Effect of caffeine on in vivo processing of alkylated bases in proliferating plant cells.

DNA damage was induced by either 2 mM ethylmethanesulfonate or 1 Gy of gamma-irradiation in Allium cepa L. root meristems. The percentage of DNA that migrated towards the anode during microelectrophoresis after alkali denaturation (pH approximately 13.5) of the isolated nuclei (comet assay) reflects the amount of single strand breaks present in them. There was some DNA migration (12.8+/-2.4%) in untreated roots. This percentage doubled at the end of 1.5 h treatment with the mono-functional alkylating agent 2 mM ethylmethanesulfonate, and trebled after a single exposure to 1 Gy of gamma-rays. A proportion of the DNA migration caused by these two treatments was reversed (repaired) by a 2 h long period of in vivo recovery. However, when 5 mM caffeine was applied after removal of the alkylating agent, the amount of DNA migrating to the comet tail over the same 2 h period was almost double that at the onset of recovery. In both control and irradiated nuclei, caffeine also increased the initial level of DNA migration in the comet assay, but to a lesser extent. These results indicate that caffeine increases the DNA damage that accumulates during the processing of alkylated bases and, to a lesser extent, of the DNA bases damaged by gamma-irradiation. Thus, the potentiation effect of caffeine on induced chromosomal damage may not just be due to caffeine-induced cancellation of the G2 checkpoint, but also to a direct effect this methylxantine has on the processing of DNA damage.

Ataxia telangiectasia: G2 checkpoint and chromosomal damage in proliferating lymphocytes.

There is a checkpoint pathway in eukaryotic cells that depends on ATM (ataxia telangiectasia mutated) kinase which activates the processes leading to the repair of DNA damage and also lengthens the G(2) stage of the cell cycle. In cells from ataxia telangiectasia patients, due to their lack of active ATM kinase, an increase in chromosomal aberrations and a failure to induce G(2) lengthening could be expected. However, the basal G(2) timing in ataxia telangiectasia cells was longer than in controls and was further extended after X-ray irradiation (0.4 Gy), although to a lesser extent than in controls. Moreover, in control cells caffeine shortened G(2) and increased chromosomal damage 7-fold, while in ataxia telangiectasia cells caffeine only trebled aberration yield without shortening G(2). As caffeine is an inhibitor of ATM kinase, these results suggest the existence of some redundant ATM-independent checkpoint in G(2) of ataxia telangiectasia cells. The differential response to caffeine of ataxia telangiectasia and control lymphocytes may be explained by the presence of two different subpathways in the G(2) checkpoint: one regulating the processing and repair of damaged DNA and the other controlling G(2) timing. While in controls both subpathways may be mediated by ATM kinase, in ataxia telangiectasia cells caffeine-sensitive ATR kinase and the caffeine-insensitive DNA-PK kinases might be responsible for DNA repair and the G(2) delay subpathways, respectively. Confirmation of this model in ataxia telangiectasia cells with another cell type in which both subpathways are mediated by DNA-PK should define whether a metylxanthine such as caffeine may also have an additional direct inhibitory effect on DNA repair.

Effect of vitamin E on chromosomal aberrations in lymphocytes from patients with Down's syndrome.

A possible protective effect of vitamin E (DL-alpha-tocopherol) on chromosomal damage was evaluated in lymphocytes from patients with Down's syndrome (DS) and from controls. This included the analysis of the basal and G2 chromosomal aberration frequencies in lymphocytes cultured with and without 100 microM vitamin E. The chromosomal damage in G2 was determined by scoring the number of chromosomal aberrations in lymphocyte cultures treated with 5 mM caffeine, 2 h before harvesting. Vitamin E treatment decreased the basal and G2 chromosomal aberrations both in control and DS lymphocytes. In DS cells, this protective effect, expressed as a decrease in the chromosomal damage, was greater (50%) than in controls (30%). These results suggest that the increment in basal and G2 aberrations yield in DS lymphocytes may be related to the increase in oxidative damage reported in these patients.

G2 repair and chromosomal damage in lymphocytes from workers occupationally exposed to low-level ionizing radiation.

The effect of the G2 repair of chromosomal damage in lymphocytes from workers exposed to low levels of X- or gamma-rays was evaluated. Samples of peripheral blood were collected from 15 radiation workers, 20 subjects working in radiodiagnostics, and 30 healthy control donors. Chromosomal aberrations (CA) were evaluated by scoring the presence of chromatid and isochromatid breaks, dicentric and ring chromosomes in lymphocytes with/without 5 mM caffeine plus 3 mM-aminobenzamide (3-AB) treatment during G2. Our results showed that the mean value of basal aberrations in lymphocytes from exposed workers was higher than in control cells (p < 0.001). The chromosomal damage in G2, detected with caffeine plus 3-AB treatment was higher than the basal damage (untreated conditions), both in control and exposed populations (p < 0.05). In the exposed workers group, the mean value of chromosomal abnormalities in G2 was higher than in the control (p < 0.0001). No correlation was found between the frequency of chromosome type of aberrations (basal or in G2), and the absorbed dose. Nevertheless, significant correlation coefficients (p < 0.05) between absorbed dose and basal aberrations yield (r = 0.430) or in G2 (r = 0.448) were detected when chromatid breaks were included in the total aberrations yield. Under this latter condition no significant effect of age, years of employment or smoking habit on the chromosomal aberrations yield was detected. However, analysis of the relationship between basal aberrations yield and the efficiency of G2 repair mechanisms, defined as the percentage of chromosomal lesions repaired in G2, showed a significant correlation coefficient (r = -0.802; p < 0.001). These results suggest that in addition to the absorbed dose, the individual G2 repair efficiency may be another important factor affecting the chromosomal aberrations yield detected in workers exposed to low-level ionizing radiation.

G2 repair and chromosomal damage in lymphocytes from workers occupationally exposed to low-level ionizing radiation

The effect of the G2 repair of chromosomal damage in lymphocytes from workers exposed to low levels of X- or g-rays was evaluated. Samples of peripheral blood were collected from 15 radiation workers, 20 subjects working in radiodiagnostics, and 30 healthy control donors. Chromosomal aberrations (CA) were evaluated by scoring the presence of chromatid and isochromatid breaks, dicentric and ring chromosomes in lymphocytes with/without 5mM caffeine plus 3mM-aminobenzamide (3-AB) treatment during G2. Our results showed that the mean value of basal aberrations in lymphocytes from exposed workers was higher than in control cells (p< 0.001). The chromosomal damage in G2, detected with caffeine plus 3-AB treatment was higher than the basal damage (untreated conditions), both in control and exposed populations (p< 0.05). In the exposed workers group, the mean value of chromosomal abnormalities in G2 was higher than in the control (p< 0.0001). No correlation was found between the frequency of chromosome type of aberrations (basal or in G2), and the absorbed dose. Nevertheless, significant correlation coefficients (p< 0.05) between absorbed dose and basal aberrations yield (r = 0.430) or in G2 (r = 0.448) were detected when chromatid breaks were included in the total aberrations yield. Under this latter condition no significant effect of age, years of employment or smoking habit on the chromosomal aberrations yield was detected. However, analysis of the relationship between basal aberrations yield and the efficiency of G2 repair mechanisms, defined as the percentage of chromosomal lesions repaired in G2, showed a significant correlation coefficient (r = -0.802; p< 0.001). These results suggest that in addition to the absorbed dose, the individual G2 repair efficiency may be another important factor affecting the chromosomal aberrations yield detected in workers exposed to low-level ionizing radiation

In vivo response of mouse liver to gamma-radiation assessed by the comet assay.

The alkaline comet assay was used to measure DNA damage induced in liver cells of mice irradiated with gamma-radiation, as well as the repair competency of these cells. A simplified procedure for the isolation of nuclei from cells in solid tissues was developed. This simplified method allows nuclei to be processed into lysis only 5 min after briefly chilling the tissue to depress any enzymatic activity. The nuclei were spontaneously released by a sharp cut of the tissue and exposure of the cut to a drop of 50 mM sodium-phosphate buffer at pH 7.2, immediately before adding the low melting agarose. Thus, the procedure minimizes time-dependent modification of the endogenous level of damage by reducing additional strand breaks or repair produced during processing. The induction of DNA damage by gamma-radiation behaved as a one-hit event in the liver cells, as there was a positive linear correlation between the radiation dose and the fraction of DNA migrated into the comet tails. The level of DNA damage produced by gamma-radiation was highly significant at doses of 0.5 and 1 Gy. Based on the mean extent of DNA migration, the level of damage was not reduced following only one hour of repair time however, after two hours, there was a significant reduction in DNA migration. To increase the resolution of the statistical analysis, the nuclei of each sample were distributed in five types of comets, according to the percentage of DNA in the tail. To compare the frequency distributions of these types of comets between different experimental situations, a Pearson chi-square statistical analysis was applied. It was found, by this analysis, that the DNA repair which occurred 1 h after 1 Gy of gamma-irradiation is significant and that, after 2 h, more DNA repair occurs, but a significant residual damage still persists when comparing this sample with the control.

The positional control of mitosis and cytokinesis in higher-plant cells.

The present work establishes a correlation between cell length and patterns of mitotic microtubular assemblies in Allium cepa L. root meristems. Binucleate cells were formed by a short caffeine treatment which aborted the formation of the phragmoplast during telophase. The largest binucleate cells (about 50 microns in length) behaved as two contiguous mononucleate cells in their next mitosis: they developed two preprophase bands (PPBs), one around each nucleus, where two spindles and two phragmoplasts were subsequently formed. On the other hand, the shortest binucleate cells (about 36 microns in length) formed a single PPB at the site of the aborted phragmoplast and, in the medium-sized cells (about 44 microns) in which the single PPB formed around the nucleus possessing the largest cytoplasmic environment, the two mitotic spindles and the new phragmoplasts moved to, or were assembled in the position of the phragmoplast that had been aborted one cycle earlier. Some rules derive from these observations. First of all, the aborted phragmoplast left a signal for microtubule positioning which was still operative one cycle later, in two-thirds of the bimitoses. Also, that formation of the PPB is dispensable. Moreover, its development does not always predict the future division plane, because of the presence of competing old signals which are stronger than those shed by the PPB in the same mitosis, but which fade away with distance. Finally, the positional signals were reinforced when the ratio of monomeric to fibrillar actin was increased by cytochalasin D during their shedding. When this drug was given simultaneously with caffeine, the frequency of bimitoses which, one cycle later, developed spindles and phragmoplasts in the positions of the old phragmoplast increased. On the other hand, those frequencies dropped in relation to control when the cytochalasin D treatment took place during bimitosis, indicating that at this time the treatment reinforced the signals produced in bimitosis itself.

A fast comet assay variant for solid tissue cells. The assessment of DNA damage in higher plants.

The single-cell gel electrophoresis or comet assay, under high alkaline conditions, detects low levels of DNA damage. In it, broken DNA migrates from the nucleus to the anode providing images similar to comets. To adapt this assay to solid tissue cells, nuclei were directly obtained from Allium cepa L. roots. The surface of each single fresh sharply cut meristem was exposed to a small drop of 50 mM Sörensen buffer at pH 6.8, placed on a regular agarose-coated slide. By immediately adding low melting point agarose at 30 degrees C, nuclei resulted embedded in agarose. A final layer of this agarose ended the preparative steps. Conventionaly prepared leukocytes were used as a control. The treatment with detergent (lysis step of the conventional assay) proved to be unnecessary for the nude nuclei. A 20 min-long electrophoresis (at 0.65 V. cm-1, 230 mA and 10 degrees C) was more sensitive than a 10 min-long one for detecting the differential response of plant nuclei to 2 and 4 Gy of gamma-irradiation. A short fixation in methanol transformed the preparations into semi-permanent ones, without altering their later DNA staining by ethidium bromide. The use of instantaneously isolated nuclei simplifies and expands the use of this technique to any eukaryotic cell from solid tissues.

G2 repair and evaluation of the cytogenetic damage induced by low doses of X-irradiation during G0 in human lymphocytes.

In the present study two cytogenetic parameters were used to evaluate the DNA damage induced by low doses (1 up to 40 rad) of X-ray irradiation in G0 human lymphocytes. These parameters were the frequency of chromosomal lesions in G2 and the length of this cell cycle phase. The frequency of chromosomal lesions in G2 was determined by scoring the number of chromosomal aberrations in G0 irradiated lymphocytes post treated with two inhibitors of G2 repair mechanisms: caffeine and 3-aminobenzamide. A dose-dependent increase in chromosomal aberrations yield was detected in G0 lymphocytes X-ray irradiated with or without post treatment with these two DNA repair inhibitors during G2. Nevertheless, the dose response in this latter condition was higher than the one detected in control cells, indicating that the increase of irradiation dose in G0 lymphocytes produces an increment in the number of DNA lesions arriving to be repaired in G2. The analysis of the dose-response relationships for G2 length showed an statistically significant X-ray dose-dependent increase (G2 delay) from 2.5 up to 40 rad and a positive correlation between G2 delay and the frequency of chromosomal lesions in G2. These results suggest that the DNA lesions induced by low doses of X-irradiation in G0 lymphocytes may be higher than that detected by the standard method (control conditions) and may be responsible for an increase in G2 length. We propose, therefore, that an analysis of these two cytogenetic parameters can improve the evaluation of the DNA damage induced by low doses of X-rays irradiation in G0 cells.

G2 repair and evaluation of the cytogenetic damage induced by low doses of X-irradiation during G0 in human lymphocytes [published erratum appears in Biol Res 1996; 29(1): 165]

In the present study two cytogenetic parameters were used to evaluate the DNA damage induced by low doses (1 up to 40 rad) of X-ray irradiation in G0 human lymphocytes. These parameters were the frequency of chromosomal lesions in G2 and the length of this cell cycle phase. The frequency of chromosomal lesions in G2 was determined by scoring the number of chromosomal aberrations in G0 irradiated lymphocytes post treated with two inhibitors of G2 repair mechanisms: caffeine and 3-aminobenzamide. A dose-dependent increase in chromosomal aberrations yield was detected in G0 lymphocytes X-ray irradiated with or without post treatment with these two DNA repair inhibitors during G2. Nevertheless, the dose response in this latter condition was higher than the one detected in control cells, indicating that the increase of irradiation dose in G0 lymphocytes produces an increment in the number of DNA lesions arriving to be repaired in G2. The analysis of the dose-response relationships for G2 length showed an statistically significant X-ray dose-dependent increase (G2 delay) from 2.5 up to 40 rad and a positive correlation between G2 delay and the frequency of chromosomal lesions in G2. These results suggest that the DNA lesions induced by low doses of X-irradiation in G0 lymphocytes may be higher than that detected by the standard method (control conditions) and may be responsible for an increase in G2 length. We propose, therefore, that an analysis of these two cytogenetic parameters can improve the evaluation of the DNA damage induced by low doses of X-rays irradiation in G0 cells

Defective G2 repair in Down syndrome: effect of caffeine, adenosine and niacinamide in control and X-ray irradiated lymphocytes.

Lymphocytes from both Down syndrome (DS) patients and age-matched control donors have been investigated to identify a possible disturbance in chromosomal G2 repair. Analyses of caffeine treatments during G2 have shown that the frequency of chromosomal aberrations is higher in DS lymphocytes than in normal lymphocytes. Likewise, G2 duration is longer in DS cells than in normal cells. In both control and DS lymphocytes, caffeine treatments increase the frequencies of chromatid breakages and decrease the average of G2 duration. The reversal of the caffeine potentiation effect by adenosine and niacinamide is higher in DS cells than in normal cells. Furthermore, ATP content per cell in DS lymphocytes is one third of that estimated in normal lymphocytes. The increase of ATP level produced by adenosine or niacinamide generally correlates with the reversal of the caffeine effect on chromosome aberrations. Under the experimental conditions tested, a good negative exponential correlation between ATP level and chromosome aberrations has been detected in both normal and DS lymphocytes which were or were not X-irradiated. Finally, we postulate a decrease in G2 repair capability of DS lymphocytes caused by a low availability of ATP and/or some other factor correlating with it.

G2 repair and aging: influence of donor age on chromosomal aberrations in human lymphocytes.

The caffeine effects on chromosomal aberration frequency and mean G2 duration were studied in human lymphocytes in vitro from three age groups of normal donors (I: 1-5 years old; II: 30-40 years old; III: 60-70 years old). Under control conditions, the three age groups showed a similar frequency of chromosomal aberrations. All three age groups exhibited a linear dose response for aberrations with caffeine treatments. However, lymphocytes from aged individuals (groups II and III) showed higher chromosomal aberration frequencies and longer G2 duration than cells from young individuals (group I). The caffeine effect in reducing G2 length was rather similar in every age group. The reversion of caffeine effects by adenosine or niacinamide in lymphocytes from older individuals was higher than in cells from group I. The different caffeine effects and G2 values between lymphocytes from old and young individuals are most likely due to a higher number of DNA lesions reaching G2 phase and/or a decrease of the G2 repair capability of lymphocytes from older individuals.

The probability of G1 cells to enter into S increases with their size while S length decreases with cell enlargement in Allium cepa.

The distribution of cell surface area projection (cell size) has been measured at birth and at initiation of DNA synthesis in steady-state populations of Allium cepa root meristems. The conditional probability, P(I/G1), that initiation occurs given that the event of being in G1 also occurs has been estimated from these data. P(I/G1) was found to increase when cells became larger. The distribution of G1 duration has been constructed from indicated cell size distributions. The absolute frequencies of G1 times showed a maximum in the zone of cells with short G1 periods; about 14% of cells appear to enter into S with G1 congruent to 1 h. These results suggest that the increase of P(I/G1) was due to cell enlargement and not to cell aging. By comparing the cell size distribution at initiation of S and at the end of this period, a drastic reduction of cell size variability during DNA replication was observed and both curves were seen as rather similar in shape although they obviously had different modal points. These observations support that there is a negative correlation between the initiation size and the duration of genome duplication, and that cells which initiate DNA synthesis with the same size have a similar replication time. From this hypothesis, a plot of S duration versus cell size at initiation of this period was constructed by comparing the distributions of cell size at start and end of replication; this plot was also consistent with the existence of a negative correlation between cell initiation size and S length.

Cell size of proliferating plant cells increases with temperature: implications in the control of cell division.

As chemical reactions related to the regulation of cell proliferation are governed by availability, amount, and concentration of relevant molecules, it has been suggested that cell size is an important factor in the control of cell cycle. We have measured the size of proliferating cells of Allium cepa roots in which growth rate was modified by changes in growth temperature. Two independent cell size parameters have been measured by cytophotometry: cell surface area projection and cell protein content. Average cell sizes of both the proliferating cell population and the subpopulation at the end of mitosis show that cell size increases with growth rate. Calculation of cell size at initiation of DNA replication clearly indicates that average cell size at this point is not growth invariant but positively correlated with growth rate.

Exponential pattern of cell age distribution in dividing cells of plant meristems.

In order to determine the pattern of cell age distribution in proliferating cells of Allium cepa roots we have measured by cytophotometry two cell size parameters, protein content and surface area projection, in cells that correspond to the entire proliferating population or only to the ana-telophase subpopulation. The size values of ana-telophase cells have been employed to construct theoretical size distributions for the entire proliferating cell population of the root meristem by assuming either a uniform or an exponential cell age distribution. Statistical comparison of theoretical distributions with the experimental one rules out a uniform cell age distribution and strongly favours an exponential age distribution similar to that found in bacteria.

Regulation of G2 by cell size contributes to maintaining cell size variability within certain limits in higher plants.

The variability of (1) surface area projection (size) at which cells terminate DNA replication, (2) the area at which they initiate mitosis, (3) the area at which they divide, (4) the duration of G2, and (5) the duration of G2 plus mitosis (in fact, prophase + metaphase + anaphase) has been estimated in steady-state cell populations of Allium cepa root meristems. The coefficient of variation of cell area at termination of DNA synthesis was found to be 14% while the coefficient of variation of cell area at mitosis initiation was 13%. As there is also a substantial variability of G2 (the coefficient of variation was estimated to be 38%), the combination of these data indicates that cell size regulation of G2 contributes to maintaining cell size variability (and therefore DNA concentration) within certain limits. Mitosis also varies but less than G2 (the coefficient of variation of G2 + mitosis was found to be 31%). As the coefficient of variation of cell area at division (14%) is hardly larger than the coefficient of variation of cell area at initiation of mitosis, it can be suggested that coordination between cell size and mitosis duration helps to avoid a significant increase in the variability of cell size at the end of the division cycle.

The effect of partial protein synthesis inhibition on cell proliferation in higher plants.

Meristematic cells from Allium cepa L. roots can attain a steady state of growth in the presence of anisomycin at concentrations that effectively reduce the rate of protein synthesis. Under these conditions the lengths of cell cycle periods increase but not in the same proportion as the generation time (t). Mitosis is hardly affected and S period is slightly lengthened. G2 increases less in proportion to t, while G1 is extended much higher in proportion to t. Natural synchronous populations have been used to study cell cycle parameters during transition from the physiological steady state to the new one created by the presence of the drug. G2 was the same during transition as during steady-state growth. G1 was much shorter during transition. Average cell mass at division was reduced, and a negative correlation was observed between the length of G2 and the size of the cell at termination of DNA synthesis. We propose that in higher plants, G2 length is regulated by cell mass at completion of DNA synthesis (G2 being shorter in big cells than in small cells), though there is no cell size requirement for mitosis.

DNA segregation in Escherichia coli cells with 5-bromodeoxyuridine-substituted nucleoids.

The pattern of segregation of DNA in Escherichia coli K-12 was analyzed by labeling replicating DNA with 5-bromodeoxyuridine followed by differential staining of nucleoids. Three types of visible arrangement were found in four-nucleoid groups derived from a native nucleoid after two replication rounds. Type A, segregation of both old strands toward cell poles, appeared with the highest frequency (0.6 to 0.8). Type B, segregation of one old strand toward the cell pole and the other toward the cell center, was twice as frequent as type C, segregation of both old strands toward the cell center. These results confirm previous data showing that DNA segregation in E. coli is nonrandom while presenting a certain degree of randomness. The proportions of the three indicated types of arrangement suggest a new probabilistic model to explain the observed segregation pattern. It is proposed that DNA strands segregate either nonrandomly, with a probability of between 0 and 1, or randomly. In nonrandom segregation, both old strands are always directed toward cell poles. Experimental data reported here or by other authors fit better with the predictions of this model than with those of other previously proposed proposed deterministic or probabilistic models.

Partial elimination of G1 and G2 periods in higher plant cells by increasing the S period.

Meristematic cells from Allium cepa L roots can attain a steady-state of growth at both 15 and 25 degrees C in the presence of drugs, hydroxyurea and 5-amino-uracil, which reduce the rate of DNA synthesis. These drugs, at used concentrations, significantly lengthen the S period without altering the cell growth rate, as indicated by the maintenance of the generation time. It has been observed that steady-state populations respond to a gradual increase in S by a reduction of G2 until a minimum value; with larger lengthening of S, both G1 and G2 are reduced. Natural synchronous populations have been used to study cell cycle parameters during transition from the physiological steady-state to the new one created by the presence of the drug. G2 (but not G1) is reduced during transition even in the presence of maximum drug concentrations that do not alter the cell growth rate. Both the S period and the division time are lengthened during transition. These observations support the concept that certain fractions of G1 and G2 are expendable, because they have no role in the DNA-division sequence of cell cycle events. We conclude that cell size regulates the length of these fractions by means of a negative correlation.

On the mechanism of differential Giemsa staining of BrdU-substituted chromatids.

This paper analyses the effect of acid hydrolysis on the differential Giemsa staining of 5-bromo-2'deoxyuridine (BrdU) substituted chromatids in human and plant chromosomes, after treatment with a fluorochrome and light. Human lymphocytes and Allium cepa L. root tips were grown in BrdU for two or three cell cycles. Lymphocytes spreadings and meristem squashes were treated with fluorochrome Hoechst 33258, exposed to sunlight, hydrolysed with 5N HCl and stained with giemsa. This acid hydrolysis improves the differential staining of BrdU substituted and non-substituted chromatin. It also allows the differentiation of sister chromatids with the DNA specific dye azure-A.