage. The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCEs) were measured in workers and the data compared with those of a control group consisting of 7 non-fabrication workers from the same plant and 8 age-matched community controls. In addition, the relative rates of lymphocyte proliferation were estimated for each group by analyzing the percentages of first-, second- and third-division mitotic cells after 72 h of culture. The results of the chromosomal studies show that tool and die workers have significantly increased frequencies of aberrations whether engaged in fabrication work or not, compared to control subjects. The frequency of SCEs and the frequencies of 1st, 2nd and 3rd division figures are not different among the study groups. Among workers who are engaged in fabrication, including those who are cancer patients, the frequency of more complex aberrations (i.e., interstitial deletions and small acentric fragments) is increased. In a five-year follow-up of these workers, 2 of the 13 workers with these aberrations developed some form of colon cancer. Whether the presence of interstitial deletions and small acentric fragments is related to the occupation of these workers, or is tangentially related to the development of cancer, is presently under consideration.     

Occurrence of 1st division metaphases in human lymphocyte cultures

Summary Human lymphocytes were cultured for 40, 42 and 44h and analysed for the number of 1st and 2nd division metaphases using the BrdU-Giemsa technique. The most suitable time to obtain the majority of cells in their 1st division concomittant with an adequate mitotic index was 42h. It is recommended that laboratories use sister chromatid differential staining to determine the most suitable culture time for the analysis of 1st division metaphases. This will permit accurate comparisons between individuals and laboratories to be made.     

Possible mutagenic action of a tularemia vaccine

No increase in the number of chromosome aberrations in the bone marrow cells of the Wistar rats was observed on the 1st, 2nd, 7th and 15th days after epidermal and intradermal immunization by tularemic live vaccine. Subcutaneous injection of great quantities of tularemic microbic cells which were not used in practice increased the number of cells with chromosome aberrations only on the second day.     

Chromosomal aberrations and sister-chromatid exchanges in Chinese hamster cells treated in vitro with hexavalent chromium compounds.

Chinese hamster cells (CHO line) were treated in vitro for 30--39 h with hexavalent chromium compounds (K2Cr2O7 and Na2CrO7), at concentrations ranging from 0.1 to 1.0 microgram of Cr6+ per ml, in medium containing BUdr. Chromosomal aberrations and sister-chromatid exchanges were scored on BUdr-labelled 2nd division metaphases, collected at the end of treatment and stained with Giemsa. Treatment with mitomycin C 0.009--0.030 microgram/ml) was carried out as a control for the responsiveness of the cell system to chromosomal damage. Both chromium compounds induced marked mitotic delays. Chromosomal aberrations were increased about 10-fold by exposure to Cr6+ (1.0 microgram/ml). The principal aberrations observed were single chromatid gaps, breaks and interchanges, whose frequencies increased proportionally to the concentration of chromium. Dicentric chromosomes, isochromatid breaks, chromosome and chromatid rings were also induced. The frequenyc of sister-chromatid exchanges was hardly doubled 30 h after exposure to Cr6+ at 0.3 microgram/ml, whereas it was trebled 39 h after treatment, in the cells whose division cycle had been slowed down by chromium.     

Analysis of human lymphocyte cell cycle time in culture measured by sister chromatid differential staining

Lymphocyte cell cycle time was measured by the BUdR-Giemsa method for demonstrating sister chromatid differential staining. All 48 h cultures showed metaphases which were in their second division. This finding indicates that the recommended culture time of between 48–54 h for the analysis of 1st division metaphases in lymphocyte cultures is too long, and that a culture time of 38–40 h would be preferable. The 48 h cultures also showed a significantly higher mitotic index than the 72 h cultures suggesting that the continuous incorporation of BUdR may have a toxic effect. The majority of 72 h cultures showed 1st, 2nd and 3rd division metaphases, but there was considerable variation among donors. There was a positive correlation between the number of 2nd division metaphases and the mitotic index.     

Response of X-ray-sensitive CHO mutant cells (xrs-6c) to radiation. II. Relationship between cell survival and the induction of chromosomal damage with low doses of alpha particles

The induction of cytotoxicity, chromosomal aberrations, and sister chromatid exchanges (SCEs) was measured in CHO K-1c cells and in isogenic X-ray-sensitive mutant xrs-6c cells that had been irradiated with X rays and alpha particles in isoleucine-deficient alpha-minimal essential medium in G1 phase of the cell cycle. There was a noticeable shoulder region on the survival curve for CHO K-1c cells irradiated with very low doses of alpha particles, whereas this feature was absent for xrs-6c cells with alpha-particle doses as low as 0.5 cGy. Higher frequencies of chromatid-type aberrations were induced in G1-phase xrs-6c cells than in G1-phase CHO K-1c cells by both gamma- and alpha-particle irradiation. Induction of nonlethal chromosomal aberrations was observed following exposure to 2-6 cGy of alpha particles, doses yielding 97-100% cell survival. Irradiation with 0.5 cGy of alpha particles induced SCE; nearly 60% of irradiated cells contained significantly increased levels of SCE. However, only 3% of the nuclei of cells exposed to 0.5 cGy of alpha-particle radiation were actually traversed by an alpha particle. The observation that a large fraction of cells apparently survive exposure to very low doses of alpha-particle radiation with persistent genetic damage manifested by both chromosomal aberrations and SCEs may have important implications for the carcinogenic hazards of high-LET radiation.     

p53‐dependent G1 arrest in 1st or 2nd cell cycle may protect human cancer cells from cell death after treatment with ionizing radiation and Chk1 inhibitors

Objectives: This study was performed to explore the strategy of combining Chk1 inhibitors with ionizing radiation (IR) to selectively target p53‐deficient cancer cells. Materials and methods: Survival and cell cycle progression were measured in response to IR and the Chk1 inhibitors, UCN‐01 and CEP‐3891, in colon carcinoma HCT116 p53+/+ and p53?/? cells, and in osteosarcoma U2OS‐VP16 cells with conditional expression of dominant‐negative p53 (p53DD). Results: Clonogenic survival was selectively reduced in HCT116 p53?/? compared to p53+/+ cells after treatment with UCN‐01 and IR, and HCT116 p53+/+ cells also displayed strong p53‐dependent G₁ arrest in the 1st cell cycle after IR. In contrast, clonogenic survival was affected similarly in U2OS‐VP16 cells with and without expression of p53DD. However, death of U2OS‐VP16 cells was p53 dependent as assessed by cell viability assay at 72 h, and this was associated with p53‐dependent G₁ arrest in the 2nd cell cycle after treatment. Notably, HCT116 cells were overall more resistant than U2OS cells to cytotoxic effects of Chk1 inhibitors. Conclusion: Our results suggest that p53‐dependent G₁ arrest in both 1st and 2nd cell cycles may protect human cancer cells from cell death after treatment with IR and Chk1 inhibitors. However, a challenge for future clinical use will be that different cancers display different intrinsic sensitivity to such inhibitors.     

Flow cytometric analysis of micronuclei found in cells after irradiation

Exposure of mammalian cells to either ionizing radiation or mutagenic and carcinogenic substances can induce chromosome aberrations. These aberrations in turn may give rise to micronuclei which can be found in cells during the interphase after division. A two-step method is presented that allows separation of micronuclei from cell nuclei. They can then be measured and analysed according to their DNA content in a flow cytometer. The method involves an initial detergent treatment of cells followed by a second treatment with sucrose and citric acid. Micronuclei with DNA content larger than 2% of the G1-nuclei can be measured. The method is tested and compared with microscopic observations of micronucleated cells in irradiated, asynchronous, and synchronized Ehrlich ascites tumour cells growing in vitro. The agreement between the flow cytometric technique and microscopic observations is excellent when the dose-dependent number of micronuclei per cell is taken into consideration.     

Different effects of 1-beta-D-arabinofuranosylcytosine and aphidicolin in S-phase cells--chromosome aberrations, cell-cycle delay and cytotoxicity

Some effects of a 2-h exposure to either aphidicolin (APC) or cytosine arabinoside (ara-C) on S-phase cells of the cell line JU56 have been measured. At a concentration of 1.5 X 10(-5) M of either drug, incorporation of tritiated thymidine into log-phase cultured was reduced by 97-99%. A 2-h exposure to either drug at the same concentration induced chromosome aberrations in cells in S when they subsequently reached mitosis. However, exposure to ara-C induced small numbers of aberrations per damaged cells, and most cells were undamaged. Exposure to APC induced gross chromosomal damage (pulverized chromosomes) in damaged cells. More cells were delayed, and for longer, after exposure to APC than after exposure to ara-C. The results of clonal assays were consistent with the assumption that chromosome aberrations are the proximal cause of reproductive cell death. In the case of ara-C, the results of this and a previous study are consistent with the assumption that cell death and chromosome aberrations are correlated with incorporation of ara-C into DNA in S-phase cells, but that these biological effects manifest themselves only with doses when inhibition of semi-conservative DNA synthesis is greater than 97%.     

Cytogenetic activities of tobacco particulate matter (TPM) derived from a low to middle tar British cigarette

Tobacco particulate matter (TPM) derived from an experimental low to middle tar cigarette was tested for its cytogenetic activity upon a low passage number Chinese hamster pulmonary cell line. Examination of the mitotic profiles (after one cell cycle) revealed no interference by the agent with mitotic spindle formation and/or function. However, complete chromosomes (or parts of them) were seen to dislocate from the mitotic spindles. Such an event was probably the result of the chromosome aberrations, substantial numbers of which were observed in second division cells, or through a process of centromere inactivation. In second division cells there was a reduction in the number of diploid cells accompanied by an increase in both hypodiploidy and polyploidy and there was also a non-dose-related increase in endoreduplication. The results demonstrate that TPM was capable of inducing both structural and numerical chromosome aberrations in cultured mammalian cells.     

The use of harlequin staining to measure delay in the human lymphocyte cell cycle induced by in vitro X-irradiation

Samples of human peripheral blood were given X-ray doses of 1, 2, 3 or 4 Gy at 37°C with a further sample remaining unirradiated. Lymphocytes were then stimulated to divide in cultures containing BrdU for 40–72 h. After harlequin staining the metaphases were recorded as being in their 1st, 2nd or 3rd post-irradiation division. It was confirmed that irradiation delays the proliferation of lymphocytes in culture. A linear relationship between dose and mitotic delay of approximately 1 h per Gray was obtained.This finding of a small effect on cell proliferation is particularly important for biological dosimetry. All in vivo exposures are more or less non-uniform and the lymphocytes in a blood sample therefore possess a spectrum of induced delay characteristics. However, in the great majority of overdose investigations it should not be necessary to increase the normal culture time for the most highly irradiated cells to reach metaphase.The trend towards using harlequin preparations to ensure that only first-division cells are analysed is briefly discussed and it is noted that in this experiment 2nd-cycle metaphases accounted for a maximum of 14% of the cells scored after 48 h in culture.     

Karyologic research on murine B-cell hybridomas

Karyotypes of 10 murine hybridomas producing monoclonal antibodies to microbal antigenes were examined using chromosome slides stained with Azur-eosine. Hybrid origin of all the cell clones was confirmed. The cultures differed from each other in modal chromosome numbers, in novel markers that were absent from cells of the parental myeloma X63.Ag8.653, in the frequency of metaphases with double minute chromosomes and in the level of cells with chromosome aberrations. The results obtained enable us to recommend a cytogenetic analysis for the identification of hybridomas. The following observations point out to a relative instability of the chromosomal apparatus of hybridomas: chromosome numbers varied significantly from cell to cell within one and the same clone; modal chromosome counts decreased in 3 of 5 hybridomas that were studied repeatedly within 1-2 months; in some hybridomas unstable chromosome aberrations were found in 18-38% of cells.     

In vivo and in vitro radioprotective effects of the prostaglandin E1 analogue misoprostol in DNA repair-proficient and -deficient rodent cell systems.

The radioprotective effect of a stable prostaglandin E(1) analogue, misoprostol, was studied in cells from mice with severe combined immunodeficiency (SCID) and in normal cells using X-ray-induced chromosomal aberrations and/or cell killing as the end points. The results clearly show misoprostol-induced radioprotective effects in spermatocytes of the first meiotic division when analyzed for X-ray-induced chromosomal aberrations. The protective effect was independent of Trp53 (formerly known as p53) status. Since spermatocytes are relatively easy to isolate, this appears to be a suitable in vivo model that will allow biochemical studies of the mechanisms involved in radioprotection mediated by misoprostol. Using transfected CHO-K1 cells that stably express a PGE(2) receptor (CPE cells), significant radioprotection mediated by misoprostol from both chromosome breakage and cell death could be demonstrated under in vitro conditions. In addition, evidence was obtained indicating that the degree of radioprotection was dependent on the cell cycle and that S-phase cells were less responsive to misoprostol-mediated radioprotection. These results suggest that CPE cells may be a suitable in vitro model for further studies on the cellular pathways involved in radioprotection by misoprostol in particular and prostaglandins in general.     

DNA polymerase zeta is required for proliferation of normal mammalian cells

Unique among translesion synthesis (TLS) DNA polymerases, pol ζ is essential during embryogenesis. To determine whether pol ζ is necessary for proliferation of normal cells, primary mouse fibroblasts were established in which Rev3L could be conditionally inactivated by Cre recombinase. Cells were grown in 2% O(2) to prevent oxidative stress-induced senescence. Cells rapidly became senescent or apoptotic and ceased growth within 3-4 population doublings. Within one population doubling following Rev3L deletion, DNA double-strand breaks and chromatid aberrations were found in 30-50% of cells. These breaks were replication dependent, and found in G1 and G2 phase cells. Double-strand breaks were reduced when cells were treated with the reactive oxygen species scavenger N-acetyl-cysteine, but this did not rescue the cell proliferation defect, indicating that several classes of endogenously formed DNA lesions require Rev3L for tolerance or repair. T-antigen immortalization of cells allowed cell growth. In summary, even in the absence of external challenges to DNA, pol ζ is essential for preventing replication-dependent DNA breaks in every division of normal mammalian cells. Loss of pol ζ in slowly proliferating mouse cells in vivo may allow accumulation of chromosomal aberrations that could lead to tumorigenesis. Pol ζ is unique amongst TLS polymerases for its essential role in cell proliferation.