Effect of UV light on DNA replication and chain elongation in Chinese hamster UV61 cells

Exposure of eukaryotic cells to ultraviolet light results in a temporary inhibition of DNA replication as well as a temporary blockage of DNA fork progression. Recently there has been considerable debate as to whether the (5-6)cyclobutane pyrimidine dimer, the pyrimidine(6-4)pyrimidone lesion or both are responsible for these effects. Using cell lines that repair both of these lesions (CHO AA8), only (6-4) lesions (CHO UV61) or neither (CHO UV5), we have shown that in rodent cells both lesions appear to play a role in both the inhibition of thymidine incorporation and the blockage of DNA fork progression. Specifically, after exposure to 2.5 J/m2, AA8 cells recover normal rates of DNA replication within 5 h after exposure, while UV5 cells exhibit a greater depression in thymidine incorporation for at least 10 h. UV61 cells, on the other hand, show an intermediate response, both with respect to the extent of the initial depression and the rate of recovery of thymidine incorporation. UV61 cells also exhibit an intermediate response with respect to blockage of DNA fork progression. In previous publications we have shown that UV5 cells exhibit extensive blockage of DNA fork progression and only limited recovery of this effect within the first 5 h after exposure to UV. In this report we show that UV61 cells exhibit a more extensive blockage of fork progression than is observed in AA8 cells. These blocks also appear to be removed (or overcome) more slowly than in the AA8 cells, but more rapidly than in UV5 cells. Taken together we conclude that both lesions appear to be involved in the initial depression in thymidine incorporation and the initial blockage of DNA fork progression in rodent cells. These data also indicate that (6-4) lesions may be responsible for the prolonged depression in thymidine incorporation and the prolonged blockage of DNA fork progression observed in UV5 cells.     

Kinetics of replicon initiation during S phase of Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells were synchronized by a thymidine-hydroxyurea block. At different times after release from the block, cells were treated with trioxsalen and long-wavelength ultraviolet light to crosslink DNA in vivo and were labelled with [³H]thymidine for 30 min. This technique permits labelling of only the short nescent DNA fragments initiated between crosslinks. The amount of radioactivity incorporated in these fragments during the labelling period reflects the number of replicon initiation events and allows us to follow the replicon initiation pattern after removing the inhibitor. It was shown that the rate of initiation was high at the beginning of S phase and then steadily decreased.     

Effects of UV light on DNA chain growth and replicon initiation in human cells

Exposure of mammalian cells to 254 nm UV light produces lesions that block DNA polymerases at least on the leading strand. For rodent cells the extent and duration of this blockage is both cell line- and fluence-dependent. Using DNA fiber autoradiography we report here similar findings for human cells. Wild-type human cells did not exhibit significant blockage following exposure to 2.5 J/m2. After exposure to 5.0 J/m2, there was significant blockage immediately after exposure, but by 5 h segment lengths returned to control values. Excision-deficient human cells, on the other hand, exhibited significant blockage both immediately and 5.0 h after exposure to 2.5 J/m2. Exposure of rodent cells to UV light is also known to activate alternative sites of replication. Such activation would enable cells to replicate areas of DNA which do not contain a 'normal' site of initiation, yet contain blocking lesions both upstream and downstream. We have previously shown (Griffiths and Ling, 1987) that this activation is more pronounced and long-lived in excision-deficient Chinese hamster ovary (CHO) cells than it is in wild-type CHO cells. We report here that excision-deficient human cells also exhibited a marked and prolonged activation of alternative sites of replicon initiation. Wild-type human cells, on the other hand, exhibited little if any activation.     

Continued initiation of DNA synthesis in arginine-deprived Chinese hamster ovary cells

When exponentially growing CHO cells were deprived of arginine (Arg), cell multiplication ceased after 12 h, but initiation of DNA synthesis continued: after 48 h of starvation with continuous [3H]thymidine exposure, 85% of the population had incorporated label, as detected autoradiographically. Consideration of the distribution of exponential cells in the various cell cycle phases leads to a calculation that most cells in G1 at the time that Arg was removed, as well as those in S, engaged in some DNA synthesis during starvation. In contrast, isoleucine (Ile)-starved cells did not initiate DNA synthesis, as has been reported by others. Experiments with cells synchronized by mitotic selection confirmed this difference in Arg- and Ile- deprived behavior, but also showed that cells which underwent the mitosis leads to G1 transition during Arg starvation remained arrested in G1 (G0?). The results suggest that Arg-deprived cells continue to maintain some proliferative function(s) while Ile-deprived cells do not.     

Effect of UVC light on growth, incorporation of thymidine, and DNA chain elongation in cells derived from the Indian meal moth and the cabbage looper.

After exposure to 10 or 20 J/m2 UVC light, cells of the UMN-PIE-1181 line, an embryonic cell line derived from the Indian meal moth, Plodia interpunctella, exhibited a rapid and prolonged depression in the rate of incorporation of [3H]thymidine, whereas cells of the TN-368 line, an ovarian cell line derived from Trichoplusia ni, the cabbage looper, showed only a slight drop in incorporation and a rapid recovery after exposure to 10 or 40 J/m2 UVC light. The extent of this depression was not correlated to the amount of cell killing by UVC light in these cell lines or in IAL-PID2 cells. Blockage of fork progression was correlated to the depression in thymidine incorporation. TN-368 cells exhibited little blockage after exposure to 10 or 20 J/m2 UVC light, whereas UMN-PIE-1181 cells exhibited significant blockage at these fluences. Photoreactivation did not entirely relieve blockage, depression in thymidine incorporation, or cell killing, indicating that, although the (5-6) dimer appears to be the major lesion responsible for these effects, other lesions such as the (6-4) photoproduct may play a role.     

Independence of centriole formation and initiation of DNA synthesis in Chinese hamster ovary cells

The relationship between centriole formation and DNA synthesis was investigated by examining the effect of taxol on the centriole cycle and the initiation of DNA synthesis in synchronized cells. The centriole cycle was monitored by electron microscopy of whole-mount preparations [Kuriyama and Borisy, J. Cell Biol., 1981, 91:814-821]. A short daughter centriole appeared in perpendicular orientation to each parent during late G1 or early S and elongated slowly during S to G2. Addition of 5-20 micrograms/ml taxol to a synchronous population of cells in S phase did not inhibit centriole elongation; rather, elongation was accelerated. In contrast, when taxol was added to M phase or early G1 cells, centriole duplication was completely inhibited. The taxol block was reversible since nucleation and elongation of centrioles resumed as soon as the drug was removed. Cells exposed to taxol progressed through the cell cycle and became blocked in mitosis, as indicated by an increase in the mitotic index, but eventually the mitotic arrest was overcome, resulting in formation of multinucleated cells. A peak in mitotic index was seen in the following generation, indicating that chromosomes duplicated in the presence of taxol. Incorporation of 3H-thymidine followed by autoradiography confirmed that DNA synthesis was initiated in the presence of taxol even though formation of daughter centrioles was inhibited. It seems, therefore, that centriole duplication is not a prerequisite for entry into S phase. Since DNA synthesis has already been demonstrated not to be necessary for centriole duplication, these two events, normally coordinated in time, appear to be independent of each other.     

Effect of UV light on sister-chromatid exchanges, activation of alternative sites of replicon initiation and thymidine incorporation in CHO AA8, UV61 and UV5 cells.

The relative importance of the UV-induced pyrimidine(5-6)pyrimidine and the pyrimidine(6-4)-pyrimidone lesions in sister-chromatid exchanges (SCEs), activation of alternative sites of replicon initiation and thymidine incorporation were examined using wild-type Chinese hamster ovary (CHO) AA8 cells which remove both lesions, mutant CHO UV61 cells which remove only the (6-4) lesion and mutant CHO UV5 cells which remove neither lesion. Our data suggest that both lesions play a role in each end point examined. The relative importance of these lesions is dependent on the end point studied as well as the fluence used. For SCE induction and the activation of alternative sites of replicon initiation, the (6-4) lesion appears to play a predominant role, while for the thymidine incorporation studies the (6-4) lesion appears to play the predominant role at low fluences while the role of the (5-6) lesion increases at higher fluences.     

DNA base sequence changes induced by ultraviolet light mutagenesis of a gene on a chromosome in Chinese hamster ovary cells

The DNA base sequence changes induced by mutagenesis with ultraviolet light have been determined in a gene on a chromosome of cultured Chinese hamster ovary (CHO) cells. The gene was the Escherichia coli gpt gene, of which a single copy was stably incorporated and expressed in the CHO cell genome. The cells were irradiated with ultraviolet light and gpt- colonies were selected by resistance to 6-thioguanine. The gpt gene was amplified from chromosomal DNA by use of the polymerase chain reaction (PCR), and the amplified DNA sequenced directly by the dideoxy method. Of the 58 sequenced mutants of independent origin 53 were base change mutations. Forty-one base substitutions were single base changes, ten had two adjacent (or tandem) base changes, and one had two base changes separated by a single base-pair. Only one mutant had a multiple base change mutation with two or more well separated base changes. In contrast much higher levels of such mutations were reported in ultraviolet mutagenesis of genes on a shuttle vector in primate cells. Two deletions of a single base-pair were observed and three deletions ranging from 6 to 37 base-pairs. The mutation spectrum in the gpt gene had similarities to the ultraviolet mutation spectra for several genes in prokaryotes, which suggests similarities in mutational mechanisms in prokaryotes and eukaryotes.     

Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells

Acidification of endocytic compartments is necessary for the proper sorting and processing of many ligands and their receptors. Robbins and co-workers have obtained Chinese hamster ovary (CHO) cell mutants that are pleiotropically defective in endocytosis and deficient in ATP- dependent acidification of endosomes isolated by density centrifugation (Robbins, A. R., S. S. Peng, and J. L. Marshall. 1983. J. Cell Biol. 96:1064-1071; Robbins, A. R., C. Oliver, J. L. Bateman, S. S. Krag, C. J. Galloway, and I. Mellman. 1984. J. Cell Biol. 99:1296-1308). In this and the following paper (Yamashiro, D. J., and F. R. Maxfield. 1987. J. Cell Biol. 105:2723-2733) we describe detailed studies of endosome acidification in the mutant and wild-type CHO cells. Here we describe a new microspectrofluorometry method based on changes in fluorescein fluorescence when all cellular compartments are equilibrated to the same pH value. Using this method we measured the pH of endocytic compartments during the first minutes of endocytosis. We found in wild- type CHO cells that after 3 min, fluorescein-labeled dextran (F-Dex) was in endosomes having an average pH of 6.3. By 10 min, both F-Dex and fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) had reached acidic endosomes having an average pH of 6.0 or below. In contrast, endosome acidification in the CHO mutants DTG 1-5-4 and DTF 1-5-1 was markedly slowed. The average endosomal pH after 5 min was 6.7 in both mutant cell lines. At least 15 min was required for F-Dex and F-alpha 2M to reach an average pH of 6.0 in DTG 1-5-4. Acidification of early endocytic compartments is defective in the CHO mutants DTG 1-5-4 and DTF 1-5-1, but pH regulation of later compartments on both the recycling pathway and lysosomal pathway is nearly normal. The properties of the mutant cells suggest that proper functioning of pH regulatory mechanisms in early endocytic compartments is critical for many pH-mediated processes of endocytosis.     

Influence of exogenous thymidine on killing and mutation of Chinese hamster V-79 cells by X-rays, ultraviolet light and N-methyl-N'-nitro-N-nitrosoguanidine

V-79 cells when exposed to thymidine (5 micrograms/ml) in growth medium after treatment with X-rays, UV light and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), responded differently depending upon the agent. For treatment with X-rays and UV light, only induction of mutation was potentiated, but for MNNG treatment, both killing and mutation induction were potentiated. The increase in killing of MNNG exposed cells could be reversed by simultaneous addition of deoxycytidine with thymidine, but, for all the three mutagenic treatments, enhancement in mutation induction could not be suppressed by deoxycytidine.     

Sodium arsenite enhances the cytotoxicity, clastogenicity, and 6-thioguanine-resistant mutagenicity of ultraviolet light in Chinese hamster ovary cells

Cytotoxicity, chromosome aberrations, and mutations to 6-thioguanine resistance were synergistically increased by incubating the ultraviolet light (UV)-irradiated Chinese hamster ovary (CHO) cells in medium containing sodium arsenite. However, the frequencies of sister-chromatid exchanges and mutations to ouabain resistance induced by UV were not synergistically increased by sodium arsenite. The synergistic effect of sodium arsenite on UV-induced chromosome aberrations varied with cell-harvesting time and decreased with increasing time intervals between UV and sodium arsenite treatments.     

Inhibition of DNA chain elongation in a purine-auxotrophic mutant of Chinese hamster

DNA synthesis has been examined in a purine-auxotrophic mutant cell line of Chinese hamster (V79 pur 1) under conditions of purine deprivation. At 6 h after the removal of purines from the growth medium, there is a decrease in semiconservative DNA replication. Alkaline velocity centrifugation of the DNA synthesized during a 1-min pulse under conditions of purine deprivation shows that approximately 50% of the newly replicated DNA is the size of Okazaki pieces. These are not incorporated into bulk DNA during a 1-h chase. If the purine supply is restored to the growth medium, these short DNA pieces are jointed to full-sized DNA within 1 h. DNA fiber autoradiolgraphy reveals a retardation in the rate of DNA replication fork movement but no apparent inhibition of initiation of synthesis on replication units within clusters actively engaged in replication. Our results indicate that purine deprivation specifically inhibits elongation of nascent dna chains.     

Effect of exogenous thymidine on sister-chromatid exchange frequency in Chinese hamster ovary cells with bromodeoxyuridine- and chlorodeoxyuridine-substituted chromosomes

There are conflicting reports on the effect of exogenous thymidine (dThd) on the frequency of sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Thymidine has been reported either to increase or to have no effect on SCE frequency under similar experimental conditions. To resolve this controversy, we have carried out a series of experiments to examine the effect of dThd on CHO cells cultured with 5-bromodeoxyuridine (BrdUrd). In addition, we have examined the effect of dThd on CHO cells cultured with 5-chlorodeoxyuridine (CldUrd), a much more potent inducer of SCEs than BrdUrd. The addition of 100 microM dThd to the culture medium caused a consistent decrease in the yield of SCEs in cells grown in BrdUrd for two cell cycles. The decrease was even greater when cells were grown in dThd and CldUrd. Analysis of twin and single SCEs indicated that dThd must be present during the first cell cycle to reduce the frequency of SCEs. Because excess dThd is thought to have an effect when DNA replicates on a template substituted with a halogenated nucleoside, dThd at concentrations from 100 microM to 9 mM was added to cultures for the second cell cycle after a first cell cycle in BrdUrd. In this experiment, the presence of dThd increased SCE frequency in a dose-dependent manner. The results suggest that if dThd competes with halogenated nucleosides and thus decreases their incorporation into DNA, SCEs are suppressed in the subsequent cell cycle, whereas if excess dThd creates a dNTP pool imbalance, SCEs can be increased.     

Ethanol and acetaldehyde potentiate the clastogenicity of ultraviolet light, methyl methanesulfonate, mitomycin C and bleomycin in Chinese hamster ovary cells

Ethanol itself did not induce any apparent chromosome aberrations in Chinese hamster ovary cells. However, posttreatment with ethanol potentiated the chromosome aberrations induced by ultraviolet light (UV), methyl methanesulfonate (MMS), mitomycin C (MMC) or bleomycin (BLM). Chromatid exchanges were predominantly increased in cultures treated with UV, MMS or MMC and then with ethanol, whereas chromosome breaks and chromatid exchange were the major types of aberrations increased in the cultures treated with BLM and ethanol. Posttreatment with acetaldehyde, the major metabolite of ethanol, also potentiated the chromosome aberrations induced by UV, MMS, MMC or BLM. The main types of aberrations potentiated by posttreatment with acetaldehyde were similar to those by posttreatment with ethanol.     

Mutagen-induced changes in cellular deoxycytidine triphosphate and thymidine triphosphate in Chinese hamster ovary cells

Deoxynucleoside triphosphate concentrations in Chinese hamster ovary cell lines, CHO-K1 and Mut 8–16, were examined following exposure of cells to UV or dimethylsulfate. Marked decreases in dCTP were observed 2 hr after exposure to both mutagens. In contrast, dTTP concentrations increased with increased cell killing after exposure to UV but not after exposure to dimethylsulfate. Examination of DNA synthesis in permeabilized cells in the presence of excess concentrations of dNTP substrates suggests that excess dCTP enhances replication while excess of dTTP inhibits replication. We therefore ask whether the increase in the $\text{dTTP}\text{dCTP}$ ration in mutagenized whole cells either contributes to or prolongs induced inhibition of replication. In addition we proposed that such an induced dNTP imbalance may also contribute to an increase in mutations by enhancing the probability for base-misincorporation.     

Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants

During endocytosis in Chinese hamster ovary (CHO) cells, Semliki Forest virus (SFV) passes through two distinct subpopulations of endosomes before reaching lysosomes. One subpopulation, defined by cell fractionation using free flow electrophoresis as "early endosomes," constitutes the major site of membrane and receptor recycling; while "late endosomes," an electrophoretically distinct endosome subpopulation, are involved in the delivery of endosomal content to lysosomes. In this paper, the pH-sensitive conformational changes of the SFV E1 spike glycoprotein were used to study the acidification of these defined endosome subpopulations in intact wild-type and acidification-defective CHO cells. Different virus strains were used to measure the kinetics at which internalized SFV was delivered to endosomes of pH less than or equal to 6.2 (the pH at which wild-type E1 becomes resistant to trypsin digestion) vs. endosomes of pH less than or equal to 5.3 (the threshold pH for E1 of the SFV mutant fus-1). By correlating the kinetics of acquisition of E1 trypsin resistance with the transfer of SFV among distinct endosome subpopulations defined by cell fractionation, we found that after a brief residence in vesicles of relatively neutral pH, internalized virus encountered pH less than or equal to 6.2 in early endosomes with a t1/2 of 5 min. Although a fraction of the virus reached a pH of less than or equal to 5.3 in early endosomes, most fus-1 SFV did not exhibit the acid-induced conformational change until arrival in late endosomes (t1/2 = 8-10 min). Thus, acidification of both endosome subpopulations was heterogeneous. However, passage of SFV through a less acidic early endosome subpopulation always preceded arrival in the more acidic late endosome subpopulation. In mutant CHO cells with temperature-sensitive defects in endosome acidification in vitro, acidification of both early and late endosomes was found to be impaired at the restrictive temperature (41 degrees C). The acidification defect was also found to be partially penetrant at the permissive temperature, resulting in the inability of any early endosomes in these cells to attain pH less than or equal to 5.3. In vitro studies of endosomes isolated from mutant cells suggested that the acidification defect is most likely in the proton pump itself. In one mutant, this defect resulted in increased sensitivity of the electrogenic H+ pump to fluctuations in the endosomal membrane potential.