Genetic heterogeneity in patients having ataxia telangiectasia

UV-irradiated Chinese hamster cells on post-irradiation treatment with caffeine in growth medium for 24 h gave rise to biphasic UV-survival curves. At caffeine concentrations between 0.001 and 0.1 mM, control and caffeine-grown cells had similar survival curves initially from 0 to 30 J/m². At fluences greater than 30 J/m², there was effectively only little further killing of caffeine-grown cells beyond that observed at 30 J/m². At concentrations of caffeine greater than 0.5 mM, there was a gradual sensitization in the early part of the survival curve with increasing caffeine concentrations; but at fluences greater than 3 J/m², the slopes in the survival curves decreased very much.It has been proposed that the initial sensitization observed at low UV fluences is due to the suppression of post-replication repair by caffeine. After high fluences of UV exposures in these excision-deficient cells, in the presence of caffeine, the possibility of an induced ‘SOS’-like repair process has been suggested. This suggestion was supported by the observation that caffeine increased the yield of the UV-induced 8-azaguanine-resistant mutants only for the cell population exposed to UV fluences greater than 30 J/m².     

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.     

Modification of the radiosensitivity of E. coli cells by factors affecting the yield of photoreactivated damage

A study was made of the influence of irradiation conditions on the yield of the photoreactivable damages in radiosensitive mutants of E. coli cells (E. coli WP2). Pyrimidine dimers were shown to occur in exrA- and recA- mutants irradiated under anoxic conditions, the survival of these mutants being modified depending on cell genotype. The processes of direct excitation of the molecules were involved in the formation of the damages observed. It can be assumed that the lesser oxygen effect observed in exrA- and partially in recA- mutants of E. coli WP2 cells is associated with a contribution of the photoreactivable damages to a lethal effect of ionizing radiation.     

Prevention of cytokine withdrawal-induced apoptosis by Mcl-1 requires interaction between Mcl-1 and Bim

Growth factor withdrawal from hemopoietic cells results in activation of the mitochondrial pathway of apoptosis. Members of the Bcl-2 family regulate this pathway, with anti-apoptotic members counteracting the effects of pro-apoptotic members. We investigated the effect on Mcl-1 function of mutation at a conserved threonine 163 residue (T163) in its proline, glutamate, serine, and threonine rich (PEST) region. Under normal growth conditions, Mcl-1 half-life increased with alteration of T163 to glutamic acid, but decreased with mutation to alanine. However, both T163 mutants exhibited greater pro-survival effects compared with the wild type, which can be explained by an increased stability of the T163A mutant in cytokine-starved conditions. Both the mutant forms exhibited prolonged binding to pro-apoptotic Bim in cytokine-deprived cells. The extent to which Mcl-1 mutants were able to exert their anti-apoptotic effects correlated with their ability to associate with Bim. We further observed that primary bone marrow derived macrophages survived following cytokine withdrawal as long as Bim and Mcl-1 remained associated. In our study, we were unable to detect a role for GSK-3-mediated regulation of Mcl-1 expression. Based on these results we propose that upon cytokine withdrawal, survival of hemopoietic cells depends on association between Mcl-1 and Bim. Furthermore, alteration of T163 of Mcl-1 may change the protein such that its association with Bim is affected, resulting in prolonged association and increased survival.     

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.     

Genome-Wide Screen of Genes Required for Caffeine Tolerance in Fission Yeast

Background

An excess of caffeine is cytotoxic to all eukaryotic cell types. We aim to study how cells become tolerant to a toxic dose of this drug, and the relationship between caffeine and oxidative stress pathways.

Methodology/Principal Findings

We searched for Schizosaccharomyces pombe mutants with inhibited growth on caffeine-containing plates. We screened a collection of 2,700 haploid mutant cells, of which 98 were sensitive to caffeine. The genes mutated in these sensitive clones were involved in a number of cellular roles including the H₂O₂-induced Pap1 and Sty1 stress pathways, the integrity and calcineurin pathways, cell morphology and chromatin remodeling. We have investigated the role of the oxidative stress pathways in sensing and promoting survival to caffeine. The Pap1 and the Sty1 pathways are both required for normal tolerance to caffeine, but only the Sty1 pathway is activated by the drug. Cells lacking Pap1 are sensitive to caffeine due to the decreased expression of the efflux pump Hba2. Indeed, ?hba2 cells are sensitive to caffeine, and constitutive activation of the Pap1 pathway enhances resistance to caffeine in an Hba2-dependent manner.

Conclusions/Significance

With our caffeine-sensitive, genome-wide screen of an S. pombe deletion collection, we have demonstrated the importance of some oxidative stress pathway components on wild-type tolerance to the drug.     

Dissociated occurrence of single-gene mutation and oncogenic transformation in C3H 10T½ cells exposed to ultraviolet light and caffeine

We examined the relationship of cytotoxicity, mutagenesis, and malignant transformation by measuring in parallel clonogenic survival, mutation to ouabain resistance, and malignant transformation in cultured C3H mouse 10T 1/2 cells. Exposure of caffeine alone for 48 hours was cytotoxic and induced transformation in a dose-dependent manner. However, this same treatment did not induce any detectable ouabain-resistant mutants. When caffeine was present for 48 hours immediately following UV irradiation, alkaline sucrose gradient sedimentation of DNA showed that postreplication repair was inhibited. This inhibition of repair was correlated with reduced survival and inhibition of mutation induction, but the transformation frequencies were either unaltered or potentiated, depending on the UV dose and caffeine concentration. Thus, these experiments demonstrate that gene mutation and malignant transformation in 10T 1/2 cells can be dissociated. We suggest that the mechanism of transformation of 10T 1/2 cells is nonmutagenic in nature.     

Common repair pathways acting upon U.V.- and X-ray induced damage in diploid cells of Saccharomyces cerevisiae

Studies on X-ray sensitive mutants of Saccharomyces cerevisiae (Benathen 1973, Benathen and Beam 1977) show that the XS6, XS8 and XS9 genes are not only involved in the repair of X-ray-induced damage but also in the repair of U.V.-induced damage. Analysis of the U.V. sensitivity of multiple xs mutants indicates the participation of three repair pathways which differ from excision repair. Under conditions which can influence repair, such as plating of the U.V.-irradiated cells in the presence of caffeine, followed or not by hyperthermic incubation, the wild type strain shows a diphasic survival curve, consisting of an exponential component for low doses and a sigmoidal one for higher doses. Comparison with the survival curves obtained for the sensitive mutants suggests that the first component of the wild type survival curve corresponds to the inhibition of the XS6 and XS8 gene products while the appearance of a radio-resistant fraction in the population relies on the induction of another repair pathway. A sequential model of repair with two branching points is proposed to explain the results.     

Caffeine-induced reduction of the survival of gamma-irradiated HeLa cells and the reversal of the caffeine effect by Escherichia coli RecA protein

It is confirmed that survival of gamma-irradiated HeLa cells is decreased by post-treatment with caffeine. The caffeine effect is believed to be the result of an inhibition of the repair of gamma-ray-induced DNA damage. In this work we show that the caffeine-induced reduction of the survival of gamma-irradiated HeLa cells is reversed when Escherichia coli RecA protein is introduced into the cells with the aid of liposomes.     

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     

The effect of caffeine on cytotoxicity, mutagenesis, and sister-chromatid exchanges in Chinese hamster cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene

The effect of caffeine on Chinese hamster V79 cells after treatment with the highly mutagenic (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-7,8,9,10-tetrahydrobenzo[a]pyrene, and the weaker mutagen (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, B[a]P-deiol-epoxide II, was studied at both the biological and molecular levels. Caffeine, at nontoxic dose levels, caused a synergistic reduction in cell survival induced by both isomers and also inhibited DNA elongation as measured by alkaline sucrose-gradient analysis of nascent DNA. However, caffeine did not affect the induction of either ouabain-resistant mutants or sister-chromatid exchanges by either isomer. These results suggest that enhanced cell killing by caffeine in benzo[a]pyrene-diol-epoxide treated V79 cells may be related to caffeine's inhibitory effect on DNA elongation. However, inhibition of DNA elongation by caffeine did not influence the resulting induced levels of mutagenesis or sister-chromatid exchanges.     

The action of caffeine on X-irradiated HeLa cells. X. Depressed recovery from potentially lethal damage in cells containing 5-bromodeoxyuridine

HeLa S3 cells were sensitized to the lethal action of 220-kV X rays by partially replacing the thymidine in their DNA with 5-bromodeoxyuridine (BrdU). To examine the expression of and recovery from potentially lethal radiation damage (PLD), both BrdU-grown and control cells were treated with 4 mM caffeine for increasing times up to 2 days, either immediately after irradiation or after increasing delays up to 28 h. When the same dose of X rays (3 Gy) was applied to BrdU-grown and control cells, the difference in survival that is found in the absence of caffeine disappeared after about 30 h of incubation in its presence; when isosurvival doses were applied (BrdU-grown cells, 2.5 Gy; control cells, 4 Gy), the control cells suffered more killing. When treatment with caffeine was delayed for progressively longer times after both groups of cells received 3 Gy, the control cells achieved a higher level of survival. These results indicate that the increased radiation sensitivity of cells containing BrdU derives from a decreased ability to repair PLD.     

Killing and mutation induction in quiescent V79 cells as influenced by nalidixic acid

Involvement of topoisomerase II in the repair of damage by N-methyl-N'-nitro-N-nitrosoguanidine, UV light and gamma-rays has been studied using quiescent V79 cells. In each case, the presence of nalidixic acid, the inhibitor of topoisomerase II, decreased the survival through suppression of potentially lethal damage repair. There was also an increase in the yield of mutants because of such suppression. The observations were in contrast with those made with exponential cells in the following aspects: (a) density-inhibited cells showed a positive response to suppression of topoisomerase II activity after treatment with UV light and gamma-rays and (b) for MNNG exposure, mutational yield increased instead of decreasing as in exponential cells. The results showed that topoisomerase II played an important part in the repair of damage of density-inhibited cells.     

Detrimental effects of cryopreservation of loach (Misgurnus fossilis) sperm on subsequent embryo development are reversed by incubating fertilised eggs in caffeine

Cryopreservation can cause changes to the genetic material of cells, but the mechanism and significance of these changes are still unknown. It has been suggested that some damage to the sperm genome could be repaired by the DNA repair system of the oocyte after fertilisation. Caffeine has been reported to be an inhibitor of such repair processes. In this study the effect of caffeine on the repair system of Loach (Misgurnus fossilis) oocytes was investigated. Loach eggs were fertilised using cryopreserved sperm. Embryos derived from cryopreserved sperm were exposed to 2.6mM caffeine for 1h after fertilisation. The experiments were carried out using 32313 embryos from four females and eight males. Embryo survival was evaluated for 46 h until the hatching stage. Reduction in embryo survival after 20th stage is generally believed to result from the failure in the genome function of embryos. Cryopreservation of sperm significantly decreased embryo survival (53.4+/-2.8% compared to 68.4+/-2.8% of control) after the 20th stage. However, the addition of caffeine to the embryos derived from cryopreserved sperm, in contrast to our expectation, significantly increased survival of loach embryos (70.9+/-2.8% compared to 53.4+/-2.8% of embryos derived from cryopreserved sperm in the absence of caffeine). The effect of individual donors of sperm and eggs on overall embryo survival was also studied. Whilst no significant differences were observed between males, the effect of individual females on embryo survival was significant. The analysis of embryo survival at different developmental stages showed that embryo survival both before and after 20th stage decreased with embryo development. When fresh sperm were used the decline of embryo survival with development was more pronounced compared with those embryos derived from cryopreserved sperm. Possible explanations of these effects are presented.     

General characteristics, molecular and genetic analysis of two new UV-sensitive mutants of Chlamydomonas reinhardtii

Two new UV-sensitive mutants of Chlamydomonas, UVS10 and UVS11, were isolated. Both behave as single nuclear mutations. UVS10 was mapped to linkage group I. UVS11 is a separate, unlinked mutation but has not yet been located to a specific linkage group. Both mutants are proficient in the excision of pyrimidine dimers from nuclear DNA. The survival of UV-irradiated UVS11 is increased when plated in the presence of 1.5 mM caffeine, similar to wild-type. Caffeine has no effect on the survival of UV-irradiated UVS10. UV-irradiated UVS11 frequently divides at least once before dying, in contrast to UVS10 or wild-type. UVS11 also exhibits a much increased frequency of mutation to streptomycin resistance after UV irradiation.     

Impact of mitochondrial function on yeast susceptibility to antifungal compounds

Saccharomyces cerevisiae pell and crd1 mutants deficient in the biosynthesis of mitochondrial phosphatidylglycerol (PG) and cardiolipin (CL) as well as Kluyveromyces lactis mutants impaired in the respiratory chain function (RCF) containing dysfunctional mitochondria show altered sensitivity to metabolic inhibitors. The S. cerevisiae pell mutant displayed increased sensitivity to cycloheximide, chloramphenicol, oligomycin and the cell-wall perturbing agents caffeine, caspofungin and hygromycin. On the other hand, the pel1 mutant was less sensitive to fluconazole, similarly as the K. lactis mutants impaired in the function of mitochondrial cytochromes. Mitochondrial dysfunction resulting either from the absence of PG and CL or impairment of the RCF presumably renders the cells more resistant to fluconazole. The increased tolerance of K. lactis respiratory chain mutants to amphotericin B, caffeine and hygromycin is probably related to a modification of the cell wall.     

Comparative mutagenic effects of ethyl methane-sulfonate, n-methyl-n'-nitro-n-nitrosoguanidine, ultraviolet radiation and caffeine on Dictyostelium discoideum.

A high frequency of morphogenetic mutants of Dictyostelium discoideum can be induced by treatment with MNNG under conditions which result in relatively low cell killing. Six temperature-sensitive growth mutants induced by this treatment were isolated by replica plating. Among these, five showed spontaneous reversion rates of 10(-4) to 10(-5). The mutagenic activity of ems, measured for the induction of both morphogenetic and temperature-sensitive mutants, was weaker than that of MNNG and UV radiation. High frequencies of morphogenetic mutants were obtained only with doses of UV irradiation that resulted in high killing of cells or spores. Caffeine, at concentrations that slightly decreased the growth rate of amoebae in axenic medium, induced morphogenetic defects and also enhanced the mutagenic effect of UV irradiation. However, all the aggregateless clones derived from caffeine treatment that were studied reverted to the wild-type phenotype after a variable number of clonal re-isolations.     

Ultraviolet Irradiation of the Vegetative Cells of Dictyostelium discoideum

Experiments on the effect of ultraviolet (UV) light on the survival of vegetative Dictyostelium discoideum cells indicate that this is a relatively UV-resistant organism. Several factors suggest the presence of some type of repair process. Experiments to test for liquid-holding recovery and simple photoreactivation yielded negative results. Acriflavine and caffeine were utilized to possibly interfere with dark repair. Acriflavine produced no UV sensitization, but caffeine did cause a concentration-dependent decrease in survival of irradiated cells. When UV-irradiated cells were illuminated with photoreactivating light while suspended in caffeine, the survival increased above that for cells treated with caffeine alone, suggesting an overlap between lesions repaired by photorepair and dark repair. Growth experiments showed that UV light induced a dose-dependent division delay, followed by a period of retarded growth characterized by the presence of a constant fraction of nonviable cells in the irradiated population. The delayed exposure of cells to caffeine after irradiation showed that the magnitude of the caffeine sensitization diminished throughout the division-delay period. An action spectrum indicated probable nucleoprotein involvement in the induction of division delay. UV light retarded ribonucleic acid and protein synthesis and temporarily blocked deoxyribonucleic acid synthesis. However, synthesis of all three accelerated prior to the end of the division-delay period and then closely paralleled the increase in cell number.     

Temperature-sensitive photosynthesis-deficient mutants ofAnabœna variabilis show enhanced ultraviolet sensitivity and loss of repair mechanism

Six temperature-sensitive mutants derived from the cyanobacteriumAnabœna variabilis exhibited differences in their photosynthetic efficiency (as evidenced by oxygen evolution studies). All the ts-mutants exhibited lower chlorophyll and phycocyanin contents at 40°C relative to the wild strain and to their control cultures at 28°C. Whole cell absorption spectra of the wild strain showed the same level of chlorophyll, phycocyanin and phycoerythrin at 28 and 40°C, while the spectra from UV irradiated cells showed a decreased content of these pigments. The UV-sensitivity, photoreactivation and dark repair of the ts-mutants indicated a four- to seven-fold increased sensitivity to UV-light as evidenced by LD₃₇ values. The ability of these six mutants to repair UV-induced lesions either by photoreactivation or dark-repair was lower than in the wild strain. The ability of ts-43 and ts-49 to mediate dark-repair appears to have been lost, as documented by the survival curves obtained after post-irradiation treatment with caffeine. These results point to a relationship between the photosynthetic efficiency and the ability to repair UV-induced lesions.     

The effect of caffeine on repair in Chlamydomonas reinhardtii: I. Enhancement of recombination repair

The effect of caffeine on repair was studied in the green alga Chlamydomonas reinhardtii. Treatment of UV-irradiated wild-type (UVS⁺) cells with a sublethal level of caffeine caused a significant increase in survival compared to untreated UV-irradiated cells. Caffeine did not affect survival in the repair-deficient strain UVSE1, which is deficient in repair of UV-induced damage carried out by enzymes associated with recombination during meiosis. A significant increase in survival in the presence of caffeine was observed in the repair-deficient strain UVSE4 in which recombination during meiosis is not affected. Treatment of zygotes homozygous for UVS⁺, UVSE1, or UVSE4 with sublethal levels of caffeine caused marked increases in recombination frequency in UVS⁺ and UVSE4 zygotes and no increase in recombination in UVSE1 zygotes. These results indicate that caffeine increases recombination in normal strains. Increased opportunity for recombination caused by caffeine would not result in increased recombination frequency in the UVSE1 strain, assuming limited-recombination enzyme activity in this strain. The observed increase in survival following UV-irradiation in the presence of caffeine in strains having normal recombination would therefore be associated with a caffeine-induced increase in opportunities for recombination repair.