A UV-sensitive human clonal cell line,RSa, which has low repair activity

The repair activity of a human transformed cell line, RSa, which was found to be highly sensitive to the lethal effects of 254 nm far-ultraviolet radiation, was compared with that of HeLa cells by evaluating the range of UV-induced incorporation of [methyl-³H]thymidine ([³H]dThd) or 5-[6-³H]bromodeoxyuridine ([³H]BrdUrd) into deoxyribonucleic acid. Direct scintillation counting was used for measuring the extent of unscheduled DNA synthesis (UDS) in UV-irradiated cells, which were treated with hydroxyurea or with arginine deprivation. More quantitative measurements were made by using the density labeling and equilibrium centrifugation method for assaying repair replication. All the amounts of UDS and repair replication in RSa cells were markedly below those in HeLa cells. The possible relationships of the low repair activity to abnormally high UV sensitivity in RSa cells are discussed.     

Enhancement of repair replication in mammalian cells by hydroxyurea

The effect of hydroxyurea on DNA repair replication has been studied in Chinese hamster ovary cells. Mitotic cells were treated with UV irradiation, methyl methanesulfonate or nitrogen mustard and incuated in the presence of each of the 4 [³H]deoxyribonucleosides plus BrdUrd and FdUrd for 2 h. The amount of repair replication was quantitated on CsCl gradients and similar values were obtained for each nucleoside. In all cases addition of HU during the incubation period increased these values approximately 2-fold. Following MMS treatment, pool sizes for each of the nucleosides were estimated by varying the amount of exogenously supplied nucleoside. They were found to be insensitive to the addition of HU and it is concluded that the increased incorporation of [³H]deoxyribonucleosides in the presence of HU reflects an increased amount of repair replication.     

Double minutes replicate once during S phase of the cell cycle

Double minutes (dm) characteristically exhibit greater numerical heterogeneity among tumor cells than do chromosomes. The biological basis of this heterogeneity was studied in human carcinoma cell line S 18. Pulse labeling of asynchronous cells with [³H]dThd, continuous labeling of synchronized cells with BrdUrd and prematurely condensed chromosome (PCC) studies of G1 and G2 phase S 18 cells indicate that dm-DNA replicates only once during S phase of the cell cycle. No evidence was found for replication of dm-DNA at G1 phase, G2 phase or mitotis. Cells observed at anaphase show imprecise distribution of dm to daughter cells. These studies suggest numerical heterogeneity of dm results from anomalous mitotic segregation rather than anomalous replication of dmDNA.     

Unscheduled DNA synthesis in rat pleural mesothelial cells treated with mineral fibres

Unscheduled DNA synthesis (UDS) was studied in confluent rat pleural mesothelial cells (RPMCs) arrested in G0/G1 with hydroxyurea (HU) and treated with various fibre types, i.e., chrysotile, crocidolite or attapulgite. In addition, the effects of UV light and of benzo[a]pyrene were determined as references. Using autoradiography after [3H]thymidine incorporation ([3H]dThd), RPMCs treated with 4 micrograms/cm2 of chrysotile fibres exhibited a low but significant enhancement of net grains compared to untreated cells. Treatment with higher doses of chrysotile was not possible because of the impairment of microscopic observation due to the presence of the fibres. Using liquid scintillation counting, RPMCs treated with chrysotile or crocidolite showed a significant dose-dependent increase in [3H]dThd incorporation compared to untreated cells. In contrast, attapulgite did not enhance [3H]dThd incorporation compared to untreated cells. Treatment of RPMCs with 1, 2 or 4 micrograms/ml of benzo[a]pyrene resulted in a significant increase in [3H]dThd incorporation. In order to discount a possible role of S cells in the augmentation of [3H]dThd incorporation, despite the presence of 5 mM HU, S cells were counted by autoradiography. Results indicated that the percentage of S cells was similar in asbestos-treated and untreated cultures. Stimulation of the S phase also seems unlikely because treatment of RPMCs with asbestos fibres in the absence of HU resulted in a reduction of [3H]dThd incorporation attributed to an impairment of the S phase by the fibres. 1-4 micrograms/ml benzo[a]pyrene or 10-50 J/m2 UV light resulted in an approximate doubling of [3H]dThd incorporation. The effects of inhibitors of DNA repair were determined in chrysotile-treated RPMCs. [3H]dThd incorporation was inhibited by cytosine arabinoside and nalidixic acid. These results show that asbestos produces UDS in RPMCs.     

Breakage of Parental DNA Strands in Haemophilus influenzae by 313 nm Radiation after Replication in the Presence of 5-Bromodeoxyuridine

Haemophilus influenzae was labeled with thymidine-³H (dThd), then grown in the presence of 5-bromodeoxyuridine (BrdUrd), and then irradiated with 313 nm light (a wavelength that selectively photolyzes DNA containing 5-bromouracil [BrUra]). Irradiation with 313 nm light induced breaks in the ³H-labeled strands in cells grown with BrdUrd at a much higher frequency than in ¹⁴C-labeled DNA of cells not exposed to BrdUrd. Breakage of the ³H-labeled strands was about 0.6% as efficient as that of fully BrUra-substituted DNA. During growth in the presence of BrdUrd, susceptibility to 313 nm-induced breakage of the ³H-labeled DNA strands increased, reaching a maximum in about one generation, and it decreased to zero during subsequent growth for one generation in medium containing dThd instead of BrdUrd. Heat denaturation of DNA extracted from dThd-³H-labeled cells grown in the presence of BrdUrd eliminated 313 nm-induced breakage of the ³H-labeled strands. It is concluded that breakage of the ³H-labeled DNA strands resulted from reaction with photoproducts in the base-paired, BrUra-containing strands, rather than from photolysis of BrdUrd incorporated into parental strands. It may be possible to utilize the phenomenon of interstrand breakage in physical studies of DNA replication.     

Simultaneous detection of DNA strand breaks and unscheduled DNA synthesis in mutagen-treated human lymphocytes in the absence of hydroxyurea

Human lymphocytes in the quiescent state were exposed to UVC radiation. After irradiation the cells were allowed to repair for various times in the presence of [3H]thymidine or [3H]deoxycytidine in the culture medium. Hydroxyurea was not used to suppress semiconservative DNA replication in the small number of growing cells. After incubation DNA strand breaks were detected by the DNA-unwinding method and the amount of 3H incorporation in DNA was measured by liquid scintillation counting. The results show that the yield of DNA strand breaks and the amount of unscheduled DNA synthesis (UDS) can be measured from the same lymphocyte sample. A low background 3H incorporation in untreated cells could be achieved even in the absence of hydroxyurea. This requires, however, that 3H incorporation is measured only in the double-stranded DNA and that [3H]dCyd is used instead of [3H]dThd as the labelled deoxynucleoside.     

Reversible permeabilization of lymphocytes destroys the incorporation of deoxythymidine but not of deoxycytidine

The total uptake, phosphorylation and incorporation of thymidine (dThd) and deoxycytidine (dCyd) were compared in intact and reversibly permeabilized human tonsillar lymphocytes. The total uptake of [3H]dThd was lower than that of [5-3H]dCyd, but almost all of [3H]dThd was incorporated into DNA. However, the main part of [5-3H]dCyd taken up by the lymphocytes was found in the pool as phosphorylated nucleoside (55%), and only a smaller part (13%) was incorporated into DNA. Phosphorylated nucleosides were determined by DEAE-cellulose sheets in the ethanol-soluble fraction of the cells. The reversible permeabilization of lymphocytes by Dextran T-150 destroys totally the [3H]dThd incorporation, while [5-3H]dCyd incorporation decreased only to 60% of intact cells. During permeabilization the phosphorylation of both nucleosides increased severalfold. After permeabilization all [3H]dThd was in dTMP form, while [5-3H]dCyd was also found in dCDP (3%) and dCTP (38%) form. In the meanwhile, 22% of thymidine kinase, 63% of deoxycytidine kinase and 98% of DNA polymerase activity were measured in permeabilized cells as compared to intact cells. The results suggest different relationships between the lymphocyte plasma membrane and the salvage pathways of the two pyrimidine nucleosides.     

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.     

Attempts to induce mutations in Haemophilus influenzae with the base analogues 5-bromodeoxyuridine and 2-aminopurine.

Attempts were made to induce mutations in Haemophilus influenzae with the base analogues 5-bromodeoxyuridine and 2-aminopurine. These attempts were unsuccessful. Incorporation studies with BrdUrd showed, in agreement with earlier studies on Escherichia coli, that BrdUrd was discriminated against when dThd was also present but was incorporated to essentially the same extent as dThd when only BrdUrd was present. In this latter case, strands fully substituted with BrdUrd was produced, but survival data suggest that bacteria deriving their DNA by replication on such fully substituted templates were inviable. However, bacteria with about 20% of the thymine substituted with bromouracil were usually viable. No mutations could be detected in the descendants of such bacteria. The reasons for this are discussed and it is concluded that in all probability the replication system in species rarely if every treats incorporated bromouracil as anything except a thymine analogue. The alternative possibility, that the negative results are a consequence of the absence of the reclex (SOS) error-prone repair system in this species, is considered much less likely.     

Nicotinamide deficiency in human lymphocytes prevents the [H]thymidine-induced adaptive response for the repair of X-ray-induced chromosomal damage

Human lymphocytes treated with [3H]thymidine ([3H]dThd) become refractory to the induction of chromosomal aberrations by subsequent doses of X rays. This adaptive response to [3H]dThd does not occur in the presence of 3-aminobenzamide (3AB). 3AB inhibits the synthesis of poly(ADP-ribose) by the enzyme adenosine diphosphate ribosyl transferase (ADPRT), which requires NAD as a substrate. 3AB also prevents chromosomal repair, as measured in X-ray dose-fractionation studies. Because 3AB might interfere with metabolic reactions other than those mediated by ADPRT, experiments were carried out to see if the adaptive response was also inhibited in nicotinamide-free medium, which prevents poly(ADP-ribosyl)ation by depleting cellular NAD. The experiments show that the incorporation of [3H]dThd has no effect on the induction of chromosomal aberrations by subsequent doses of X rays if the cells are cultured in nicotinamide-free medium. Nicotinamide deficiency mimics the effects of 3AB on both the adaptive response and chromosome repair. The results indicate that ADPRT activity itself, and not other metabolic processes affected by inhibitors of this enzyme, plays an essential role in the adaptive response.     

DNA nucleotide excision-repair synthesis is independent of perturbations of deoxynucleoside triphosphate pool size

We have investigated the effects of fluctuations in deoxynucleoside triphosphate (dNTP) pool size on DNA repair and, conversely, the effect of DNA repair on dNTP pool size. In confluent normal human skin fibroblasts, dNTP pool size was quantitated by the formation of [3H]TTP from [3H]thymidine; DNA repair was examined by repair replication in cultures irradiated with UV light. As defined by HPLC analysis, the [3H]TTP pool was formed within 30 min of the addition of [3H]thymidine and remained relatively constant for the next 6 h. Addition of 2-10 mM hydroxyurea (HU) caused a gradual 2-4-fold increase in the [3H]TTP pool as HU inhibited DNA synthesis but not TTP production. No difference was seen between the [3H]TTP pool size in cells exposed to 20 J/m2 and unirradiated controls, although DNA-repair synthesis was readily quantitated in the former. This result was observed even though the repair replication protocol caused an 8-10-fold reduction in the size of the [3H]TTP pool relative to the initial studies. In the UV excision-repair studies the presence of hydroxyurea did not alter the specific activity of [3H] thymidine 5'-monophosphate incorporated into parental DNA due to repair replication. These results suggest that fluctuations in the deoxynucleoside triphosphate pools do not limit the extent of excision-repair synthesis in human cells and demonstrate that DNA nucleotide excision-repair synthesis does not significantly diminish the size of the [3H]TTP pool.     

Tumor necrosis factor stimulates DNA synthesis in the liver of intact rats

TNF is cytotoxic to tumor cell lines but enhances growth of some nontransformed cells. Because animals administered TNF have an increase in liver size, we studied the [3H]thymidine incorporation into DNA in the liver of intact rats. A significant increase in [3H]thymidine incorporation is seen 20 hours following TNF administration and peaks at 24 hours. The lowest dose of TNF that increases DNA synthesis is 10 micrograms/200 g rat with a maximal increase occurring with 25 micrograms/200 g, considerably less than the dose required for maximally increasing plasma triglycerides. The increase in [3H]thymidine incorporation was shown to be due to an increase in DNA polymerase alpha activity (associated with the replication of DNA) rather than DNA polymerases beta (associated with DNA repair) plus gamma activity. These results indicate that TNF administration stimulates DNA replication in the liver of intact animals.     

Possible importance of PLD repair in the modulation of BrdUrd and IdUrd-mediated radiosensitization in plateau-phase C3H10T1/2 mouse embryo cells

C3H10T1/2 mouse embryo cells exhibiting strong contact inhibition of growth at confluency were grown in the presence of 5-bromodeoxyuridine (BrdUrd) or 5-iododeoxyuridine (IdUrd) (0-1.2 microM) with daily refeeding and exposed to gamma-rays (6 Gy) either in the logarithmic or the plateau phase of growth. Sensitization to radiation was observed in both growth states with increasing concentration of BrdUrd or IdUrd but the degree of sensitization achieved was lower for plateau-phase cells. Because the degree of [H3]BrdUrd incorporation was found to be similar in exponentially growing and plateau-phase cells, it is hypothesized that the radiosensitization caused by pyrimidine analogues may be affected by the physiological state of the cells at the time of irradiation. Delayed plating of plateau-phase cells (6 h) caused an increase in survival, indicating repair of potentially lethal damage (PLD). A greater increase in cell survival was observed in cells that had been grown in the presence of BrdUrd and IdUrd and it was found to increase with increasing concentrations. This analogue-concentration dependent PLD repair activity resulted in an almost complete loss of the radiosensitizing effect in delayed plated plateau-phase cells up to a concentration of about 0.6 microM of BrdUrd and IdUrd. Both compounds, but especially BrdUrd, caused a relaxation in the mechanism of contact inhibition and led to higher cell densities in the plateau phase. The results suggest that repair and/or expression of PLD might be involved in the mechanism underlying BrdUrd and IdUrd-mediated radiosensitization and point out the potential importance of PLD repair in the modulation of the radiosensitizing effect of these compounds in their clinical application.     

Repair replication in human cells. Simplified determination utilizing hydroxyurea.

A simplified and shortened procedure has been developed for the determination of repair replication of DNA in cultured mammalian cells. The procedure, using the bromodeoxyuridine density label and a radio-isotopic label has been applied to normal diploid human cells (WI38) and to their SV40 transformants (VA13). After incubation with the repair label the cells are lysed and digested for two hours at 50 degrees C with proteinase K. This digest can then be immediately subjected to alkaline cesium chloride density gradient centrifugation with no need for DNA extraction. Hydroxyurea is used to reduce the level of semi-conservative synthesis that a quantitative determination of repair replication can be accomplished by a single centrifugation. The method is not affected by variation in the effectiveness of the inhibitor although a small amount of semi-conservative synthesis normally occurs in the presence of the drug. The time course of repair replication in WI38 cells is unaffected by the drug. The apparent amount of repair synthesis in ultraviolet irradiated cells is increased 25 to 40% in the presence of hydroxyurea when thymidine is used as tracer. Under certain conditions in which the level of semiconservative synthesis is low (e.g., contact inhibited cells, high ultraviolet doses) the use of hydroxyurea is unnecessary.     

Effect of 5-fluoro-2'-deoxyuridine (FdUrd) on 5-bromo-2'-deoxyuridine (BrdUrd) incorporation into DNA measured with a monoclonal BrdUrd antibody and by the BrdUrd/Hoechst quenching effect

The purpose of this study was to improve the application of bromodeoxyuridine (BrdUrd) for the flow cytometric analysis of cell kinetics. In order to obtain a quantitative measure of the DNA synthesis rate (or the number of divided cells), BrdUrd should replace thymidine (dThd) completely in the newly synthesized DNA strands. The de novo synthesis of dThd monophosphate competing with BrdUrd incorporation was stopped by fluorodeoxyuridine (FdUrd). Cells of a human leukemic cell line (REH) were exposed to BrdUrd for either 20 min, 8 h, or 24 h. Bromodeoxyuridine incorporation was determined by a monoclonal antibody as well as by the BrdUrd/Hoechst (H) technique. Counterstaining of the DNA was performed with propidium iodide or ethidium bromide. DNA fluorescence was measured in both techniques with a two-parameter flow cytometer, the histograms being analyzed by computer. It was found that FdUrd is required in the BrdUrd/H technique for replacement of dThd at low BrdUrd concentrations and long incubation times. With short incubation periods, as used for detection by the monoclonal anti-BrdUrd antibody, FdUrd increases the incorporated BrdUrd amount when BrdUrd concentrations of 10 microM or less are applied.     

Enzymatic determination of nonrandom incorporation of 5-bromodeoxyuridine in rat DNA.

Secondary cultures of normal rat embryo cells were synchronized by a double thymidine block and pulsed with 10(-7) M 5-[3H]bromodeoxyuridine (BrdUrd) OR 10(-7) M[3H]thymidine during an entire S phase (7.5 h). To examine the pattern of [3H]thymidine, DNA was immediately extracted and purified at the completion of the S phase, CsCl density gradient centrifugation revealed that substitution for thymine by bromouracil was less than 7%. Single-strand specific nucleases obtained from Aspergillus oryzae and Neurospora crassa were allowed to react with native and partially depurinated (24-29%) [3H]BrdUrd-labeled rat DNA samples, and the products were assayed by hydroxylapatite column chromatography. Approximately 4-6% of the native, nondepurinated rat DNA was hydrolyzed by both nucleases. However, 24-28% of the partially depurinated, [3H] thymidine-labeled rat DNA was hydrolyzed by both enzymes as determined by loss of mass as well as radioactivity. Whereas comparable levels of depurinated, [3H]BrdUrd-labeled DNA were physically hydrolyzed by both nucleases, nearly 65% of the radioactivity was not recovered. Native, as well as depurinated, enzyme-treated DNA samples were sequentially and preparatively reassociated into highly repetitive, middle repetitive, and nonrepetitive nucleotide sequence components. The absolute and relative specific activities of each subfraction of native [3H]thymidine-labeled DNA were comparable. [3H]BrdUrd was differentially concentrated in the middle repetitive sequences as compared to other reiteration frequency types. When depurinated, nuclease-treated DNA samples were similarly fractionated, [3H]thymine moieties were uniformly distributed thoughout all sequences. However, a differential loss of [3H]BrdUrd moieties was detected predominantly from the middle repetitive nucleotide fraction. Melting profiles of the renatured DNA samples were characteristic of each respective DNA subfraction regardless of isotopic precursor. These results suggest that [3H]BrdUrd may be differentially incorporated into A + T rich clusters of rat DNA, especially in the moderately repeated chromosomal elements.     

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.     

Unscheduled DNA synthesis induced in mouse spermatids after combined treatment with methyl methanesulfonate and X-rays.

Unscheduled DNA synthesis (UDS), which is considered to be DNA repair, has been studied in early- to mid-spermatid stages of the mouse after combined treatments with X-rays and methyl methanesulfonate (MMS). UDS in spermatids was detected by giving testicular injections of [methyl-3H]thymidine ([3H]dThd) and making use of the fact that no scheduled DNA synthesis occurs in the germ cells after the last S period in primary spermatocytes. X-rays and MMS are each able to induce UDS in mouse spermatids. However, there was a statistically significant reduction in the amount of UDS observed when X-ray exposures of from 200 to 600 R were given 4 h before an i.p. injection of 75 mg/kg of MMS and concurrent testicular injections of [3H]dThd. This reduction in UDS is more than can be explained by the completion of repair of X-ray-induced DNA lesions. We suggest that the reduction in UDS is the result of an X-ray-produced impairment of a least a part of the repair mechanism involved in correcting MMS-induced DNA lesions. When the time interval between a 600-R X-ray exposure and MMS treatment was between 3 and 20 h (latest time interval s;udied) there was a statistically significant reduction of UDS in the spermatids. No significant decrease in UDS response occurred when the time interval between radiation exposure and MMS treatment was less than approximately 3 h.     

Strategies for the measurement of the inhibitory effects of thymidine analogs on the activity of thymidylate synthase in intact murine leukemia L1210 cells

Two strategies have been pursued to monitor the inhibition of thymidylate (dTMP) synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) by thymidine (dThd) analogs in intact murine leukemia L1210 cells. The first method was based on the determination of tritium release from 2'-deoxy[5-3H]uridine [( 5-3H]dUrd) or 2'-deoxy[5-3H]cytidine [( 5-3H]dCyd); the second method was based on an estimation of the amount of dCyd incorporated into DNA as dTMP. The validity of these procedures was assessed by evaluating the inhibition of thymidylate synthase in murine leukemia L1210 cells by a series of 18 dThd analogs. There was a strong correlation between the inhibitory effects of the dThd analogs on the proliferation of L1210 cells on the one hand, and (i) their inhibitory effects on tritium release from [5-3H]dCyd (r = 0.926) and (ii) their inhibitory effects on the incorporation of dCyd into DNA dTMP (r = 0.921), on the other hand. Evaluation of tritium release from [5-3H]dCyd proved to be the most convenient method that has been described so far to measure thymidylate synthase activity and to follow the inhibitory effects of thymidylate synthase inhibitors in intact L1210 cells, since this method is rapid and very sensitive, and since it proved superior to the evaluation of tritium release from [5-3H]dUrd because it circumvents possible interactions of the inhibitors with thymidine kinase activity.     

DNA nucleotide excision-repair synthesis is dependent of pertubations of deoxynucleoside triphosphate pool size

We have investigated the effects of fluctuations in deoxynucleoside triphosphate (dNTP) pool size on DNA repair and, conversely, the effect of DNA repair on dNTP pool size. In confluent normal human skin fibroblasts, dNTP pool size was quantitated by the formation of [³H]TTP from [³H]thymidine; DNA repair was examined by repair replication in cultures irradiated with UV light. As defined by HPLC analysis, the [³H]TTP pool was formed within 30 min of the addition of [³H]thymidine and remained relatively constant for the next 6 h. Addition of 2–10 mM hydroxyurea (HU) caused a gradual 2–4-fold increase in the [³H]TTP pool as HU inhibited DNA synthesis but not TTP production. No difference was seen between the [³H]TTP pool size in cells exposed to 20 M/m² and unrradiated controls, although DNA-repair synthesis was readily quantitated in the former. This result was observed even though the repair replication protocol caused an 8–10-fold reduction in the size of the [³H]TTP pool relative to the initial studies. In the UV excision-repair studies the precense of hydroxyurea did not alter the specific activity of [³H] thymidine 5'-monophospahte incorporated into parental DNA due to repaier replication. These results suggest that fluctuations in the deoxynucleoside triphosphate pools do not limit the extent of excision-repair sythesis in human cells and demonstrate that DNA nucleotide excision-repair synthesis does not significantly diminish the size of the [³H]TTP pool.