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.     

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     

Evidence Relating Cessation of Respiration, Cell Envelope Changes, and Death in Ultraviolet-Irradiated Escherichia coli B/r Cells

Ionic and nonionic detergents have little effect on respiring bacteria, but in cultures poisoned with KCN rapid solubilization of the cell membrane, as indicated by turbidity losses, takes place. Ultraviolet radiations cause Escherichia coli cells grown in minimal medium with glycerol as a carbon source to cease respiring and growing about 1 h after irradiation. We tested the effect of the nonionic detergent Triton X-100 on growth and cell membrane dissolution (both measured by turbidity changes), respiration, and viability of unirradiated and irradiated E. coli B/r cells. When the detergent was added to cells immediately after irradiation, a decrease in turbidity occurred only when respiration was about to cease; when it was added after cessation of respiration, the turbidity loss was immediate. In both cases the turbidity loss was about 60%, and disintegration of the cell walls did not take place. 5-Fluorouracil (FU) and thermal (42 C) treatments cause respiration of irradiated cells to be maintained and also cause viability increases. Irradiated cells treated with FU and detergent show no turbidity loss just prior to the time respiration normally ceases, but a loss does occur in irradiated cells incubated with detergent at 42 C. We conclude that FU maintains respiration for all of the cells, but that thermal treatment maintains respiration for only part of the cells. In all cases the detergent had only a negligible effect on the respiration and viability of unirradiated and irradiated cells. We conclude that Triton X-100 causes solubilization of cell membranes of only nonrespiring cells that are not destined to survive.     

Chemical radiosensitization by misonidazole: production and repair of DNA single-strand breaks in Yoshida ascites tumor cells.

Formation of strand-breaks in DNA and its repair in Yoshida ascites tumor cells exposed to gamma radiation (100-400 Gy) in presence and absence of misonidazole (10 mM) were studied. The methodology involved pre-labelling of cellular DNA by 3H-thymidine during cell proliferation in rats, irradiation of cells in vitro and analysing sedimentation profile of DNA by ultracentrifugation in alkaline sucrose density gradients. Irradiation under euoxic conditions resulted in formation of about 1.5 times greater number of strand breaks as compared to those formed during irradiation under hypoxic conditions. Misonidazole (10 mM) by its presence along with the cells during irradiation under hypoxic conditions caused a 3-fold increase in the number of single strand breaks, but under euoxic conditions of irradiation the presence of misonidazole did not enhance the strand break formation. Incubation of cells irradiated in absence of misonidazole for 1 hr in tissue culture medium at 37 degrees C resulted in repair of substantial fraction of the strand breaks while there was no repair of the DNA strand breaks in cells irradiated in the presence of the chemical.     

An Enrichment for Temperature-Sensitive Mutants in TETRAHYMENA THERMOPHILA

The parameters for the killing of Tetrahymena by 5-bromodeoxyuridine(BUdR) and near-ultraviolet light have been determined. Significant preferential killing by UV of cells that have incorporated BUdR was obtained when the cells were irradiated in a nonnutrient buffer. UV alone was found to be toxic to cells irradiated in growth medium. Mutants defective in division at a restrictive temperature were isolated from mutagenized cultures that had been treated with BUdR and UV and from mutagenized cultures that had no such treatment. Results indicate that the number of temperature sensitive (ts) growth mutants can be increase five to six times using the BUdR/UV treatment. Data are presented that indicate differences in the frequency of occurrence of various types of ts mutants, with and without enrichment. A mutant that immediately stopped macromolecular synthesis and cell division upon being placed at the restrictive temperature was more resistant to BUdR/UV treatment than wild type by 1000-fold. Using the above techniques, BUdR-resistant mutants altered in the phosphorylation of thymidine have been isolated.     

Studies on the control of cell division in a green alga, Ankistrodesmus gracilis (Chlorococcales) III. Induction synchrony with controlled division patterns

When stagnant cells of Ankistrodesmus gracilis obtained froma standard culture were inoculated into the basal medium atcell densities lower than 1.0 ? 10⁷ cells/ml, cell proliferationoccurred stepwise at time intervals of about 30 hr. At a densityof 5.5 ? 10⁴cells/ml, the increase in cell number per step wasabout 2.7-fold. When inoculated into a glutamine medium thetime interval was 24 hr, and the average increase of cell numberwas about 4-fold. When cells were preincubated at about 5.0? 10⁵ cells per ml in the basal medium for 30 hr, then transferredinto a glutamine or arginine medium at about 7.0 ? 10⁶ cells/ml,synchronous division occurred about 18 hr later with binaryfission or about 33 hr later with multiple fission, respectively. (Received May 16, 1979; )     

Induction of sister chromatid exchanges by UV light and its inhibition by caffeine

Sister chromatid exchanges in Chinese hamster chromosomes were studied after pulse-labeling cells with ³H-thymidine at various concentrations. Whereas the frequency of chromatid aberrations varied widely, depending upon tritium dose, there was no significant change in the sister chromatid exchange frequency, even with a 40-fold range of variation in the tritium concentration in the medium. When cells were exposed immediately after labeling to UV light at 40 erg/mm² and examined at the second mitosis, the frequency of sister chromatid exchanges was found to be 4 times higher than that of the unirradiated controls. A synchronization treatment utilizing 2 mM thymidine also caused a two-fold rise in the exchange frequency above the control level. Furthermore, when synchronized cells were irradiated with UV light at a dose of 40 erg/mm², the exchange frequency exceeded 5 times that of the untreated controls. However, this effect was detectable only when cells were irradiated at the earlier part of the S phase, while no change was detected when irradiated at the late S or G2 phase. A post-treatment of irradiated cells with caffeine caused a remarkable decrease in the frequency of sister chromatid exchanges. On the other hand, the frequency of chromatid aberrations of the deletion type increased strikingly after the same treatment. The results appear to suggest a certain correlation between the mechanism involved in the induction of sister chromatid exchanges and a post-replication repair of DNA damage.     

GROWTH OF THE BARLEY COLEOPTILE. I. ITS RELATIONSHIP TO CELL NUMBER AND LENGTH IN NORMAL AND γ-IRRADIATED SEEDLINGS

The number of cells per vertical column in barley coleoptiles differs in various growth classes; it is highest in tall coleoptiles, intermediate in medium ones, and lowest in short ones. In those that elongate early and grow rapidly, cells per column increased from 88–218 between 12 and 44 hr after the seeds were placed on water; in short coleoptiles they increased to only 85 per column after 115 hr because elongation and division are restricted in these. Cell number does not increase in coleoptiles from seeds irradiated with 250 krad. Variation in the growth pattern of irradiated coleoptiles was similar to that of normal ones, although the range in lengths was reduced. Although the number of cells is much higher in tall controls than in irradiated coleoptiles, the latter can become tall; therefore an increase in cell number during germination does not seem to be a prerequisite for tallness. Coleoptiles 32 mm long, from irradiated seeds, have the same number of cells per column as the shortest ones (6 mm) after 119 hr.     

Daughter Cell Repair by Mammalian Cells in Culture after Potentially Lethal Radiation Damage

COLONY formation by irradiated surface-attached mammalian cells in culture requires that at least one of the progeny produced at the first post-irradiation division retains the capacity for unlimited division. Abortive colonies result when the irradiated parent cell has experienced sufficient damage to suppress eventual colony formation, but not to prevent the production of a small number of progeny. We now present data which indicate that, if given a suitable environment, progeny from cells “lethally” damaged by X-radiation (not capable of producing a macroscopic colony) can repair damage transferred (sectored) from the parent cell.     

Postirradiation recovery dependent on the uvr-1 locus in Bacillus subtilis.

A mutant (uvr-1) of Bacillus subtilis that is deficient in excision of ultraviolet (UV)-induced pyrimidine dimers from deoxyribonucleic acid (DNA) shows a marked increase in ability to survive UV irradiation when plated on amino acid-supplemented agar medium compared with its survival ability when plated on nutrient plating medium, the effect is considered to be one of growth-dependent lethality. Irradiated stationary phase uvr-1 cells, incubated in liquid medium lacking amino acids required for growth, recover from this sensitivity to rich medium within 3 to 4 h after irradiation. Recovery is greatly reduced in the absence of glucose oiminated. Exponentially growing cells have a limited ability to recover from sensitivity to rich medium. Growth-dependent lethality can also occur in liquid medium. In nutrient broth the ability of irradiated stationary-phase uvr-1 cells to form colonies on defined agar medium decreases during postirradiation incubation, but treatmeth with chloramphenicol inhibits the loss of colony-forming ability. Recovery from sensitivity to rich media is inhibited by caffeine but not by 6-(p-hydroxyphenylazo)-uracil, and inhibitor of DNA replication. Alkaline sucrose gradient profiles show that conditions allowing recovery also favor maintaining intact DNA strands, whereas DNA strand breakage or degradation is associated with loss of viability. Recovery from sensitivity to rich medium has not been observed in the Ur+ parent or in strains carrying the mutations uvs-42 (another deficiency in dimer excision), recA1, or polA59. A uvr-1 recA1 mutants shows a higher level of recovery than does the recA1 single mutant, but a much lower level than the uvr-1 single mutant. Apparently, both the uvr-1 defect and Rec+ and PoII+ functions are essential for recovery from sensitivity to rich medium. For optimal recovery, growth immediately after irradiation must be delayed. The process requires energy, apparently involves recombination, and probably results in rejoining of DNA strands in which incision but not excision has occurred.     

3T3-L1 adipocytes promote the growth of mammary epithelium

Murine mammary epithelium grows in association with predominantly adipocyte stroma in vivo. To investigate potential growth-promoting effects of adipocytes on mammary epithelium, we developed a co-culture system of mammary epithelium and adipocytes by taking advantage of the 3T3-L1 cell line. These cells undergo adipocyte differentiation when the culture reaches confluence and growth ceases. Mid-pregnant murine mammary epithelium was plated on lethally irradiated feeder layers of 3T3-L1 adipocytes, undifferentiated 3T3-L1 cells, 3T3-C2 fibroblasts (a subclone of 3T3 cells that does not undergo adipocyte differentiation), or tissue culture plastic. Mammary epithelial colony size on adipocyte feeder layers was 2-fold larger than colonies on 3T3-C2 cells and 4-fold larger than colonies on tissue culture plastic. Measurement of tritiated thymidine [3H]TdR incorporation and labelling index in mammary cells was significantly higher on adipocytes than on other feeder layers or plastic. There was a 6-fold increase in mammary cell number after 5 days in culture when mammary epithelium was plated on substrate-attached material ('extracellular matrix') derived from 3T3-L1 cells and a 4-fold increase in cell number when plated on plastic in conditioned medium derived from 3T3-L1 adipocytes compared with growth on plastic in unconditioned medium. We conclude that interaction of mammary epithelium with adipocytes results in a marked increase in proliferation of mammary epithelium and that extracellular components may mediate this effect.     

Radiation-induced mitotic and meiotic aneuploidy in the yeast Saccharomyces cerevisiae.

A number of genetic systems are described which in yeast may be used to monitor the induction of chromosome aneuploidy during both mitotic and meiotic cell division. Using these systems we have been able to demonstrate the induction of both monosomic and trisomic cells in mitotically dividing cells and disomic spores in meiotically dividing cells after both UV light and X-ray exposure. The frequency of UV-light-induced monosomic colonies were reduced by post-treatment with photoreactivity light and both UV-light- and X-ray-induced monosomic colonies were reduced by liquid holding post-treatment under non-nutrient conditions. Both responses indicate an involvement of DNA-repair mechanisms in the removal of lesions which may lead to monosomy in yeast. This was further confirmed by the response of an excision-defective yeast strain which showed considerably increased sensitivity to the induction of monosomic colonies by UV-light treatment at low doses. Yeast cultures irradiated at different stages of growth showed variation in their responses to both UV-light and X-rays, cells at the exponential phase of growth show maximum sensitivity to the induction of monosomic colonies at low doses whereas stationary phase cultures showed maximum induction of monosomic colonies at high does. The frequencies of X-ray-induced chromosome aneuploidy during meiosis leading to the production of disomic spores was shown to be dependent upon the stage of meiosis at which the yeast cells were exposed to radiation. Cells which had proceeded beyond the DNA synthetic stage of meiosis were shown to produce disomic spores at considerably lower radiation doses than those cells which had only recently been inoculated into sporulation medium. The results obtained suggest that the yeast sustem may be suitable for the study of sensitivities of the various stages of meiotic cell division to the induction of chromosome aneuploidy after radiation exposure.     

Lack of synergistic effect between X-ray and UV irradiation on the frequency of chromosome aberrations in PHA-stimulated human lymphocytes in the G1 stage.

PHA-stimulated human lymphocytes in the G1 stage were irradiated with UV radiation and X-rays, and the cells were analyzed for chromosomal aberrations in the first mitotic division. The frequency of dicentric chromosomes after single X-irradiation in the G1 stage was about twice the yield in the G0 stage. No increase in the yield of dicentrics was observed after combined irradiation with UV and X-rays. This is contrary to the finding for G0 lymphocytes, where a 2-fold increase of chromosome aberrations was observed. UV irradiation of G1 lymphocytes induced chromatid-type aberrations whereas no significant yield of dicentric chromosomes was observed. This is in agreement with previous findings in Chinese hamster cells in the G1 stage [7]. Irradiation of G0 lymphocytes with UV radiation induce a low frequency of dicentric chromosomes. Thus, the present data indicate that the ratio between chromosome-type and chromatid-type aberrations is different in the G1 and G0 stages in human lymphocytes irradiated with UV radiation.     

Effect of Rehydration on Recovery, Repair, and Growth of Injured Freeze-Dried Salmonella anatum

From 70 to 90% of the Salmonella anatum cells that survived freeze-drying in nonfat milk solids were injured. After rehydration, these injured survivors failed to grow on a selective plating medium containing deoxycholate but could form colonies on a nonselective medium. In a suitable environment after rehydration, injury disappeared in most of these cells. The rate of this repair at 25 C was very rapid initially and, in a medium containing milk solids, was completed within 1 hr after rehydration. The repaired cells initiated growth about 1 hr later than normal cells and grew at a slower rate. In a medium containing milk solids, initial recovery, extent of repair of injury, initiation of growth, and rate of growth were not influenced by supplementation with extra nutrients in other rehydration media. Rehydration controlled by modifying the concentrations of lactose, sucrose, or milk solids in the rehydration medium influenced the recovery of cells and the time that growth was initiated. Glycerol failed to increase recovery. Higher numbers of cells were recovered by rehydrating at 15 to 25 C, but an earlier initiation of growth and more rapid growth were observed at 35 C.     

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.     

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.     

Postreplication Repair of Ultraviolet Damage to DNA, DNA-Chain Elongation, and Effects of Metabolic Inhibitors in Mouse L Cells

Alkaline sucrose sedimentation studies of DNA from mouse L cells have demonstrated the following effects of several inhibitors of nucleic acid and protein synthesis on postreplication repair of ultraviolet (UV) damage to their DNA. The DNA newly synthesized by a 2 h [³H]thymidine (dThd) label following 254 nm UV irradiation of 20 J/m² is made in smaller segments of the number average mol wt (Mn) of ~10 × 10⁶ than the control of ~40 × 10⁶. The presence of caffeine at a concentration of 2 mM during the labeling of the irradiated cells reduces the Mn value to 5.8 × 10⁶, which is nearly comparable to, but somewhat larger than the expected distance between dimers in parental DNA. Afterwards, such an interrupted DNA made in the irradiated cells is completely repaired to the present maximum Mn value of 40 × 10⁶ in the consecutive 4 h chase in unlabeled dThd. The presence of the nucleic acid inhibitor, either 2 mM hydroxyurea, 50 μM arabinofuranosyl cytosine, 2 mM excess dThd or 5 μg/ml of actinomycin D (AMD) during 2- to 24-h chase periods after a 2 h postirradiation label prevents the repair to various extents, while 2 mM caffeine completely inhibits it. In the unirradiated cells, these agents except excess dThd and caffeine also interfere severely with normal elongation of nascent DNA made by a 3 min pulse label, but do not appreciably induce single chain breaks of either newly synthesized or parental DNA. The inhibition of the repair by AMD suggests that de novo elongation of DNA to close the gaps in new DNA made in the irradiated cells requires at least a template-dependent DNA polymerase. In contrast, 100 μg/ml of cycloheximide allows to complete the gap-filling repair, while it simply reduces the rates of chain growth for the repair and normal replication. Therefore, the similar sensitivity of gap-filling repair and normal replication towards the above inhibitors indicates that a preexisting DNA polymerizing system appears to be responsible and to play a common role without new protein synthesis, as far as the repair at early time after UV is concerned.     

Post-irradiation modification of radiation-induced heteroallelic reversion in diploid yeast: Effect of nutrient broth

The effect of post-irradiation growth in complete rich medium on the expression of the reversion to arginine-independence induced by gamma and alpha radiation in a heteroallelic diploid yeast strain (Saccharomyces cerevisiae BZ34) has been studied. During the post-irradiation treatment the reversion frequency increased, reached a peak at about 90 min and decreased thereafter reaching a constant value for treatment periods exceeding 6 h. As determined by the increase in number of budding cells, extensive DNA synthesis took place in cells incubated only in the nutrient medium and not in the omission medium. Hence the observed increase in the reversion frequency is explained on the basis that post-irradiation DNA synthesis is necessary for the expression of gene conversion. The decrease in the reversion frequency for continued treatment with yeast extract, peptone, dextrose (YEPD) is related to the fact that only one daughter of the post-irradiation first cell division is a revertant.The broth effect was not lost when the irradiated cells were first incubated for 90 min in arginine-less medium and then transferred to the broth. Similarly, the broth effect persisted even at doses high enough to induce considerable division delay. These results suggest that the radiation-induced pre-conversional lesions are not susceptible to repair by alternative pathways.