385

Biochimica et Biophysica Acta, 5 6 3 ( 1 9 7 9 ) 3 8 5 - - 3 9 2 © Elsevier/North-Holland

Biomedical Press

BBA 99492

INHIBITION OF DNA REPAIR IN ULTRAVIOLET-IRRADIATED HUMAN CELLS BY HYDROXYUREA

A N D R E W A. F R A N C I S a , , , R . D E A N B L E V I N S b, W I L L I A M L. C A R R I E R S M I T H a a n d J A M E S D. R E G A N a

a, D A V I D P.

a Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 and b Biology Department, East Tennessee State University, Johnson City, TN 37601 (U.S.A.) (Received November 21st, 1978)

Key words: DNA repair inhibition; Hydroxyurea; Photolysis; DNA synthesis: Thymine dimer; (Human fibroblast)

Summary The effect on DNA repair in ultraviolet-irradiated human skin fibroblasts by hydroxyurea has been examined in this study using three independent methods for measuring DNA repair: the 5-bromodeoxyuridine photolysis assay which measures DNA repair replication, chromatographic measurement of thyminecontaining dimers, and measurement of specific ultraviolet-endonucleasesensitive sites in irradiated DNA. Little effect of hydroxyurea was observed at the concentration of 2 mM, which is often used to inhibit semiconservative DNA synthesis; however, 10 mM hydroxyurea resulted in marked inhibition (65--70%) of excision repair. This inhibition was accompanied by a possible doubling in the size of the repaired region. The accumulation of large numbers of single-strand breaks following ultraviolet irradiation and hydroxyurea inctlbation seen by other investigators was not observed with the normal skin fibroblasts used in this study. A comparison of hydroxyurea effects on the different DNA repair assays indicates inhibition of one step in DNA repair also results in varying degrees of inhibition of other steps as well. Introduction Hydroxyurea has been used for treatment of leukemia and solid tumors; it has also been used in the treatment of the skin disease psoriasis. The ability of hydroxyurea to act as a potent inhibitor of semiconservative DNA synthesis 1"o w h o m correspondence should be addressed. Aobreviation used: BrdUrd, 5-bromodeoxyuridine. By acceptance of this article, the publisher or recipient acknowledges the U.S. G o v e r n m e n t ' s right to retain a nonexclusive, royalty-free license in and to any copyright covering the article.

386 has served as an explanation of its clinical properties as well as a basis for its usefulness as a biochemical tool [1]. In the past, h y d r o x y u r e a has been reported to have little effect on DNA repair replication in HeLa cells or rejoining of strand breaks in primary cultures of bovine cells [2,3 ]. This resistance to h y d r o x y u r e a has been used to characterize repair replication and to repress semiconservative DNA synthesis to better measure repair synthesis [4,5]. There is also evidence that indicates h y d r o x y u r e a may also have an inhibitory effect on ultraviolet-induced repair synthesis [6--9]. In the light of these results, we have measured the effects of h y d r o x y u r e a on the repair of DNA damage induced by the ultraviolet irradiation of human fibroblasts. Direct chromatographic measurement of excised thymine dimers, measurement of endonuclease-sensitive sites in the DNA, and 5-bromodeoxyuridine (BrdUrd) photolysis have been used in this study to quantitate the effect of hydroxyurea on repair of irradiated DNA. The results from the use of h y d r o x y u r e a in past experiments, the size of the repaired region, and current conflicting evidence in the literature are discussed. Materials and Methods Normal human skin fibroblasts were grown on Eagle's minimal essential medium [10] containing 15% fetal calf serum. Radioactive labeling with 3 Ci/ mM [Me-3H]thymidine (Schwartz BioResearch) at 8 t~Ci/ml or with 500 mCi/ mM [ '4C]thymidine (Schwartz BioResearch) at 0.5 tzCi/ml was accomplished in Eagle's minimal essential medium containing 10% calf serum. The BrdUrd incubations were also in Eagle's minimal essential medium containing 10% calf serum. The h y d r o x y u r e a was purchased from Sigma Chemical Company; thymidine and BrdUrd was purchased from Schwartz/Mann. Cells received ultraviolet irradiation as a monolayer from a General Electric germicidal lamp with a peak emission at 254 nm. The BrdUrd photolysis is described in detail elsewhere [11]. Briefly, it involves incubation of ultraviolet-irradiated cells in the presence of BrdUrd during the first 20 h of the DNA repair period, thus allowing the incorporation of B r d U r d in the repaired regions. Irradiation of this repaired DNA with 313 nm light results in photolysis of BrdUrd-containing sites. The resulting singlestrand breaks are then analyzed on 5--20% alkaline sucrose gradients. This DNA is compared directly to that from cells allowed to repair in media containing thymidine on the same gradient. Cells prelabeled with [Me-3H]thymidine were used for the BrdUrd incubation, and ['4C]thymidine-labeled cells were used for the thymidine control. The weight-average molecular weight of the two DNA species are calculated, and the difference of the reciprocal molecular weights (A1/Mw) is plotted as a function of the 313 nm light fluence. The number o f resulting breaks is then 2 X A1/Mw, and the total number of repaired sites detected is 2 X A1/Mw at 313 nm light saturation [12]. The relative patch size can be estimated as a function of total repaired sites and initial slopes of the photolysis curves. Endonuclease-sensitive sites were determined using a partially purified pyrimidine-dimer-specific endonuclease isolated from Micrococcus luteus [13]. Cell cultures were labeled with [Me-SH]thymidine, irradiated with 20 J/m 2

387 ultraviolet light, and incubated with 0, 2, or 10 mM h y d r o x y u r e a for various time periods, after which the DNA was extracted [14]. Following incubation of the DNA with the ultraviolet endonuclease, the single-strand breaks were quantitated by analyzing the DNA on alkaline sucrose gradients. Samples containing DNA isolated from cells which received no ultraviolet irradiation were also analyzed to determine the degree of nonspecific enzyme activity. Thymidine-containing pyrimidine dimers were assayed using the method of Carrier and Setlow [15]. Cells were prelabeled by incubation with 0.5 aCi/ml [Me-3H]thymidine (55 Ci/mM from Schwartz/Mann). A dose of 20 J/m 2 ultraviolet irradiation was given and the cultures were then incubated with 0, 2, and 10 mM hydroxyurea. Samples were taken at Various times for dimer analysis. The trichloroacetic acid (5%) insoluble fraction from each sample was hydrolyzed with formic acid, and the dimer content of the DNA was analyzed chromatographically. The accumulation of single-strand DNA breaks following ultraviolet irradiation and subsequent incubation with h y d r o x y u r e a was examined using cell cultures labeled with [Me-3H]thymidine and [14C]thymidine as with the BrdUrd photolysis procedure. Both cultures w e r e given 20 J/m 2 ultraviolet light, after which the 3H-labeled cells were incubated in either 2, 5, or 10 mM h y d r o x y u r e a while the 14C-labeled cells were incubated in the absence of hydroxyurea. At 1, 6, and 20 h after ultraviolet irradiation, cultures were harvested, the 14C- and 3H-labeled cells were mixed, and their DNA was analyzed on alkaline sucrose gradients. In a similar manner, gradients were utilized to test for the effect o f h y d r o x y u r e a on DNA strand breaks in the absence of ultraviolet irradiation. The number of breaks in the single-strand DNA attributable to the h y d r o x y u r e a treatment was then calculated as 2 ×

A1/Mw. Results Cells irradiated with 20 J/m 2 ultraviolet light and allowed to repair in differer~t concentrations o f h y d r o x y u r e a show a concentration-dependent inhibition of DNA repair. Inhibition by h y d r o x y u r e a of thymine dimer excision from the DNA of ultraviolet-irradiated human fibroblasts is shown in Fig. 1. Only the thymine-containing dimers remaining in the 5% trichloroacetic acid insoluble DNA were measured. H y d r o x y u r e a at 10 mM reduces dimer excision by a b o u t 65% after a 48-h repair period. Fig. 2 shows the effect of h y d r o x y u r e a on remaining ultraviolet-endonuclease-sensitive sites in the DNA of irradiated cells after different repair periods. In this case, only those dimer-containing sites in the DNA where the incision step had n o t taken place were measured. No effect is seen with 2 mM hydroxyurea; however, 10 mM results in a b o u t a 42% reduction after 48 h. The BrdUrd-photolysis assay depends on the filling o f the gaps in the DNA by the repair polymerase following the incision and excision steps in the repair sequence. The effect of a single dose of 313 nm light on the DNA of ultraviolet-irradiated cells following repair for 20 h in the presence of BrdUrd and varying concentrations of h y d r o x y u r e a is shown in Fig. 3. A 65% inhibition is seen after only 20 h with 10 mM hydroxyurea. The effect o f h y d r o x y u r e a on

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HOURS AFTER ULTRAVIOLET EXPOSURE

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F i g . 1. E x c i s i o n o f t h y m i d i n e - c o n t a i n i n g pyrimidine dimers from the DNA of ultraviolet-irradiated cells in the p r e s e n c e o f n o h y d r o x y u r e a (o), 2 mM hydroxyurea (o), and 10 mM hydroxyurea (v) measured by the dimers remaining in the acid insoluble DNA. F i g . 2. R e m o v a l o f u l t r a v i o l e t e n d o n u c l e a s e - s e n s i t i v e s i t e s f r o m t h e D N A o f u l t r a v i o l e t - i r r a d i a t e d t h e p r e s e n c e o f n o h y d r o x y u r e a ( o ) , 2 m M h y d r o x y u r e a ( o ) , a n d 1 0 m M h y d r o x y u r e a (':').

cells in

the kinetics of photolysis is shown in Fig. 4. A series of increasing 313 nm light doses was given to cells which were allowed to repair in different concentrations of hydroxyurea. At 5 and 10 mM concentrations, rapid saturation of photolysis with 313 nm light occurred. Below 2 mM hydroxyurea there appear to be only slight changes in the photolysis kinetics. These results indicate not only fewer sites were repaired in the presence of hydroxyurea but changes in the calculated patch size have also occurred. In Fig. 5 the calculated patch size 6.,- x 1,0 8

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F i g . 3. S i n g l e - s t r a n d b r e a k s f o l l o w i n g 2 0 J / m 2 u l t r a v i o l e t i r r a d i a t i o n a n d s u b s e q u e n t D N A r e p a i r i n the presence of BrdUrd followed by photolysis with 105 J/m 2 313 nm light. The effect of hydroxyurea during repair incubation.

Fig. 4. P h o t o l y s i s o f B r d U r d - c o n t a i n i n g r e g i o n s o f D N A f o l l o w i n g 2 0 J / m 2 u l t r a v i o l e t i r r a d i a t i o n a n d repair in the presence of BrdUrd. Kinetics obtained when repair has occurred in the presence of no hydroxyurea (o), 2 mM hydroxyurea ( n ) , 5 m M h y d r o x y u r c a (A), a n d 1 0 r a M h y d r o x y u r e a ( v ) .

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o f h y d r o x y u r e a o n the calculated patch size f o l l o w i n g D N A repair in ultraviolet-

TABLE I SINGLE-STRAND BREAKS IN THE TREATED WITH HYDROXYUREA

DNA OF

ULTRAVIOLET-IRRADIATED HUMAN CELLS

The number of breaks per 109 da]tons in DNA from hydroxyurea-treated cells as compared to control cells with no hydroxyurea [2 X (1/Mw DNA from hydroxyurea-treated cells -- 1/M w DNA from control cells) = 2 X A 1 / M w = number of breaks] (see Ref. 12). HSBP, normal human skin fibroblasts.

Repair period (h)

Cell type

No u l t r a v i o l e t 10 m M hydroxyurea

20 J / m 2 u l t r a v i o l e t + 2 mM hydroxyurea

20 J / m 2 u l t r a v i o l e t + 5 mM hydroxyurea

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0.8

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0.7

--

1.1

is plotted as a function of the hydroxyurea concentration. No effect on patch size is detected at 2 mM hydroxyurea; however, at 10 mM the number of BrdUrd residues per repaired area has been doubled. The large accumulation of single-strand breaks reported by others [6--9] was not seen after 1, 6, or 20 h incubation with up to 10 mM hydroxyurea. The number o f breaks which were detected was near the limit o f resolution of our system but they, nevertheless, show a dose response to hydroxyurea concentration, appear in the first hour following ultraviolet irradiation, and diminish in 20 h (Table I). Discussion Our measurements o f DNA repair following 20 J/m 2 ultraviolet irradiation confirm that hydroxyurea in concentrations above 2 mM has a strong, concen-

390 tration-dependent inhibitory effect. This concentration is greater than that needed to inhibit semiconservative DNA synthesis [ 1]. Cleaver, measuring repair replication of DNA following ultraviolet irradiation, found no effect of h y d r o x y u r e a at 1 and 2 mM in HeLa cells [2]. Although we found a slight effect at 2 mM when DNA repair is followed by direct measurement of thymine dimers or by the BrdUrd photolysis assay, no effect was seen when measuring endonuclease-sensitive sites; and it is unlikely that the sensitivity of either assay would allow detection of inhibition with 1 mM hydroxyurea. If one compares the relative a m o u n t of inhibition by h y d r o x y u r e a of the DNA repair system shown by the three methods used in this study, differences can be seen; however all m e t h o d s show sensitivity to hydroxyurea. The BrdUrd photolysis assay, which is the most sensitive to the h y d r o x y u r e a inhibition, is directly dependent on the action of the repair polymerase. This is consistent with the evidence that indicates the h y d r o x y u r e a inhibition is acting through the repair polymerase. The inhibition measured by the thymine dimer assay depends both on the action of the endonuclease and exonuclease and shows less sensitivity to hydroxyurea. The endonuclease-sensitive site assay is least sensitive to the inhibition and depends only on the action of the endonuclease, which is the first step in the DNA repair sequence and should be least dependent on the action of the polymerase. This assay, however, is the least sensitive of the three DNA repair assays used in this study. We feel the difference seen especially between this m e t h o d and chromatographic dimer analysis is primarily due to this decreased sensitivity. Therefore, while a definite effect of h y d r o x y u r e a can be shown, calculations of repaired sites cannot meaningfully be compared with those calculated by the other methods. The inhibition of DNA repair by h y d r o x y u r e a has been inferred by an apparent accumulation of single-strand breaks in the DNA following ultraviolet irradiation and incubation with h y d r o x y u r e a [6--8]. Ben-Hur and Ben-Ishai [6] found DNA strand breaks persisting at 8 h after 25 J/m 2 ultraviolet irradiation of HeLa cells when in the presence of 2.5 mM hydroxyurea. Collins et al. [8] have shown DNA breaks occurring following ultraviolet irradiation of HeLa cells and repair for 45 min in the presence of 10 mM hydroxyurea. Collins [7] explained the accumulation of breaks which he observed in the DNA of irradiated HeLa cells when allowed to repair in the presence of 10 mM h y d r o x y u r e a as evidence that 'uncoupling' of repair sequences had occurred. The hydroxyurea, therefore, inhibited the repair polymerase, thereby blocking strand rejoining while the ultraviolet endonuclease continued to create singlestrand breaks. The accumulation of strand breaks in these studies was observed in synchronized cells following relatively high doses (100 J/m 2) of ultraviolet irradiation [ 7--9 ]. Single strand breaks have also been shown to occur in nonirradiated synchronized cells treated with 10 mM h y d r o x y u r e a b u t only during S phase [16]. The inhibition of ribonucleoside diphosphate reductase by h y d r o x y u r e a and the subsequent restriction of DNA polymerase(s) due to the lack of available deoxyribonucleotide precursors appear to be involved, b u t the exact mechanism is still unexplained. We were only able to show a relatively small number of strand breaks with our unsynchronized normal human fibroblasts and HeLa

391 cells. These breaks were seen only following ultraviolet (20 J/m ~) irradiation and showed a dose dependency on the hydroxyurea concentration. They did not accumulate with hydroxyurea exposure time but, in fact, reached a peak and then decreased, similar to the breaks observed in the nonirradiated cells. The strand breaks observed by us and those seen by Collins may reflect the relative amount of DNA repair in progress at the time of the assay and the degree of uncoupling which has occurred in the different cell types at specific times in the cell cycle. Clarkson [18] has reported a stimulation in repair synthesis in Chinese hamster ovary cells in the presence of hydroxyurea. There was no concurrent change in nucleoside pools. She, therefore, postulated an increase in patch size to explain the current conflicting results reported. The increase in patch size we have observed with the BrdUrd photolysis is in agreement with this suggestion. If the number of sites repaired is being measured, an inhibitory effect of hydroxyurea can be seen; however, incorporation of labeled deoxyribonucleosides may actually increase in the presence of hydroxyurea due to the increase in patch size. We accept this explanation with caution since restriction of available thymidine by hydroxyurea [17] would allow the incorporation of more BrdUrd in the repaired region, thereby increasing the probability of photolysis without actually increasing the size of the patch. The inhibitory action of hydroxyurea on DNA repair in ultraviolet-irradiated normal human fibroblasts results in the inhibition of the incision, excision and resynthesis steps involved in DNA excision repair. Hydroxyurea has been used to inhibit semiconservative DNA synthesis during studies on repair especially with the BrdUrd assay. The concentration of hydroxyurea (2 mM) usually used slightly lowers the total number of repaired sites measured after a repair period of 20 h but has no detectable effect on patch size. Since this assay usually utilizes cells prelabeled with DNA precursors, semiconservative DNA synthesis occurring during the repair period does not interfere with the analysis. Only the prelabeled parental DNA can be visualized on the gradients [19]. Due to possible artifacts which could be introduced, it would be better to avoid the use of hydroxyurea to repress semiconservative DNA synthesis when measuring DNA repair synthesis. If experimental conditions do not permit this, concentration, of 2 mM or less should be used with normal human fibroblasts. In cases where DNA repair is measured following chemical treatment and semiconservatire DNA synthesis must be inhibited with hydroxyurea, the appropriate controls should be applied to determine if the chemical treatment is interfering with the inhibition of semiconservative DNA synthesis. Treatment with chemical agents has been known to partially block the inhibition of semiconservative DNA synthesis when hydroxyurea is used at lower concentrations (2 mM or less) [20].

Acknowledgements This research was jointly sponsored by the National Cancer Institute Y-01CP-50200, NASA Interagency Agreement 40-565-76, and by the Division of Biomedical and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation.

392

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