Relation between the dynamics of the cell cycle and the frequency of chromosome aberrations in cultured irradiated human lymphocytes

The method of differential staining of sister chromatids was used to study the dynamics of cell divisions at different periods of fixation (48, 56, 68, 80 h) of human lymphocyte cultures in control and after gamma-irradiation in vitro in doses of 150 and 300 rad. It is shown that the cell population of lymphocytes is extremely asynchronous by the time of first and subsequent cell divisions. Administration of gamma-rays before PHA stimulation results in a mitotic delay whose duration is proportional to the irradiation dose. The frequency of radiation-induced chromosome aberrations does not reliably differ in early- and late-dividing cells.     

Evaluation of culture media for effects on cell cycle kinetics and incidence of chromosomal aberrations in human blood cells

Peripheral blood samples from 17 apparently healthy male volunteers were set up in duplicate cultures using three commercially available media: Eagle's MEM, RPMI 1640, and TC 199. BUdR (5-bromo,2-deoxyuridine) (10 micrograms/mL) was added to one of the cultures from each person in each medium after 24 h of culture initiation. All cultures were harvested at 72 h of incubation in the presence of colcemid. RPMI 1640 stimulated the highest mitotic activity in both BUdR-treated and untreated cultures. Higher numbers of first division metaphases corresponded with the higher frequency of chromosome-type aberrations in cultures with Eagle's MEM as compared with RPMI 1640 media. On the other hand, higher numbers of chromatid-type aberrations were present in cultures with TC 199 as compared with those with Eagle's MEM. When the chromosome- and chromatid-type aberration data were pooled to score total cytogenetic abnormalities, an influence of the medium was demonstrable. While cultures with Eagle's MEM and TC 199 had the greater number of first division cells, third of subsequent division cells were most prevalent in RPMI 1640 cultures. It is inferred that the length of the cell cycle, the mitotic index, and to some degree the incidence of spontaneous cytogenetic abnormalities are variable attributes of culture media.     

A simple and convenient method for gaining pure populations of lymphocytes at the first mitotic division in vitro.

This paper describes a simple method for obtaining pure populations of human lymphocytes at the first in vitro mitotic division (M-1) by continuous treatment with colchicine or colcemid. When colchicine (7.2 x 10(-8) M) or colcemid (4.8 x 10(-8) M) was added to cultures 0-24 h after initiation, cultures harvested at 54 h had more than 99.9% of mitotic cells in the first metaphase (M-1). We showed that not only more analyzable M-1 cells may be obtained, but staining for sister-chromatid differentiation may be avoided.     

Aneugenic effect of sodium arsenite on human lymphocytes in vitro: an individual susceptibility effect detected

Arsenic is a well known carcinogenic environmental pollutant although its mechanism of action remains unknown. Since alterations in chromosome segregation have been observed in individuals exposed to high concentrations of arsenic in the drinking water, the aneuploidogenic potential of arsenic was evaluated in vitro. Whole blood cultures were incubated for 72 h and treated with various concentrations of sodium arsenite for the last 24 h. Cells were harvested and samples were processed specially for aneuploidy evaluation. The number of chromosomes in 200 metaphases of first and second division cells was scored. A dose-related effect was observed: the highest concentration (10⁻² μM) induces 28.33% and 22.4% hyperploid cells in first and second division respectively and 29% tetraploid cells. The colchicine-like effect of arsenic was also evaluated. Mitotic arrest was evaluated in cultures treated for the last 2 h. Sodium arsenite can produce 40.24% and 12.93% of the colcemid effect (mitotic arrestant effect at 10⁻² μM and 10⁻¹⁰ μM respectively). A different individual susceptibility effect was observed in both parameters and confirmed with the chromosome aberrations levels induced by arsenic in the same donors. Data indicate that sodium arsenite has an aneuploidogenic and a mitotic arrestant effect.     

G2 cell cycle arrest induced by glycopeptides isolated from the bovine cerebral cortex

The ability of glycopeptides, isolated from bovine cerebral cortex, to alter cell division was studied by cell-cycle analyses. The results showed that glycopeptides arrested baby hamster kidney (BHK)-21 cells and Chinese hamster ovary (CHO) cells in the G2 phase of the cell cycle. Upon removal of the growth inhibition from arrested BHK-21 cells, the mitotic index in colchicine-treated cultures increased from 5 to 40% within 6 h and the increase in mitotic activity was accompanied by a complete doubling of all arrested cells within this 6- h time period. Determination of DNA content in growth-arrested BHK-21 cells showed that growth-arrested cells contained about twice the DNA of control cell cultures. Although CHO cells treated in a like manner with growth inhibitor could not be arrested for the same length of time as BHK-21 cells (18 h vs. 72 h before initiation of escape) and to the same degree (60% of the cell population vs. 99% of BHK-21 cells), the escape kinetics of CHO cells did indicate a G2 arrest. Approximately 3.5 h after escape began, CHO cell numbers in treated cultures attained the cell numbers found in control cultures. This rapid growth phase occurring in less than 4 h indicated that the growth inhibitor induced a G2 arrest-point in CHO cells that was not lethal since the entire arrested cell population divided.     

Time-Lapse Cinemicrographic Studies of X-Irradiated HeLa S3 Cells: II. Cell Fusion

Analysis of time-lapse cinemicrographs of X-irradiated HeLa S3 cells has shown that the incidence of cell fusion was increased from 0.9% (following 1267 divisions) in control cells to an average of 22% (following 655 divisions) in cells irradiated with 500 rad doses of 220 kv X-rays. The incidence depended on the stage of the generation cycle at which the parent cells were irradiated. It was nearly constant in the first three postirradiation generations. Fusion occurred at all stages of the generation cycle, but preferentially during the first 20%. Cells undergoing fusion progressed more slowly through the generation cycle and had a higher probability of disintegrating than did irradiated cells that did not fuse. The occurrence of fusion was clonally distributed in the population. It took place only between sister (or closely related) cells. Protoplasmic bridges were often visible between sister cells prior to fusion. Giant cells arose only as a result of fusion. The incidence of multipolar divisions, though higher than in unirradiated cells, was only 5.5% in cultures irradiated with 500 rads. Fusion occurred following 85% of the multipolar divisions and was often followed by a multipolar division.     

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.     

Sister-chromatid exchanges and cell-cycle kinetics in human lymphocyte cultures exposed to alkylating mutagens: apparent deformity in dose-response relationships

Experiments have been carried out using human whole-blood cultures to determine the effects of sampling times and of the duration of 5-bromodeoxyuridine (BrdUrd) treatment before fixation on sister-chromatid exchange (SCE) frequencies following exposure to mitomycin C (MMC). Cells were pulse treated for 1 h with 3 X 10(-6) M MMC at G1, and then sampled at 4-h intervals up to 88 h after stimulation of cultures with phytohemagglutinin (PHA). Results showed that this MMC treatment induced a 5-6 h proliferation delay per cell cycle, and that SCE frequencies first increased with time of fixation, peaking at 68 h, and then decreased. When cells were similarly treated with MMC, but subsequently exposed to BrdUrd for various times before fixation of cultures at 72 h, the SCE frequencies markedly increased with increasing durations of BrdUrd incubation times. These data indicate that, in mutagen-treated cultures, lymphocytes having relatively longer cell-cycle times show a higher mean frequency of SCEs. In a subsequent experiment, cells were treated for 1 h with increasing doses of MMC or 4-nitroquinoline 1-oxide (4NQO) at 0, 24, or 48 h, and then fixed at 72 h after PHA stimulation. Results showed that the optimal treatment times at which the agents could most efficiently produce SCEs were different for MMC and 4NQO, and that the dose-response curves tended to 'bend down' at very high doses; that is, treatments with very high doses induced smaller than expected numbers of SCEs. However, cells similarly treated with very high doses showed a higher, expected frequency of SCEs when sampled at 84 h, but again had a lower than expected SCE frequency when fixed at 96 h. The results indicate that there is an optimal time for sampling at which one can observe the maximum increase in SCE frequencies following mutagen exposure, and strongly suggest that the higher the dose, the later the optimal sampling time. Because of the apparent deformity of dose-response curves obtained after various treatments and sampling times, it seems necessary that extra fixation-time points be included in test protocols so as to avoid false negatives or confirm possible positives.     

Kinetics of division of human and rabbit lymphocytes stimulated by phytohemagglutinin, studied by means of the bromodeoxyuridine (BUDR) technic]

The BUDR-Giemsa technique has been used to distinguish the first from later divisions for man and rabbit lymphocytes stimulated by treatment with phytohemagglutinin. At 48 hours, 99% of human lymphocytes exposed to 200 rads of X-rays are in first division whereas 45% of the rabbit lymphocytes are already in second division and 28% in third division.     

Perturbation of cell division by acrylamide in vitro and in vivo

Exposure of V79 Chinese hamster cells to acrylamide (AA) caused a concentration-dependent increase in the incidence of spindle disturbances. A c-mitotic effect with the appearance of C-metaphases, a mitotic block and the concomitant disappearance of ana-telophase figures, was observed after 6 h of treatment with concentrations ranging from 0.01 to 1.0 mg/ml of AA.Intraperitoneal injection of male mice with the highest tolerated dose of 120 mg/kg of AA showed no mitotic arrest in bone marrow cells. However, 1 h and 3 h after treatment the frequencies of cells with highly condensed and separated chromatids was reduced indicating an effect on mitotic progression.In spermatocytes of mice AA caused a meiotic delay from 2 h to 22 h after treatment determined by a reduced ratio of second/first meiotic divisions. The meiotic delay was predominantly due to a prolongation of interkinesis.The present results show that AA causes disturbances of cell division in vitro and in vivo. They suggest that AA might induce aneuploidy in mammalian cells in vitro by interfering with proper functioning of the spindle similar to the effect of colchicine. In vivo, particularly in spermatocytes, the progression of cell division was altered by AA. It cannot be explained simply by an effect on spindle function however, this alteration may also cause errors in chromosome segregation.     

Cell division induced by mechanical stimulation in starved Tetrahymena thermophila: cell cycle without synthesis of macronuclear DNA

Starved Tetrahymena thermophila cells underwent synchronous cell division 2 h after a mechanical stimulation. The macronucleus showed no obvious increase in DNA content before the cell division in the starvation medium, and the DNA content was decreased after the cell division. On the other hand, when the starved cells were given nutrient-supplied medium immediately after the mechanical stimulation, cell division was delayed for 3 h. This period was almost the same as that for G1 cells in the stationary culture to first division after transfer to fresh nutrient medium. These results suggest that the mechanical stimulation induces an early division of starved cells, skipping the macronuclear S-phase with the starved cells probably becoming trapped in G1. Starved cells that had finished division soon formed mating pairs with cells of the opposite type. These observations lead us to propose that cell division in starvation conditions may be necessary to reduce macronuclear DNA content prior to the mating of T. thermophila.     

Phase response versus positive and negative division delay in animal cells

The time of median cell division in V79 Chinese hamster cells following high serum pulses was determined for two synchronous cell generations following mitotic selection. Differences in cell cycle time for each pair of pulse and control cultures were computed and plotted as a function of time of serum pulse. This phase response curve for hamster cells with an 8.5 h cell cycle shows a characteristic biphasic pattern. Beginning 0.5 h after mitotic selection, pulses with serum produce delays in the midpoint of the subsequent mitotic waves. Delay is maximum at 1.5 h. Delays give way abruptly to advances at 2.5 h and the amount of advance then decreases as pulses are given between 3 and 5 h into the cycle. At 5 h decreasing advances become delays, with increasing delays due to serum pulses occurring between 5 and 6 h. Delays again give way abruptly to advances at 6 h and again the amount of advance decreases through the late portion of the cycle. Pulses very late in the cycle appear to generate phase delays. This biphasic response to serum is interpreted as an expression of an underlying time-keeping oscillator whose period is nominally of 4 h duration.     

Influence of mitotic delay on the results of biological dosimetry for high doses of ionizing radiation

The purpose of this study was to systematically investigate how high doses of sparsely and densely ionizing radiations influence the proliferation time of lymphocytes in short-term cultures and, consequently, the observed frequencies of dicentric and centric ring chromosomes. Peripheral blood samples from five volunteers were irradiated with high doses of 200 kV X-rays and with neutrons with a mean energy of <E _n>=2.1 MeV. First division metaphase cells were collected after different culture times of 48, 56, and 72 h and dicentrics, centric ring chromosomes, and acentric fragments were determined. The data hint at considerable mitotic delay. The main increase in the number of chromosome aberrations occurred between 48 and 72 h after an X-ray exposure and between 56 and 72 h after neutron exposure. When the data were used for a calibration of aberration frequency versus dose, subsequent dose estimations resulted, however, in comparable values. Thus, in spite of the influence of mitotic delay on observable chromosome aberrations, at least for the radiation types investigated here, a culture time of 48 h is acceptable for biological dosimetry.     

Rate of progress of the 1st post-stimulation division of the mitotic cycle by cells of ascitic hepatoma 22A of different ages]

A study was made of the progress rate of cells of the ascitic hepatoma 22A of different age during the iirst mitotic cycle after the stimulation of division. The "ageing" (11-day), terminal (14-day), and "delayed" (4 days older than the terminal stage) ascitic fluids were used. The maximal values of the labeled nuclei index was found to be reached by 9--12 hours (it was mainly due to the transtion of the quiescent to the S-period) and the maximal mitotic index--by 18--21 hours after the inoculation, independently of the tumour age. These results suggest that the duration of both the prereplicative (G1) period and of the whole first mitotic cycle after the stimulation were independent of the time during which the cells of the ascitic hepatoma 22A were at the resting stage or at the very prolonged G1-period.     

Chromosome constitution and cell division in in vitro cultures ofZea mays L. endosperm

Summary Cultures of maize (Zea mays L.) endosperm grown in vitro for over 3 years were examined cytologically. Conditions of aneuploidy and polyploidy were noted. Chromosome numbers ranged from 21 to over 200, with 30 to 60 being observed most often. Although a few extra large cells with polyploid nuclei were scattered throughout the smear preparation, a large proportion of the interphase nuclei appeared similar in volume and probably contained a near normal complement of chromosomes. Anaphase bridges were the most commonly observed chromosome aberration. No cell divisions were observed the first 24 hr after transfer. From 2 to 8 days after transfer the proportion of cells in division was relatively constant with a mitotic index of approximately 5.5%. The proportion of cells in division began to decline 8 days after transfer and in the final sample taken after 13 days only 2.6% of the cells were in division. Examples of localized synchrony were observed and mitotic indices for individual cell clumps ranged from 0 to 17%. Authorized for publication on October 16, 1973 as paper number 4552 in the Journal Series of the Pennsylvania State Agricultural Experiment Station.     

A quantitative theory of liver regeneration in the rat. II: matching an improved mitotic inhibitor model to the data

A model of liver regeneration is put forward in which the rate of liver growth is controlled both by a liver-produced mitotic inhibitor and by the availability of parenchymal cells to enter the mitotic cycle. The model can be expressed as a pair of coupled differential equations, the first describing the dependance of inhibitor concentration on liver size and inhibitor decay and the second specifying the dependance of liver growth on inhibitor concentration and entry of cells into the mitotic cycle. The model is tested by comparing its solutions to the published data on mitotic indices following partial hepatectomy. For such a comparison, it is necessary to specify the cell-cycle time and the inhibitor dose-response function and half-life. If a negative exponential dose-response function, an inhibitor half-life of 11·4 h, and a cycle time of 18·25 h are postulated, the solutions match the data of Fabrikant (1968) who found that there were two waves of mitosis with a period of quiescence between them. The data of Grisham (1962), characterized by a single peak of mitosis, is matched by the theory using similar inhibitor properties but a shorter cell-cycle time (13·25 h); this causes the two peaks to overlap. In both cases, a better fit is obtained if the second cell cycle is longer than the first by 2–3 h. This suggests that cells enter a G₀ period after mitosis. A mechanism for littoral cell division, which occurs some 24 h after parenchymal cell division, is put forward in which the former cells depend on the enlargement of the latter for the stimulus to divide.     

Chromosome damage and micronucleus formation in human blood lymphocytes exposed in vitro to radiofrequency radiation at a cellular telephone frequency (847.74 MHz, CDMA)

Peripheral blood samples collected from four healthy nonsmoking human volunteers were diluted with tissue culture medium and exposed in vitro for 24 h to 847.74 MHz radiofrequency (RF) radiation (continuous wave), a frequency employed for cellular telephone communications. A code division multiple access (CDMA) technology was used with a nominal net forward power of 75 W and a nominal power density of 950 W/m(2) (95 mW/cm(2)). The mean specific absorption rate (SAR) was 4.9 or 5.5 W/kg. Blood aliquots that were sham-exposed or exposed in vitro to an acute dose of 1.5 Gy of gamma radiation were included in the study as controls. The temperatures of the medium during RF-radiation and sham exposures in the Radial Transmission Line facility were controlled at 37 +/- 0.3 degrees C. Immediately after the exposures, lymphocytes were cultured at 37 +/- 1 degrees C for 48 or 72 h. The extent of genetic damage was assessed from the incidence of chromosome aberrations and micronuclei. The kinetics of cell proliferation was determined from the mitotic indices in 48-h cultures and from the incidence of binucleate cells in 72-h cultures. The data indicated no significant differences between RF-radiation-exposed and sham-exposed lymphocytes with respect to mitotic indices, frequencies of exchange aberrations, excess fragments, binucleate cells, and micronuclei. The response of gamma-irradiated lymphocytes was significantly different from that of both RF-radiation-exposed and sham-exposed cells for all of these indices. Thus there was no evidence for induction of chromosome aberrations and micronuclei in human blood lymphocytes exposed in vitro for 24 h to 847.74 MHz RF radiation (CDMA) at SARs of 4.9 or 5.5 W/kg.     

Inter- and intra-chromosomal distribution of chromatid breaks induced by X-rays during G2 in human lymphocytes

Cultures of blood from healthy adults were irradiated 48 h after stimulation with 240 R of X-rays and fixed after various time intervals (0–2 h, 2–4 h, 4–6 h). ³HTdR was added to several cultures after irradiation. Mitotic and labelling indices were used to distinguish between two cell samples inside the irradiated G₂ population: D − cells reaching mitosis without mitotic delay and a high frequency of chromatic breaks and D + cells with mitotic delay and which, during the delay, repair most of the damage produced. After R banding 450 chromatid deletions were located in each of the two cell samples. The D + cells showed a higher frequency of breaks than the D − cells with decreasing chromosome size, in the telomeric and centromeric region and in the junction between the R + and R − bands. These results can be interpreted as indicative of a non-random distribution of repair processes both between and within chromosomes.