G2 effects of DNA-repair inhibitors on chromatid-type aberrations in root-tip cells treated with maleic hydrazide and mitomycin C

In recent years the existence of a DNA-repair process in G2 has been proposed to explain the potentiating effects of DNA-repair inhibitors given in G2 on chromatid aberrations (CA) induced by S-dependent as well as S-independent DNA-damaging agents. In the present report, root-tip cells of Allium cepa were exposed to maleic hydrazide (MH) or mitomycin C (MMC) and post-treated in G2 with caffeine (Caff) and various inhibitors of DNA synthesis. No enhancement of chromosome damage was observed when Caff was present in G2, but hydroxyurea (HU) or 5-fluorodeoxyuridine (FdUrd) potentiated the frequencies of CA. A slight additional increase of CA frequencies was observed following treatment with Ara C and excess thymidine in G2. When MH-damaged cells were pulse-treated with Caff earlier during recovery, the yield of CA was enhanced. The earlier Caff was present following MH treatment, the stronger was the potentiation.     

Effect of caffeine on radiation-induced mitotic delay: delayed expression of G2 arrest

In the presence of 5 mM caffeine, irradiated (1.5 Gy) S and G2 cells progressed to mitosis in register and without arrest in G2. Caffeine (5 mM) markedly reduced mitotic delay even after radiation doses up to 20 Gy. When caffeine was removed from irradiated (1.5 Gy) and caffeine-treated cells, a period of G2 arrest followed, similar in length to that produced by radiation alone. The arrest expressed was independent of the duration of the caffeine treatment for exposures up to 3 hr. The similarity of the response to the cited effects of caffeine on S-phase delay suggests a common basis for delay induction in S and G2 phases.     

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.     

Caffeine eliminates gamma-ray-induced G2-phase delay in human tumor cells but not in normal cells.

It has been known for many years that caffeine reduces or eliminates the G2-phase cell cycle delay normally seen in human HeLa cells or Chinese hamster ovary (CHO) cells after exposure to X or gamma rays. In light of our recent demonstration of a consistent difference between human normal and tumor cells in a G2-phase checkpoint response in the presence of microtubule-active drugs, we examined the effect of caffeine on the G2-phase delays after exposure to gamma rays for cells of three human normal cell lines (GM2149, GM4626, AG1522) and three human tumor cell lines (HeLa, MCF7, OVGI). The G2-phase delays after a dose of 1 Gy were similar for all six cell lines. In agreement with the above-mentioned reports for HeLa and CHO cells, we also observed that the G2-phase delays were eliminated by caffeine in the tumor cell lines. In sharp contrast, caffeine did not eliminate or even reduce the gamma-ray-induced G2-phase delays in any of the human normal cell lines. Since caffeine has several effects in cells, including the inhibition of cAMP and cGMP phosphodiesterases, as well as causing a release of Ca(++) from intracellular stores, we evaluated the effects of other drugs affecting these processes on radiation-induced G2-phase delays in the tumor cell lines. Drugs that inhibit cAMP or cGMP phosphodiesterases did not eliminate the radiation-induced G2-phase delay either separately or in combination. The ability of caffeine to eliminate radiation-induced G2-phase delay was, however, partially reduced by ryanodine and eliminated by thapsigargin, both of which can modulate intracellular calcium, but by different mechanisms. To determine if caffeine was acting through the release of calcium from intracellular stores, calcium was monitored in living cells using a fluorescent calcium indicator, furaII, before and after the addition of caffeine. No calcium release was seen after the addition of caffeine in either OVGI tumor cells or GM2149 normal cells, even though a large calcium release was measured in parallel experiments with ciliary neurons. Thus it is likely that caffeine is eliminating the radiation-induced G2-phase delay through a Ca(++)-independent mechanism, such as the inhibition of a cell cycle-regulating kinase.     

On the identity of the damage expressed by treatment of X-irradiated HeLa cells with different agents

To determine whether different agents that enhance the expression of potentially lethal X-ray damage (PLD) interact with the same or different lesions (or spectrum of lesions), cell killing was measured in three kinds of experiments: (1) When cells were irradiated in G1 phase and treated with caffeine or hydroxyurea at concentrations that yield maximal response, the same survival plateaus were reached. (2) Treatment of cells irradiated in G1 phase either with caffeine or with hydroxyurea so as to yield survival levels that differed twofold after 4 h incubation, followed by treatment with caffeine to allow expression of PLD in G2 phase, resulted eventually in the same level of survival. (3) When cells were irradiated and treated with caffeine, hydroxyurea, or 9-beta-D-arabinofuranosyladenine (araA) after progressively longer delays, to trace the time course of recovery from the PLD, the responses obtained with caffeine and araA were similar; sensitivity to hydroxyurea was lost more rapidly. The results are consistent with the possibility that these three agents interact with the same lesions, but that different steps in the repair process are inhibited by caffeine or araA than by hydroxyurea.     

Effects of caffeine and cycloheximide during G2 prophase in control and X-ray-irradiated human lymphocytes

The effect of caffeine and cycloheximide during the G2 phase on frequency of chromosomal aberrations and G2 duration was studied in control and X-ray-irradiated human lymphocytes in vitro. Caffeine treatments alone increase the frequencies of chromatid breakage and decrease the average G2 duration in control and X-ray-irradiated lymphocytes (40 R). Both caffeine effects are reversed by 0.5 micrograms/ml cycloheximide in combination treatments. Cycloheximide treatments alone prolong G2 duration in control as well as in X-ray-irradiated lymphocytes although no improvement in chromosome repairing by this inhibitor of protein synthesis was observed under the conditions of our experiments. We propose that the cycloheximide effect is associated with a low level of mitotic factors, required for the entrance into mitosis, which is maintained at a higher level in caffeine treatment alone. Finally, G2 delay has generally been associated with certain genome damage. The fact that the caffeine and cycloheximide effects on X-irradiated lymphocytes are also present in control lymphocytes (without X-rays) suggests that control of the G2 duration constitutes one of the mechanisms involved in DNA repair operating during the G2 phase.     

Effect of caffeine and adenosine on G2 repair: mitotic delay and chromosome damage

Proliferating plant cells treated during the late S period with 5-aminouracil (AU), give the typical response that DNA-damaging agents induce, characterized by: an important mitotic delay, and a potentiation of the chromosome damage by caffeine post-treatment. The study of labelled prophases, after a tritiated thymidine pulse, allowed evaluation of the mitotic delay induced by AU as well as its reversion by caffeine, while chromosome damage was estimated by the percentage of anaphases and telophases showing chromosomal aberrations. Post-treatment with adenosine alone has shown no effect on mitotic delay or chromosomal damage. However, when cells after AU were incubated in caffeine plus adenosine, the chromosome damage potentiation was abolished without affecting the caffeine action on mitotic delay. As a consequence, we postulate that caffeine could have two effects on G2 cells with damaged DNA: the first, to cancel their mitotic delay and the second to inhibit some DNA-repair pathway(s). Only this last effect could be reversed by adenosine.     

Genistein regulates the IL-1 beta induced activation of MAPKs in human periodontal ligament cells through G protein-coupled receptor 30

Periodontal ligament (PDL) cells are fibroblasts that play key roles in tissue integrity, periodontal inflammation and tissue regeneration in the periodontium. The periodontal tissue destruction in periodontitis is mediated by host tissue-produced inflammatory cytokines, including interleukin-1β (IL-1β). Here, we report the expression of G protein-coupled receptor 30 (GPR30, also known as G protein-coupled estrogen receptor 1 GPER) in human PDL cells and its regulation by IL-1β. IL-1β-induced GPR30 expression in human PDL cells leads to the activation of multiple signaling pathways, including MAPK, NF-κB and PI3K. In contrast, genistein, an estrogen receptor ligand, postpones the activation of MAPKs induced by IL-1β. Moreover, the inhibition of GPR30 by G15, a GPR30-specific antagonist, eliminates this delay. Thus, genistein plays a role in the regulation of MAPK activation via GPR30, and GPR30 represents a novel target regulated by steroid hormones in PDL cells.     

Periodontal ligament cell kinetics following orthodontic tooth movement.

The population of periodontal ligament (PDL) fibroblasts examined in this study may include osteogenic progenitor cells. PDL fibroblast and osteoblast kinetics in the periodontal ligament of the rat were measured following orthodontic stimulation of bone formation. Both single and multiple injections of tritiated thymidine (3H-TdR) were used. In single injection experiments, the peak percentage of PDL fibroblasts labeled with 3H-TdR is 15% at 22 hr post-stimulation. In multiple injection experiments, the total percentage of fibroblasts in the PDL which respond by synthesizing DNA is 50%. 3H-TdR-labeled osteoblasts appear at the same rate as, but with a time delay after, the labeled fibroblasts. Following stimulation, the most likely source of osteoblasts at the bbone forming site is not only fibroblasts which make DNA, divide, then differentiate, but also fibroblasts which either are differentiated to osteoblasts without DNA synthesis and cell division, or are released from G2 block by the orthodontic stimulation.     

Reduction of ultraviolet-induced mitotic delay by caffeine in G2-phase irradiated plasmodia of Physarum polycephalum

Synchronously mitotic surface Plasmodia ofPhysarum polycephalum were ultra-violet-irradiated at different times during G2-phase (—4 h to —20 min with respect to metaphase), and treated immediately thereafter with varying concentrations of caffeine. It was observed that ultraviolet-induced mitotic delay is reduced significantly by this methylxanthine. In plasmodia irradiated between —4 and —1 h with respect to metaphase, the effect was concentration-dependent and the need for a certain threshold dose for obtaining the reduction in delay was apparent. However, higher doses than this were fairly toxic when applied at this part of the cycle and led to more mitotic delay than that obtained with UV alone. The most striking observation made during this study was the phase-specific precipitous effect seen in those plasmodia irradiated at about 20 min before mitosis which almost eliminated the long delay due to ultraviolet-irradiation. These results are discussed in the context of some of the known effects of ultraviolet and caffeine on a mitosis-promoting factor. It is proposed that the significant reduction of ultraviolet-induced mitotic delay reported here is due to the reactivation of the ultraviolet-inactivated mitosis-promoting factor by caffeine. Alternatively, it is possible that caffeine may prevent the inactivation of this factor by ultraviolet.     

The effect of caffeine posttreatment of X-ray-induced chromosomal aberrations in human blood lymphocytes in vitro

Summary The potentiating effect of caffeine on X-ray-induced chromosomal aberrations in human blood lymphocytes has been investigated, with special reference to cell cycle stages (G0 and G2). Both quantitative and qualitative differences in the yield of chromosomal aberrations were detected in caffeine-posttreated cells, depending on the cell stage irradiated. The studies on caffeine potentiating effects on X-irradiated G0 lymphocytes from normal adults, newborns, Down syndrome patients, and an ataxia telangiectasia patient pointed to interindividual variations in the response to caffeine potentiation among normal probands and a very profound effect in ataxia cells.     

Comparative effects of caffeine on radiation- and heat-induced alterations in cell cycle progression

The effects of 3 mM caffeine on cell cycle progression of HeLa S3 cells exponentially and asynchronously growing in suspension culture were studied following exposure to 6.8 Gy gamma irradiation or 30 min at 45 degrees C hyperthermia. The stathmokinetic method, in which cells are grown in the presence of colcemid for the duration of experiment, in combination with two flow cytometric techniques, propidium iodide staining of DNA and acridine orange staining following acid denaturation of chromatin, were used to determine the fraction of cells in four cell cycle compartments, G1, S, G2, and M. Radiation and caffeine acted in a complementary manner, in which radiation reduced the caffeine-induced delays in cell cycle progression and caffeine prevented completely the radiation-induced accumulation of cells in G2 and mitotic delay. Heat and caffeine had additive effects on alterations in cell cycle progression. Cells containing spontaneous prematurely condensed chromatin were observed transiently immediately following heat exposure. These cells appeared to be in G2 and late S phase.     

Cytological characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. I. Induction of chromosomal aberrations by X-irradiation and its modulation with 3-aminobenzamide and caffeine

We have studied two X-ray-sensitive mutants xrs 5 and xrs 6 (derived from the CHO-K1 cell line), known to be defective in repair of double-strand breaks, for cell killing and frequency of the chromosomal aberrations induced by X-irradiation. The survival experiments showed that mutants are very sensitive to X-rays, the D0, for the wild-type CHO-K1 was 6-fold higher than D0 value for the mutants. The modal number of chromosomes (2 n = 23) and the frequency of spontaneously occurring chromosomal aberrations were similar in all 3 cell lines. X-Irradiation of synchronized mutant cells in G1-phase significantly induced both chromosome- and chromatid-type of aberrations. The frequency of aberrations in xrs mutants was 12-fold more than in the wild-type CHO-K1 cells. X-Irradiation of G2-phase cells also yielded higher frequency of aberrations in the mutants, namely 7-8-fold in xrs 5 and about 3.5-fold in xrs 6 compared to the wild-type CHO-K1 cells. There was a good correlation between relative inability to repair of DNA double-strand breaks and induction of aberrations. The effect of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthetase on the frequency of X-ray-induced chromosomal aberrations in these 3 cell lines was also studied. 3AB potentiated the frequency of aberrations in G1 and G2 in all the cell types. In the mutants, 3AB had a potentiating effect on the frequency of X-ray-induced chromosomal aberrations only at low doses. X-Ray-induced G2 arrest and its release by caffeine was studied by cytofluorometric methods. The relative speed with which irradiated S-G2 cells progressed into mitosis in the presence of caffeine was CHO-K1 greater than xrs 5 greater than xrs 6. Caffeine could counteract G2 delay induced by X-rays in CHO-K1 and xrs 5 but not in xrs 6. Large differences in potentiation by caffeine were observed among these cells subjected to X-rays and caffeine post-treatment for different durations. These responses and possible reasons for the increased radiosensitivity of xrs mutants are discussed and compared to ataxia telangiectasia (A-T) cells and a radiosensitive mutant mouse lymphoma cell line.     

Caffeine and human DNA metabolism: the magic and the mystery

The ability of caffeine to reverse cell cycle checkpoint function and enhance genotoxicity after DNA damage was examined in telomerase-expressing human fibroblasts. Caffeine reversed the ATM-dependent S and G2 checkpoint responses to DNA damage induced by ionizing radiation (IR), as well as the ATR- and Chk1-dependent S checkpoint response to ultraviolet radiation (UVC). Remarkably, under conditions in which IR-induced G2 delay was reversed by caffeine, IR-induced G1 arrest was not. Incubation in caffeine did not increase the percentage of cells entering the S phase 6-8h after irradiation; ATM-dependent phosphorylation of p53 and transactivation of p21(Cip1/Waf1) post-IR were resistant to caffeine. Caffeine alone induced a concentration- and time-dependent inhibition of DNA synthesis. It inhibited the entry of human fibroblasts into S phase by 70-80% regardless of the presence or absence of wildtype ATM or p53. Caffeine also enhanced the inhibition of cell proliferation induced by UVC in XP variant fibroblasts. This effect was reversed by expression of DNA polymerase eta, indicating that translesion synthesis of UVC-induced pyrimidine dimers by DNA pol eta protects human fibroblasts against UVC genotoxic effects even when other DNA repair functions are compromised by caffeine.     

Degradation of ATM-independent decatenation checkpoint function in human cells is secondary to inactivation of p53 and correlated with chromosomal destabilization

DNA topoisomerase II is required in the cell cycle to decatenate intertwined daughter chromatids prior to mitosis. To study the mechanisms that cells use to accomplish timely chromatid decatenation, the activity of a catenation-responsive checkpoint was monitored in human skin fibroblasts with inherited or acquired defects in the DNA damage G2 checkpoint. G2 delay was quantified shortly after a brief incubation with ICRF-193, which blocks the ability of topoisomerase II to decatenate chromatids, or treatment with ionizing radiation (IR), which damages DNA. Both treatments induced G2 delay in normal human fibroblasts. Ataxia telangiectasia fibroblasts with defective G2 checkpoint response to IR displayed normal G2 delay after treatment with ICRF-193, demonstrating that ATM kinase was not required for signaling when chromatid decatenation was blocked. The G2 delay induced by ICRF-193 was reversed by caffeine, indicating that active checkpoint signaling was involved. ICRF-193-induced G2 delay also was independent of p53 function, being evident in cells expressing HPV16E6 to inactivate p53. However, as fibroblasts expressing HPV16E6 aged in culture, they lost the ability to delay entry to mitosis, both after DNA damage and when decatenation was blocked. This age-related loss of G2 delay in response to ICRF-193 and IR in E6-expressing cells was blocked by induction of telomerase. Expression of telomerase also prevented chromosomal destabilization in aging E6-expressing cells. These observations lead to a new model of genetic instability, in which attenuation of G2 decatenatory checkpoint function permits cells to enter mitosis with insufficiently decatenated chromatids, leading to aneuploidy and polyploidy.     

Studies on the microbial production of theobromine and heteroxanthine from caffeine

Summary From soil a caffeine degrading bacterium was isolated which is able to grow on media containing up to 2% caffeine as the sole source of carbon and nitrogen. The organism was identified as Pseudomonas putida and referred to as Pseudomonas putida WS. Mutants of this strain converted caffeine and were shown to accumulate a mixture of theobromine and heteroxanthine during resting cells experiments.The highest yield in accumulation products was obtained with the mutant strain H8, however the production rate with resting cells was too small for commercial purposes. The yield was significantly increased by growth of the mutant on diluted complex media. With this technique a yield of 50% based on the amount of caffeine could be obtained for heteroxanthine. The concentration maximum is reached when caffeine is completely converted and only traces of theobromine are present.Dedicated to Professor G. Braunitzer on the occasion of his 65th birthday     

Functions of beta2-adrenergic receptor in human periodontal ligament cells

Adrenergic receptors (ARs) are receptors of noradrenalin and adrenalin, of which there are nine different subtypes. In particular, β2 adrenergic receptor (β2-AR) is known to be related to the restoration and maintenance of homeostasis in bone and cardiac tissues; however, the functional role of signaling through β2-AR in periodontal ligament (PDL) tissue has not been fully examined. In this report, we investigated that β2-AR expression in PDL tissues and their features in PDL cells. β2-AR expressed in rat PDL tissues and human PDL cells (HPDLCs) derived from two different patients (HPDLCs-2G and -3S). Rat PDL tissue with occlusal loading showed high β2-AR expression, while its expression was downregulated in that without loading. In HPDLCs, β2-AR expression was increased exposed to stretch loading. The gene expression of PDL-related molecules was investigated in PDL clone cells (2-23 cells) overexpressing β2-AR. Their gene expression and intracellular cyclic adenosine monophosphate (cAMP) levels were also investigated in HPDLCs treated with a specific β2-AR agonist, fenoterol (FEN). Overexpression of β2-AR significantly promoted the gene expression of PDL-related molecules in 2 to 23 cells. FEN led to an upregulation in the expression of PDL-related molecules and increased intracellular cAMP levels in HPDLCs. In both HPDLCs, inhibition of cAMP signaling by using protein kinase A inhibitor suppressed the FEN-induced gene expression of α-smooth muscle actin. Our findings suggest that the occlusal force is important for β2-AR expression in PDL tissue and β2-AR is involved in fibroblastic differentiation and collagen synthesis of PDL cells. The signaling through β2-AR might be important for restoration and homeostasis of PDL tissue.     

Degradation of ATM-Independent Decatenation Checkpoint Function in Human Cells is Secondary to Inactivation of p53 and Correlated with Chromosomal Destabilization

DNA topoisomerase II is required in the cell cycle to decatenate intertwined daughter chromatids prior to mitosis. To study the mechanisms that cells use to accomplish timely chromatid decatenation, the activity of a catenation-responsive checkpoint was monitored in human skin fibroblasts with inherited or acquired defects in the DNA damage G2 checkpoint. G2 delay was quantified shortly after a brief incubation with ICRF-193, which blocks the ability of topoisomerase II to decatenate chromatids, or treatment with ionizing radiation (IR), which damages DNA. Both treatments induced G2 delay in normal human fibroblasts. Ataxia telangiectasia fibroblasts with defective G2 checkpoint response to IR displayed normal G2 delay after treatment with ICRF-193, demonstrating that ATM kinase was not required for signaling when chromatid decatenation was blocked. The G2 delay induced by ICRF-193 was reversed by caffeine, indicating that active checkpoint signaling was involved. ICRF-193-induced G2 delay also was independent of p53 function, being evident in cells expressing HPV16E6 to inactivate p53. However, as fibroblasts expressing HPV16E6 aged in culture, they lost the ability to delay entry to mitosis, both after DNA damage and when decatenation was blocked. This age-related loss of G2 delay in response to ICRF-193 and IR in E6-expressing cells was blocked by induction of telomerase. Expression of telomerase also prevented chromosomal destabilization in aging E6-expressing cells. These observations lead to a new model of genetic instability, in which attenuation of G2 decatenatory checkpoint function permits cells to enter mitosis with insufficiently decatenated chromatids, leading to aneuploidy and polyploidy.

Key Words:

Checkpoints, DNA damage, Decatenation, Topoisomerase II, ICRF-193, Radiation     

The effects of in vitro age and culture state on histone variant synthesis in human diploid fibroblasts

Histone variant synthesis patterns from human diploid fibroblast-like cells of different in vitro ages were determined during exponential growth, at confluence, and during low serum arrest. The results are reported as the ratios of H2A variant synthesis (H2A.1 and H2A.2/H2A.x and H2A.z) and H3 variant synthesis (H3.1 and H3.2/H3.3) that have been used to characterize individual cell cycle states. Hydroxyurea was employed in some experiments to reduce S phase cells. The results indicate that high population doubling level (PDL) cells move through the G1 phase of the division cycle during exponential growth and exist in the G0 cell cycle state at confluence and during low serum arrest. Low PDL cells, however, exist in the G1 cell cycle state at confluence and revert to a G0 state only after maintenance as quiescent populations. This would suggest that when stimulated high PDL cells cannot enter into S phase, they revert to a GO cell cycle state.     

Effects of UV-irradiation of carp sperm and its modification by caffeine

In connection with the development of UV-induced mutagenesis in carp, the effects of UV-irradiation of sperms have been studied in the range of doses 0.3-40 J/m2. The irradiation did not cause reduced fertilization ability and cleavage delay. Small doses of irradiation (0.6 J/m2) produced stimulating effect on embryo survival, the larvae yield decreasing and the amount of aberrant anaphases increasing, as the doses are increased. LD50 is 6.0 J/m2 for embryonal period of carp development, photoreactivation increases it up to 23.5 J/m2. Correlation between embryo viability and their resistance to UV has been found (r = +0.68 +/- 0.20). Modification of the effect of sperms irradiation with caffeine has been also studied. Strengthening of lethal and cytogenetic effects was only observed in case when embryos were placed in the caffeine solution, prior to onset of the first DNA replication in the male pronucleus. This may indicate the existence of caffeine-dependent prereplicative repair in carp embryos.