UV survival of human mycoplasmas: evidence of dark reactivation in Mycoplasma buccale.

The inactivation by ultraviolet (UV) light irradiation of mycoplasma cells of five human strains was monitored by investigating the colony-forming ability. The survival curves of five strains tested indicated that the cells of Mycoplasma buccale only are single and homogenously susceptible to UV light. The effect of the repair inhibitor, caffeine, on the colony-forming ability of UV-irradiated cells was investigated with M. buccale because of its homogenous susceptibility to UV light. The colony formation of irradiated cells was markedly depressed by post-irradiation treatment with caffeine at concentrations that had little or no effect on the colony formation of unirradiated cells. The colony-forming units (CFU) of UV-irradiated cells which were kept in broth without caffeine in the dark increased without a lag as the time in the dark increased. The colony-forming ability of the irradiated cells completely recovered after 3 hr in the dark. However, when irradiated cells were kept in the presence of caffeine, no increase in their CFU was observed. The mode of action of caffeine on UV-irradiated cells closely resembles that described for other organisms which possess dark reactivation systems for UV-induced damage in deoxyribonucleic acid (DNA). Thus, the results obtained provide evidence for the existence of a dark repair function in M. buccale.     

The effect of post-treatment with caffeine on survival and UV-induced mutation frequencies in Chinese hamster and mouse lymphoma cells in vitro

The effect of post-treatment with caffeine on the survival of a number of cell lines after UV-irradiation has been studied. The mouse lymphoma cell lines P388 and L5178YS were sensitized by caffeine but only after UV doses of 50 erg/mm² and above. V79 cells also showed sensitization by caffeine but CHO cells and two cell lines YS and YR derived from Yoshida sarcoma of rats, sensitive and resistant to UV radiation, respectively, showed no effect.P388 and V79 cells were both mutable by UV, and caffeine, when studied at a single expression time (42–48 h) and at a single dose level (0.5 M and 0.75 M, respectively) suppressed the UV-induced mutation frequency in both cell lines. L51788YS cells although sensitized by caffeine showed no increase in frequency of thymidine-resistant (TdR^r) colonies when irradiated with UV.On more detaled examination, caffeine was found to delay the expression of UV-induced mutations inV79 cells, and the delay was dependent on the dose of caffine used. The effect on expression time was less when caffeine was present 0–48 h than when it was present throughout the post-irradiation incubation period. Similar results were obtained in P388 cells.The data are discussed in relation to those of other workers and to the concept that caffeine inhibits an error prone post-replication repair process in mammalian cells     

Death Through Respiratory Failure of a Fraction of Ultraviolet-Irradiated Escherichia coli B/r Cells

Escherichia coli B/r cells grown on a glycerol-containing medium and ultraviolet (UV)-irradiated to about 0.5% survival respire for about 1 hr and then cease for several hours. The cells that have completed repair and recovery processes begin to divide about 120 min after UV treatment, but this division is completely inhibited in liquid medium by caffeine, which delays repair of the irradiated deoxyribonucleic acid (DNA). When 5-fluorouracil (FU) is used to maintain respiration, the number of cells which form colonies when plated increases about 60-fold within 1 hr after irradiation. At least part of this increase does not involve repair while the cells are in the liquid medium because when caffeine is present there is still a 20-fold increase in colony formation. We conclude that many irradiated cells, although capable of carrying out complete and accurate repair of their DNA, die of respiratory failure; only when continuance of respiration is favored by FU treatment is their colony-forming potential realized. After an early increase, the number of cells able to form colonies in medium that contains FU remains constant while the completion of repair and recovery occurs. After these processes are completed, the number of cells able to form colonies increases slowly, except in the presence of caffeine, presumably because the late increase requires that repair steps take place while the cells are in liquid medium prior to cell division.     

Evidence for repair of ultraviolet-induced damage in Cystobacter species (Myxobacterales).

Cystobacter species strain CK 1 does not grow with more than 0.2 microgram/ml acriflavine. Spontaneous two-step mutants growing with 2 microgram acriflavine per ml have been selected. One mutant (strain CK3) was used to investigate the effect of repair inhibitors. Both strains exhibit pronounced shoulders in their UV dose curves of inactivation. Acriflavine (AF), coumarin (CU), and caffeine (CA) when incorporated in the post-irradiation plating medium decreased survival of irradiated cells. Post-treatment with 2 microgram acriflavine/ml abolished the shoulder of the curve. Caffeine (1600 microgram/ml) and coumarin (350 microgram/ml) reduced it only to about 40%. It is concluded that probably two repair mechanisms are present. Pre-treatment of the cells with 2 microgram acriflavine/ml for two hours before UV-irradiation resulted in a constant dose enhancement factor of 1.9. The protective effect is increased with the time of treatment with acriflavine. This may indicate that pyrimidine dimers are responsible for UV-inactivation.     

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.     

Effects of Caffeine on Radiation-Induced Phenomena Associated with Cell-Cycle Traverse of Mammalian Cells

Caffeine induced a state of G₁ arrest when added to an exponentially growing culture of Chinese hamster cells (line CHO). In addition to its effect on cell-cycle traverse, caffeine ameliorated a number of the responses of cells to ionizing radiation. The duration of the division delay period following X-irradiation of caffeine-treated cells was reduced, and the magnitude of reduction was dependent on caffeine concentration. Cells irradiated during the DNA synthetic phase in the presence of caffeine were delayed less in their exit from S, measured autoradiographically, and the radiation-induced reduction of radioactive thymidine incorporation into DNA was lessened. Cells synchronized by isoleucine deprivation, while being generally less sensitive to the effects of ionizing radiation than mitotically synchronized cells, were equally responsive to the effects of caffeine. The X-ray-induced reduction of phosphorylation of lysine-rich histone F1 was less in caffeine-treated cells than in untreated cells. Finally, survival after irradiation was only slightly reduced in caffeine-treated cells. A possible role of cyclic AMP in cell-cycle traverse of irradiated cells is discussed.     

Effect of acriflavine on ultraviolet inactivation of Acholeplasma laidlawii

An increased sensitivity to inactivation was observed when ultraviolet light-irradiated Acholeplasma laidlawiiAn increase sensitivity to inactivation was observed when ultraviolet light-irradiated Acholeplasma laidlawii cells were plated on medium containing either acriflavine or chloramphenicol. Chloramphenicol reduced liquid holding recovery (dark repair) to about 10 percent of that in untreated irradiated cells. In acriflavine treated cells no dark repair could be observed and there was a progressive degradation of cell DNA during holding. While the primary effect of acriflavine may be to inhibit excision repair, since ultraviolet-irradiated Mycoplasma gallisepticum (cells which lack an excision repair mechanism) show a slight increase in inactivation when plated on medium containing acriflavine, the dye must also have some other effects on ultraviolet repair processes. Acriflavine treatment of A. laidlawii cells before ultraviolet irradiation has a protective effect, as seen by an increased cell survival.     

Inhibition of post-replication repair of alkylated DNA by caffeine in Chinese hamster cells but not HeLa cells

Caffeine has been found to potentiate the lethal effects of sulphur mustard (SM) and N-methyl-N-nitrosourea (MNU) in a line of Chinese hamster cells but not in a line of HeLa cells. The sensitization of SM-treated cells by caffeine was S phase specific, and persisted for up to 24 h after alkylation of asynchronous cell cultures. The sensitization of MNU-treated cells, however, was not S phase specific but persisted for up to 50 h after the initial alkylation. Possible explanations for this difference between these two types of alkylating agent were discussed. Previously, evidence was presented which suggested that the alkylation-induced delay in the time of the peak rate of DNA synthesis in Chinese hamster cells was associated with the operation of post-DNA replication repair mechanism in these cells. Caffeine has now been found to reverse this alkylation-induced delay of DNA synthesis in both SM- and MNU-alkylated Chinese hamster cells. It is therefore proposed that caffeine sensitizes alkylated cells by inhibition of a post-replication DNA repair mechanism. No support was obtained for the alternative possibility that caffeine inhibits alkylation-induced excision repair of damaged DNA. The role of DNA repair in the production of the lethal mutagenic and cytological effects of alkylating agents is discussed.     

Evidence for caffeine-sensitive damage in methylazoxymethanol acetate-treated L5178Y cells.

The effects of methylazoxymethanol (MAM) acetate on colony survival, cell proliferation and DNA synthesis of murine lymphoma L5178Y cells are studied. Decreased sensitivity and immediate depression of cell proliferation and DNA synthesis were found in L5178Y cells in contrast to the reports on HeLa cells. Pre-labelling with 5-bromodeoxyuridine (BUdR) did not enhance significantly the carcinogen-induced cell lethality. Post-treatment with caffeine greatly enhanced cell lethality and depression of cell proliferation. These effects of caffeine were diminished when the cells had passed through two generations following the MAM acetate treatment. Experiments with synchronized cells showed that the action of caffeine was located primarily in S phase following the MAM acetate-treatment. These results strongly suggest that in L5178Y cells, MAM acetate induces damage, which is repaired by a mechanism analogous to post-replication repair of UV light-induced damage.     

Enhanced UV Sensitivity of Thiobacillus ferrooxidans Resulting from Caffeine and Acriflavine Treatment of Irradiated Cells

This study was aimed at identifying the roles of caffeine and acriflavine, two repair inhibitors, on UV sensitivity of iron-oxidizing Thiobacillus ferrooxidans ATCC 13728. The UV-dose response survival curve was inflected in nature, suggesting the population heterogeneity of the isolate. Caffeine and acriflavine potentiated the UV-induced killing of the organism. With the increase in concentrations of these compounds, the extent of survival decreased. Similarly, the inhibitory effects of caffeine and acriflavine increased with the increase in dose of UV-irradiation. The cells irradiated with 10 s (equivalent to 5.6 × 10⁻⁵ J/m²/s) of UV-exposure tended to become resistant to the inhibitory effects of caffeine and acriflavine, as evidenced by the time course study of recovery. The cells appear to stage a dramatic recovery from UV damage in the presence of caffeine (3.0 mg/ml) and acriflavine (20 μg/ml) over a period of 25–30h and 35–40h respectively, when grown in the presence of energy sources. Received: 4 December 2000/Accepted: 10 January 2001     

Protection of Normal, Lysogenic, and Pyocinogenic Strains Against Ultraviolet Radiation by Bound Acriflavine

The presence of bound acriflavine protects bacteria against the lethal effects of ultraviolet (UV) light, presumably because pyrimidine dimer formation is inhibited. Although acriflavine present in plating medium usually results in reduced viable counts from irradiated bacteria, no enhancement of lethal effects is observed when acriflavine is added to irradiated bacteria left in suspending buffer for 45 min before plating. Acriflavine remaining bound to the deoxyribonucleic acid of irradiated bacteria at the time they are plated likewise does not affect their survival. Protection is precisely dose-modifying unless some killing of bacteria by UV results from induction of prophage, against which bound acriflavine is less protective, or from induction of pyocin, against which there is no protection at all. It is inferred that prophage induction proceeds in part, and pyocin induction wholly, by virtue of effects of UV other than pyrimidine dimerization. The response of Escherichia coli strain B to radiation has been postulated to be attributable in part to induction of a prophage or a lethal protein; but exact dose modification was observed for this strain, to about the same extent, whether or not the irradiated organisms were grown in conditions thought to enhance the expected contribution to killing if such a mechanism were involved. Our results support the hypothesis that the inhibition by acriflavine of dimer formation is attributable to energy transfer mechanisms. They fail to support the hypothesis that shapes of survival curves (in particular the manifestation of "shoulders") can be attributed to inactivation by radiation of repair enzymes.     

Requirement for Protein Synthesis in rec-Dependent Repair of Deoxyribonucleic Acid in Escherichia coli after Ultraviolet or X Irradiation

Deprivation of amino acids required for growth or treatment with chloramphenicol or puromycin after irradiation reduced the survival of Rec(+) cells of Escherichia coli K-12 which had been exposed to either ultraviolet (UV) or X radiation. In contrast, these treatments caused little or no reduction in the survival of irradiated recA or recB mutants. The effect of chloramphenicol on the survival of X-irradiated cells was correlated with an inhibition of repair of single-strand breaks in irradiated deoxyribonucleic acid (DNA), previously shown to be controlled by recA and recB. In UV-irradiated cells no effect of chloramphenicol was detected on the repair of single-strand discontinuities in DNA replicated from UV-damaged templates, a process controlled by recA but not by recB. From this we concluded that inhibiting protein synthesis in UV or X-irradiated cells may interfere with some biochemical step in repair dependent upon the recB gene. When irradiated Rec(+) cells were cultured for a sufficient period of time in minimal growth medium before chloramphenicol treatment their survival was no longer decreased by the drug. After X irradiation this occurred in less than one generation time of the unirradiated control cells. After UV irradiation it occurred more slowly and was only complete after several generation times of the unirradiated controls. These observations indicated that replication of the entire irradiated genome was probably not required for rec-dependent repair of X-irradiated cells, although it might be required for rec-dependent repair of UV-irradiated cells.     

Mechanism of Caffeine Enhancement of Mutations Induced by Sublethal Ultraviolet Dosages

Certain chemical compounds increase mutation frequency of Escherichia coli B/r significantly when used in conjunction with nonlethal ultraviolet (UV) dosages. Studies were done to elucidate the mechanism of this enhancing mutational effect. Dark survival curves showed that 500 μg of caffeine per ml in the postirradiation medium markedly decreased survival to 60 ergs/mm² of UV in strain B/r. Caffeine did not markedly decrease survival to UV in strain B/r WP-2 hcr⁻. At least 90% of the mutations induced to streptomycin resistance by UV and 85% of those induced by UV with caffeine could be photoreversed. Experiments with thymine analogues suggested that thymine dimerization at the streptomycin locus was the primary premutational photoproduct induced by sublethal UV dosages. Caffeine did not interfere with the photoreversal of induced mutants, indicating that it probably does not bind to the photoreactivating enzyme or to a UV-induced lesion in the DNA. Addition of DNA or irradiated DNA with 500 μg of caffeine per ml resulted in no loss of the caffeine activity. The excision of UV-induced thymine-containing dimers from E. coli B/r T⁻ was investigated in the presence and absence of caffeine. Our results indicated that caffeine prevents excision of thymine dimers, presumably by binding to the excising enzyme. This binding results in an impairment of repair, which produces the increase in mutant numbers.     

Synthesis of inducible enzymes in irradiated yeast: UV effects on regulation and activity per cell

Summary The synthesis of two inducible enzymes in UV irradiated cells was determined during an 8 h postirradiation incubation. In contrast to the reduction of synthesis shortly after irradiation the effect after a longer period of incubation depends on the radiation sensitivity of the strain. Since exposure inhibits division leading to different cell number in controls and irradiated samples the data are also analysed on a per cell basis. A considerable increase of the activity per cell was observed. A maximum is reached at the end of the division delay.Part of the Ph.D. thesis of E.G. (D26)     

Host cell and ultraviolet reactivation of ultraviolet-irradiated Mycoplasmaviruses.

The mycoplasma Acholeplasma laidlawii was shown to have mechanisms for both host cell and ultraviolet (UV) reactivation of UV-irradiated mycoplasmaviruses. Host cell reactivation was examined by comparing the survival abilities of UV-irradiated double-stranded deoxyribonucleic acid mycoplasmavirus plated on both untreated and on acriflavine-treated cells. Acriflavine treatment inhibited cell exision repair. Decreased survival on the acriflavine-treated cells demonstrated host cell reactivation. UV reactivation was studied by comparing the survival of UV-irradiated virus plated on untreated cells with its survival on cells that received a small UV dose before plating. The UV-irradiated cells gave increased virus survival, showing UV reactivation. Similar experiments with a single-stranded deoxyribonucleic acid mycoplasmavirus showed that this virus could be UV reactivated, but not host cell reactivated.     

Characterization of Postreplication Repair in Mutagen-Sensitive Strains of DROSOPHILA MELANOGASTER

Mutants of Drosophila melanogaster, with suspected repair deficiencies, were analyzed for their capacity to repair damage induced by X-rays and UV radiation. Analysis was performed on cell cultures derived from embryos of homozygous mutant stocks. Postreplication repair following UV radiation has been analyzed in mutant stocks derived from a total of ten complementation groups. Cultures were irradiated, pulse-labeled, and incubated in the dark prior to analysis by alkaline sucrose gradient centrifugation. Kinetics of the molecular weight increase in newly synthesized DNA were assayed after cells had been incubated in the presence or absence of caffeine. Two separate pathways of postreplication repair have been tentatively identified by mutants derived from four complementation groups. The proposed caffeine sensitive pathway (CAS) is defined by mutants which also disrupt meiosis. The second pathway (CIS) is caffeine insensitive and is not yet associated with meiotic functions. All mutants deficient in postreplication repair are also sensitive to nitrogen mustard. The mutants investigated display a normal capacity to repair single-strand breaks induced in DNA by X-rays, although two may possess a reduced capacity to repair damage caused by localized incorporation of high specific activity thymidine-³H. The data have been employed to construct a model for repair of UV-induced damage in Drosophila DNA. Implications of the model for DNA repair in mammals are discussed.     

Analysis of Minimal Functions of Simian Virus 40 III. Evidence for “Host Cell Repair” of Oncogenicity and Infectivity of UV-Irradiated Simian Virus 40

The in vitro transforming capacity of simian virus 40 (SV40) for Syrian hamster cells is highly resistant to inactivation by UV light in comparison to infectivity. In the same cell system, we demonstrated a "host cell repair mechanism" sensitive to caffeine which is, to a large extent, responsible for the high resistance to UV inactivation of the transforming capacity of SV40. The survival of infectivity of UV-irradiated SV40 in CV-1 cells was also sensitive to caffeine, again indicating host cell repair. On the other hand, depression of normal cell DNA synthesis by hydroxyurea during the first 24 h postinfection only modestly reduced, and to a similar extent, the transforming capacity of UV-irradiated and nonirradiated SV40.     

Repair of deoxyribonucleic acid in ultraviolet light-sensitive and -resistant Dictyostelium discoideum strains.

Some responses of the cellular slime mold Dictyostelium discoideum to ultraviolet light (UV) irradiation were investigated by analyzing two aspects of deoxyribonucleic acid (DNA) excision repair in the vegetative cells: (i) the fate of thymine-containing dimers and (ii) the production and rejoining of single-strand breaks. Experiments were done with the parental, radiation-resistant NC-4 strain and with the radiation-sensitive gammas-13 strain. The majority (greater than 85%) of the thymine-containing dimers produced in both strains by an energy fluence of 100/Jm2 were removed from the acid-insoluble DNA fraction within the first 3 to 4 h of reincubation in the dark. Moreover, as measured by alkaline sucrose gradients, single-strand breaks appeared in the DNA of both NC-4 and gammas-13 irradiated cells very rapidly and at low temperatures. This was presumed to be a result of the incision (nicking) step of excision repair as performed by UV-specific endonuclease(s). In NC-4 the time required for dimer excision correlated with the sealing of breaks as well as with the UV-induced division delays. In gammas-13 the single-strand breaks were closed at a slower rate than in NC-4. However, this was not accompanied by more extensive division delays.     

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.