INT. J . RADIAT . BIOL .,
1977,
VOL .
31,
NO .
2, 101-111
DNA replication and repair in a human melanoma cell-line resistant to ultra-violet-radiation MARTIN F . LAVIN, GILLIAN M . WILLETT, A. H . CHALMERS and CHEV KIDSON
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Department of Biochemistry, University of Queensland, Brisbane, 4067, Australia (Received 28 October 1976 ; accepted 3 December 1976)
The effect of ultra-violet (U .V.)-irradiation on DNA replication was studied in a U .V .-resistant, human melanoma cell-line (MM96) . Semi-conservative synthesis of DNA was decreased about five-fold by a U .V.-dose of 100 ergs/mm 2 . The size of DNA fragments synthesized in irradiated cells at short times after U .V . was smaller than those synthesized in unirradiated cells . Elongation of these fragments occurred with time, and 6 hours after irradiation cells synthesized DNA in fragments of the same size as obtained in unirradiated cells . In this post-replication repair process, elongation appeared to involve de novo synthesis and was not inhibited by theophylline .
1 . Introduction Several human melanoma cell-lines have been shown to be highly resistant to ultraviolet (U .V .)-radiation (Chalmers, Lavin, Atisoontornkul, Mansbridge and Kidson 1976). In view of the relationship of the incidence of melanoma to U .V .-exposure (Lee and Merrill 1971, Beardmore 1972), it is important to explore the basis of the radioresistance and its relevance to the biological behaviour of melanoma . Resistance to U .V . does not appear to be due to rapid removal of pyrimidine dimers, since rates of removal are similar to those observed in more sensitive cells (Chalmers et al . 1976) . In a variety of cells, semi-conservative synthesis of DNA is either inhibited or depressed by U.V .-irradiation (Cleaver 1965, Klimek and Vlasinova 1966, Djordsevic and Tolmach 1967, Domon and Rauth 1968) . The molecular weight of newly-synthesized DNA in mammalian cells after U .V .-irradiation has been shown to be lower than that synthesized in unirradiated cells (Cleaver and Thomas 1969, Buhl, Stilmann, Setlow and Regan 1972, Lehmann 1972) . Discontinuities in newly-synthesized mammalian DNA after irradiation (Cleaver and Thomas 1969) are subsequently filled in by a postreplication repair mechanism (Lehmann 1972) . We have examined the effect of U .V . irradiation on semi-conservative synthesis of DNA and on repair functions associated with DNA replication in one (MM96) of the series human melanoma cell-lines shown to be resistant to U .V. 2.
Materials and methods
2.1 . Cells
The melanoma cell-line, MM96, and the U .V .-sensitive HeLa cell-line, HeLa-QB1, used in these studies have been described previously, as have the methods for growing log-phase, unsynchronized cultures (Chalmers et al. 1976) .
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2.2 . Labelling o f cells In double-label experiments, cells were grown for 24 hours at 37 ° C in medium containing [14 C] thymidine, 0 . 5 µCi/ml, 56 mCi/mmole (Nuclear Dynamics Inc ., El Monte, California) . Cells were then washed with prewarmed Hanks' solution before U .V .-irradiation, which was administered by a U .V.-lamp (Mineral light UVS 12) at a dose rate of 10 ergs/mm 2 /sec . The DNA was then pulse-labelled by adding [ 3 H]thymidine, 50 µCi/ml, 20 Ci/mmole (Nuclear Dynamics Inc ., El Monte, California) for prescribed times . After labelling, the cells were washed with ice-cold isotonic EDTA solution (Setlow, Regan, German and Carrier 1969) and removed by scraping . The cells were then centrifuged and resuspended in EDTA solution containing 2 per cent sodium lauryl sarcosine . Samples were placed on Whatman glass-fibre filters (GF/C), washed according to the method of Bollum (1966) and counted in a toluene scintillator . In pulse-chase experiments, cells were washed in Hanks' solution before irradiation and pulsed immediately with [ 3H]thymidine, 50 µCi/ml . At appropriate times, thymidine, 2 . 5 µg/ml, and deoxycytidine, 2 . 5 µg/ml, were added to the medium for stated periods .
2.3 . Sedimentation Cells were harvested as described above and resuspended in ice-cold isotonic EDTA solution . Lysis (3-5 x 104 cells/gradient) was carried out during 30 min in a 0 . 1 ml overlay of 1 M NaOH containing 0. 01 M EDTA on alkaline sucrose gradients (5-20 per cent) containing 0 . 9 M NaCl, 0 . 01 M EDTA and 0 . 01 M NaOH . Centrifugation Was for 3 hours at 30 000 r .p .m. in an SW 56 rotor of a Beckman L2-65B centrifuge . Fractions were collected and assayed by the paper strip method of Carrier and Setlow (1971) .
2 .4. Estimation of molecular weight o f DNA Gradient centrifugation in a 5-20 per cent sucrose gradient was used to define the weight-average sedimentation coefficients of newly-synthesized DNA fragments (Martin and Ames 1961) . Weight-average molecular weight (Mw) was employed since calculation of number-average molecular weight (Mn) is subject to error owing to low-molecular-weight material near the top of the gradient. However, when the estimation of distance between gaps in newlysynthesized DNA was calculated, Mn over the major peak was determined, neglecting the smaller peak at the top of the gradient (figure 4) . Molecular weights were calculated with the aid of a computer, from the sedimentation coefficients estimated by the method of Martin and Ames (1961), using the empirical calibration plots of Studier (1965) . Bacteriophage T3 DNA was used as a standard.
2.5 . CsCl isopycnic gradient analysis Cells were grown in 5 µg/ml 5-bromodeoxyuridine (BUdR) for 1 hour and rinsed with Hanks' solution before U . V .-irradiation . The cells were then incubated in fresh medium containing BUdR, 5 µg/ml and [3 H]thymidine, 10 µCi/ml, for 6 hours . After labelling, cells were harvested and DNA was extracted according to Kidson (1966), except that sodium triisopropylnaphthalene sulphonate, 1 per cent, was used as detergent, and residual phenol was removed
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by ether extraction, followed by dialysis . DNA was analysed by alkaline CsCl isopycnic gradients by adjusting the initial density of the solution to 1 . 76 g/ml . Centrifugation was for 60 hours at 35 000 r.p .m . in a 50Ti head in a Beckman L2-65B centrifuge . Gradients were dripped and samples taken to determine density from measurement of the refractive index, and radioactivity by scintillation spectrometry . 2 .6 . Autoradiography Cultures on cover-slips were irradiated and labelled with 3 [H]thymidine, 10 µCi/ml, for 4 hours . The cells were then rinsed with Hanks' solution and grown for a further 1 hour in medium supplemented with thymidine, 2 . 5 µg/ml, and deoxycytidine, 2 . 5 µg/ml, before fixation in methanol for 5 min . Fixed cultures were dipped in Ilford K2 liquid emulsion for 50 sec and exposed for 21 days before development . Autoradiographs were developed with Kodak D19, fixed, and stained through the emulsion with Giemsa before counting . 2.7 . Degradation o f B UdR-substituted DNA In experiments utilizing BUdR to fill in gaps after U .V.-irradiation, monolayer cultures were rinsed on plates with isotonic EDTA solution . Culture plates containing 5 ml of ice-cold saline EDTA solution were then exposed to a long-wave-length U .V .-source (Ultra-Violet Products, Inc ., San Gabriel, California) at an exposure rate of 300 ergs/mm 2 /sec for 30 min. 3. Results 3 .1 . Synthesis of DNA after U . V. -irradiation When cultures of HeLa and MM96 were U .V .-irradiated with a dose of 250 ergs/mm 2 and 3 [H]thymidine incorporation in the subsequent 4 hours measured by autoradiography, the number of heavily-labelled HeLa nuclei dropped to 16 per cent of the unirradiated value while the number of heavilylabelled nuclei for MM96 remained essentially unchanged at this U .V .-dose . Unscheduled synthesis was apparent in both cases as evidenced by the presence of lightly-labelled nuclei . These data suggested that MM96 had the ability to continue semi-conservative DNA synthesis after a U .V .-dose of 250 ergs/mm 2 at a substantial rate, whereas DNA synthesis in HeLa cells was severely restricted. Accordingly, we used the method of Pettijohn and Hanawalt (1964), using BUdR-labelled DNA as a means of distinguishing semi-conservative DNA synthesis and repair replication . Figure 1 shows that semi-conservative replication continued in MM96 immediately after a U .V.-dose of 250 ergs/mm2 ; repair replication was also apparent . However, in the case of HeLa-QB1, most of the label incorporated into DNA was as repair replication . These findings are consistent with the autoradiographic results, which show a considerable decrease in the number of cells carrying out semi-conservative synthesis at this U .V .-dose in the case of HeLa-QB1 but not of MM96 . 3 .2 . Size o f newly-synthesized DNA after U . V. -irradiation The molecular weight of pulse-labelled DNA from U .V.-irradiated human cells has been shown to be lower than that from unirradiated cells (Buhl et al. 1972) . Figure 2 shows that this also is the case with U .V .-resistant human
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FRACTION NUMBER Figure 1 . Nature of DNA synthesis after U .V .-irradiation (250 ergs/mm 2 ) . Melanoma and HeLa cells were incubated in the presence of BUdR (5 µg/ml) and [ 3H]thymidine (10 µCi/ml) for 6 hours as described in the text, before extraction and density analysis of DNA on alkaline CsCl gradients .
melanoma cells, a decrease in the molecular weight of newly-synthesized DNA being obtained after U .V .-irradiation . The decrease in molecular weight was dose-dependent over the range tested, 50-200 ergs/mm 2 (figure 2) . This decrease in molecular weight with increasing U .V .-dose is due either to the presence of discontinuities in the newly-synthesized DNA, because of the presence of pyrimidine dimers in, or other damage to the parental template, or it may arise from a slower rate of synthesis in irradiated cells . To distinguish between these possibilities cells were prelabelled with [14 C]thymidine for 24 hours and pulsed with [ 3 H]thymidine after U .V .-irradiation . The relative rates of synthesis in irradiated and unirradiated cells are represented in figure 3 . The rate of DNA synthesis in cells irradiated with 100 ergs/mm 2 was approximately one-fifth of that in unirradiated cells . The molecular weight of DNA from irradiated and unirradiated cells, having the same 3 H/ 14 C ratio, that is the same amount of DNA synthesized, was determined by sedimentation analysis (figure 4) . Under these conditions, the weight-average molecular weight of DNA from irradiated cells was 1 . 1 x 10', while that from unirradiated cells was 2 . 3 x 10' . This suggests that after U .V.-irradiation, in the extra period of time required to synthesize the same amount of DNA as in unirradiated cells, synthesis is initiated at many more sites giving rise to short pieces . When the irradiated cells were incubated for a further 1 . 5 hours, the molecular weight of the DNA (2 . 1 x 10') had reached approximately that of DNA from the pulse-labelled unirradiated cells . Six hours after U .V .-irradiation, cells which had received a dose of 100 ergs/mm 2
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Figure 2 . Sedimentation profile of DNA synthesized in MM96 immediately after U .V.irradiation . Cells were pulse-labelled with [ 3 H]thymidine (50 µCi/ml) for 40 min immediately after U .V . -irradiation, lysed on top of alkaline sucrose gradients in 1 m NaOH containing 0.01 M EDTA for 30 min, and centrifuged for 3 hours at 30 000 r .p .m . in an SW56 rotor . Fractions were collected and analysed for radioactivity : (A) unirradiated cells, Mw=4 .0 x 10' ; (B) cells irradiated with 50 ergs/mm2, Mw=1 . 4 x 10' ; (C) 100 ergs/mm 2 , Mw=1 . 1 x 10' ; (D) 200 ergs/mm2, Mw= 9 . 5 x 10 6 .
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Figure 3 . Effect of U .V .-irradiation on DNA synthesis immediately after irradiation . Cells pre-labelled with [ 14 C]thymidine for 24 hours as described in the text and pulsed with [3H]thymidine for the times indicated after U .V .-irradiation . Unirradiated cells (.), cells irradiated with 100 ergs/mm 2 (o) .
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DISTANCE SEDIMENTED Figure 4 . Elongation of DNA fragments synthesized immediately after U .V .-irradiation . Distance between gaps in newly-synthesized DNA was determined using numberaverage molecular weight (Mn), see methods : (A) unirradiated cells pulse-labelled with [ 3H]thymidine (50 µCi/ml) for 10 min ; Mw=2 . 3 x 10', Mn=9 . 9 x 10 8 ; (B) cells irradiated with 100 ergs/mm 2 and pulse-labelled for 50 min ; Mw=1 . 1 x 10', Mn=3 . 0 x 10 8 ; ( C) cells irradiated, pulse-labelled for 50 min and chased with thymidine (2 . 5µg/ml) for 90 min ; Mw=2 . 1 x 10 7, Mn=6 . 0 x 10 8 . Lysis and centrifugation were as described in figure 2 .
were capable of synthesizing DNA of molecular weight equal to that of unirradiated cells when the same pulse times were used (figure 5) . When HeLa-QB1 cells were treated under the same conditions (figure 5) DNA of molecular weight
of the same size as that obtained in unirradiated cells was found, but a high proportion of material of lower molecular weight was still present .
3 .3 . Gap filling To examine the post-replication repair process, cells were U .V .-irradiated, then pulse labelled with [ 3H]thymidine so that the same amount of DNA synthesis occurred in U .V .-irradiated and unirradiated cells . Under these conditions, gaps existed in the DNA of U .V .-irradiated cells (figure 4) . Label was washed out and the cells were further incubated in BUdR . In the case of
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Figure 5 . Sedimentation profile of DNA synthesized in MM96 and HeLa cells 6 hours after U .V .-irradiation . Cells were irradiated (100 ergs/mm 2) and pulse-labelled with [ 3H]thymidine (50 µCi/ml) : (A) unirradiated MM96 pulsed for 20 min, Mw=3 . 3 x 101 ; ( B) irradiated MM96 pulsed for 20 min, Mw=3 . 0 x 10' ; (C) unirradiated HeLa cells pulsed for 20 min, Mw=2 . 4 x 10' ; (D) irradiated HeLa cells pulsed for 20 min, Mw =1 . 0 x 10 1 ; lysis and centrifugation were as described in figure 2 .
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Figure 6 . Sedimentation profile of DNA from U .V .-irradiated cells exposed to longwave-length, high-intensity U .V.-light. Cells were irradiated (100 ergs/mm 2 ), pulse-labelled with [ 3H]thymidine (50 µCi/ml) for 40 min, chased with BUdR (5µg/ml) and exposed to long-wave-length U .V.-light (5 x 10 1, ergs/mm 2 ) . Cells were lysed as described in figure 2 and sedimentation was for 3 hours at 30 000 r .p .m . in a Beckman SW56 rotor : (A) irradiated cells, 40 min pulse, Mw=1 . 1 x 10' ; (B) irradiated cells, 40 min pulse, 5 hour chase ; (C) irradiated cells pulsed, chased and exposed to long-wave-length U .V ., Mw=1 . 6 x 10' ; (D) unirradiated cells 8 min pulse, Mw=3 . 3 x 10' ; (E) unirradiated cells 8 min pulse, 2-hour chase ; (F) unirradiated cells pulsed, chased and exposed to long-wave-length U .V., Mw=3 . 9 x 10' .
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the U .V .-irradiated cells, the process of elongation of the shorter pieces of DNA involved filling in of the existing gaps with MR . Since bromouracil containing sections of DNA are selectively photolysed by longwave length U .V .-light (Hutchinson and Hales 1970), this provides a means of determining whether gap-filling which involves de novo synthesis plays an important part in the elongation process involved in post-replication repair . Figure 6 shows that the DNA fragments of low molecular weight produced after U .V . -irradiation increased in molecular weight when the cells were further incubated in the presence of BUdR . Exposure of these cells to long-wave length U .V .-light led to a reversion to approximately the initial low molecular weight DNA . A reduction in molecular weight was also observed in unirradiated, BUdR-substituted cells but only to that obtained in unirradiated cells pulsed with [ 3H]thymidine (figure 6) . A control using cells in which DNA was not substituted with BUdR but was exposed to long-wave-length U .V .-light showed no reduction in molecular weight (data not shown) . This suggests that the gaps produced after the synthesis of DNA in U .V .-irradiated cells are filled in by a mechanism involving de novo synthesis .
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Figure 7 . The effect of theophylline on DNA synthesis in irradiated cells . Cells were U .V .-irradiated with 100 ergs/mm2 , pulsed with [3H]thymidine (50 µCi/ml) for 50 min in the presence or absence of theophylline (0 . 3 mg/ml) and chased for 2 hours in cold medium containing thymidine (2. 5 pg/ml) . Sedimentation and lysis as in figure 2 : (A) unirradiated cells pulsed for 10 min, chased for 60 min in absence of theophylline, Mw=B •0 x 107 ; ( B) in presence of theophylline (0 . 3 mg/ml), Mw=7 . 8 x 10 7 ; (C) irradiated cells, in absence of theophylline, Mw=3 . 5 x 10 7 ; (D) in presence of theophylline (0 . 3 mg/ml), Mw= 3-4 x 107 .
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3 .4 . Effect o f theophylline Theophylline inhibits the filling-in of gaps in mouse L5178Y cells (Lehmann and Kirk-Bell 1972) . When MM96 cells were exposed to U .V.-light and allowed to synthesize DNA in the presence and absence of theophylline, no difference in the rate of elongation was observed (figure 7) . The results indicate that theophylline does not interfere with the process of gap-filling opposite U .V .-damage in human melanoma cells, in keeping with data on some human cells (Lehmann, Kirk-Bell, Arlett, Paterson, Lohman, de Weerd-Kastelein and Bootsma 1975), although it has been reported that caffeine, a structural analogue, can bring about post- U . V .-sensitization in other human cells (Schroy and Todd 1976) . 4.
Discussion The results presented have shown that, although there was an initial decrease in the rate of DNA synthesis in U .V .-resistant human melanoma cells after U .V. -irradiation (figure 3), these cells continued to carry out semi-conservative replication after a U .V.-dose of 250 ergs/mm 2 . In contrast, HeLa-QB1, a U .V .-sensitive cell-line, showed a considerable decrease in capacity to carry out semi-conservative synthesis at this dose although it retained the capacity to do so at 100 ergs/mm 2 . When the same amount of radioactive nucleotide had been incorporated into the DNA, the molecular weight of the DNA initially synthesized in U .V .irradiated cells was smaller than that in unirradiated cells . Elongation of these smaller pieces of DNA occurred on further incubation of the irradiated cells . This indicates that gaps exist in the newly-synthesized material of U .V.-irradiated cells and that these gaps are subsequently filled by a post-replication repair process (Buhl et al. 1972) . When bromodeoxyuridine was present during gapfilling, and the DNA was subsequently photo-degraded, a reversion to the initial molecular-weight distribution occurred . This reversibility of gap-filling by photo-degradation suggests that de novo DNA synthesis is required . These data would be consistent with either de novo synthesis alone (Lehmann 1972), or a recombination repair mechanism, which has been reported to occur in mammalian cells (Buhl and Regan 1973, Meneghini and Hanawalt 1976) . If a small amount of de novo synthesis is required in the recombination process, BUdR would be incorporated at the ends of the recombinant parental segment, which would then be excised by photo-degradation . Calculations based on the methods of Lehmann (1972) show that, at short times after irradiation (100 ergs/mm 2), the size of the DNA fragments is approximately equal to the interdimer distance . This calculation takes into account the low level of dimer removal in this time (Chalmers et al. 1976) . The numberaverage molecular weight of the DNA pieces obtained at short times after U .V.irradiation was of the order of 3 x l0 6 daltons (figure 4), and the distance between dimers on single strands at this dose was of the same order (calculated from the data of Chalmers et al. 1976) . It has been demonstrated in mouse L5178Y cells (Lehmann 1972) and some other human cells (Buhl, Setlow and Regan 1973) that the size of the DNA pieces formed after U . V .-irradiation is approximately equal to the interdimer distance . On the other hand, fragments of a size greater than the interdimer distance were obtained in Chinese hamster cells (Meyn and Humphrey 1971) and mouse L cells (Chiu and Rauth 1972) .
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The elongation rate of newly-synthesized DNA after U .V .-irradiation in MM96 is somewhat more rapid than that of the bulk of newly-synthesized DNA in HeLa-QB1 (figure 5). Although it is possible, on this basis and on the basis of DNA degradation in the latter line (Chalmers et al . 1976), to suggest reasons why HeLa-QB1 is more U .V .-sensitive than MM96, the extreme U .V .-resistance of MM96 and other human melanoma lines is not explained fully by the present data, since the DNA elongation rate in MM96 is similar to those reported in some other cell-lines that are less sensitive than HeLa-QB 1 but much more sensitive than MM96 (Buhl et al . 1972, Lehmann 1972, Buhl et al. 1973) . The rate of DNA elongation does not comment on the fidelity of this process in melanoma cells ; a qualitative assessment of the accuracy of replication and post-replication repair after U .V .-irradiation may well contribute further to the understanding of the basis of the resistance . This is presently being examined by measurement of mutation frequency . ACKNOWLEDGMENTS
This work was supported in part by the Queensland Cancer Fund, the Cancer Research Fund of the University of Queensland and the National Health and Medical Research Council, Australia . The authors wish to thank Dr . Alan R . Lehmann for his critical reading of the manuscript and his helpful suggestions . We also thank P . Chen, L . Holder and K . Baker for technical assistance .
On a etudie l'effet du rayonnement ultraviolet (U .V .) sur la replication de dADN de cellules de melanome humain, U .V.-resistantes (lignee MM96) . Par une irradiation de 100 ergs/mm2 , on diminue d'environ cinq fois la synthese semi-conservatrice de dADN . Les fragments d'ADN synthetises par les cellules peu apres l'irradiation U .V . sont plus courts que ceux synthetises par des cellules non sujettes au rayonnement U .V . Il y a elongation de ces fragments en fonction du temps et, 6 heures apres irradiation, les cellules synthetisent des fragments d'ADN de la meme taille que ceux obtenus avec des cellules non irradiees . Dans ce systeme de reparation post-replicatrice, it apparait que le mode d'elongation a besoin d'une synthese de novo et n'est pas inhibe par la theophylline .
Die Wirkung ultravioletter (U .V .) Strahlung auf die DNS-Replikation in einer U .V .resistenten menschlichen Melanom-Zellinie (MM96) wurde untersucht . Die semikonservative DNS-Synthese wurde durch eine U .V .-Dosis von 100 erg/mm2 etwa filnffach reduziert . Die GroBe der DNS-Bruchstucke, die in bestrahlten Zellen in kurzen Abstanden nach der U .V .-Bestrahlung synthetisiert wurden, war kleiner als jene, die in unbestrahlten Zellen gebildet wurden . Diese Bruchstucke wurden mit der Zeit verlangert ; sechs Stunden nach Bestrahlung bauten die Zellen DNS-Bruchstucke derselben GrS13e auf wie jene, die von unbestrahlten Zellen erreicht wurden . Es zeigte sich in diesem Post-ReplikationsreparaturprozeB, dad die Verlangerung de novo Synthese erfordert and nicht durch Theophyllin gehemmt werden kann .
REFERENCES
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DNA replication and repair in a human melanoma cell-line resistant to ultra-violet-radiation MARTIN F . LAVIN, GILLIAN M . WILLETT, A. H . CHALMERS and CHEV KIDSON
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Department of Biochemistry, University of Queensland, Brisbane, 4067, Australia (Received 28 October 1976 ; accepted 3 December 1976)
The effect of ultra-violet (U .V.)-irradiation on DNA replication was studied in a U .V .-resistant, human melanoma cell-line (MM96) . Semi-conservative synthesis of DNA was decreased about five-fold by a U .V.-dose of 100 ergs/mm 2 . The size of DNA fragments synthesized in irradiated cells at short times after U .V . was smaller than those synthesized in unirradiated cells . Elongation of these fragments occurred with time, and 6 hours after irradiation cells synthesized DNA in fragments of the same size as obtained in unirradiated cells . In this post-replication repair process, elongation appeared to involve de novo synthesis and was not inhibited by theophylline .
1 . Introduction Several human melanoma cell-lines have been shown to be highly resistant to ultraviolet (U .V .)-radiation (Chalmers, Lavin, Atisoontornkul, Mansbridge and Kidson 1976). In view of the relationship of the incidence of melanoma to U .V .-exposure (Lee and Merrill 1971, Beardmore 1972), it is important to explore the basis of the radioresistance and its relevance to the biological behaviour of melanoma . Resistance to U .V . does not appear to be due to rapid removal of pyrimidine dimers, since rates of removal are similar to those observed in more sensitive cells (Chalmers et al . 1976) . In a variety of cells, semi-conservative synthesis of DNA is either inhibited or depressed by U.V .-irradiation (Cleaver 1965, Klimek and Vlasinova 1966, Djordsevic and Tolmach 1967, Domon and Rauth 1968) . The molecular weight of newly-synthesized DNA in mammalian cells after U .V .-irradiation has been shown to be lower than that synthesized in unirradiated cells (Cleaver and Thomas 1969, Buhl, Stilmann, Setlow and Regan 1972, Lehmann 1972) . Discontinuities in newly-synthesized mammalian DNA after irradiation (Cleaver and Thomas 1969) are subsequently filled in by a postreplication repair mechanism (Lehmann 1972) . We have examined the effect of U .V . irradiation on semi-conservative synthesis of DNA and on repair functions associated with DNA replication in one (MM96) of the series human melanoma cell-lines shown to be resistant to U .V. 2.
Materials and methods
2.1 . Cells
The melanoma cell-line, MM96, and the U .V .-sensitive HeLa cell-line, HeLa-QB1, used in these studies have been described previously, as have the methods for growing log-phase, unsynchronized cultures (Chalmers et al. 1976) .
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2.2 . Labelling o f cells In double-label experiments, cells were grown for 24 hours at 37 ° C in medium containing [14 C] thymidine, 0 . 5 µCi/ml, 56 mCi/mmole (Nuclear Dynamics Inc ., El Monte, California) . Cells were then washed with prewarmed Hanks' solution before U .V .-irradiation, which was administered by a U .V.-lamp (Mineral light UVS 12) at a dose rate of 10 ergs/mm 2 /sec . The DNA was then pulse-labelled by adding [ 3 H]thymidine, 50 µCi/ml, 20 Ci/mmole (Nuclear Dynamics Inc ., El Monte, California) for prescribed times . After labelling, the cells were washed with ice-cold isotonic EDTA solution (Setlow, Regan, German and Carrier 1969) and removed by scraping . The cells were then centrifuged and resuspended in EDTA solution containing 2 per cent sodium lauryl sarcosine . Samples were placed on Whatman glass-fibre filters (GF/C), washed according to the method of Bollum (1966) and counted in a toluene scintillator . In pulse-chase experiments, cells were washed in Hanks' solution before irradiation and pulsed immediately with [ 3H]thymidine, 50 µCi/ml . At appropriate times, thymidine, 2 . 5 µg/ml, and deoxycytidine, 2 . 5 µg/ml, were added to the medium for stated periods .
2.3 . Sedimentation Cells were harvested as described above and resuspended in ice-cold isotonic EDTA solution . Lysis (3-5 x 104 cells/gradient) was carried out during 30 min in a 0 . 1 ml overlay of 1 M NaOH containing 0. 01 M EDTA on alkaline sucrose gradients (5-20 per cent) containing 0 . 9 M NaCl, 0 . 01 M EDTA and 0 . 01 M NaOH . Centrifugation Was for 3 hours at 30 000 r .p .m. in an SW 56 rotor of a Beckman L2-65B centrifuge . Fractions were collected and assayed by the paper strip method of Carrier and Setlow (1971) .
2 .4. Estimation of molecular weight o f DNA Gradient centrifugation in a 5-20 per cent sucrose gradient was used to define the weight-average sedimentation coefficients of newly-synthesized DNA fragments (Martin and Ames 1961) . Weight-average molecular weight (Mw) was employed since calculation of number-average molecular weight (Mn) is subject to error owing to low-molecular-weight material near the top of the gradient. However, when the estimation of distance between gaps in newlysynthesized DNA was calculated, Mn over the major peak was determined, neglecting the smaller peak at the top of the gradient (figure 4) . Molecular weights were calculated with the aid of a computer, from the sedimentation coefficients estimated by the method of Martin and Ames (1961), using the empirical calibration plots of Studier (1965) . Bacteriophage T3 DNA was used as a standard.
2.5 . CsCl isopycnic gradient analysis Cells were grown in 5 µg/ml 5-bromodeoxyuridine (BUdR) for 1 hour and rinsed with Hanks' solution before U . V .-irradiation . The cells were then incubated in fresh medium containing BUdR, 5 µg/ml and [3 H]thymidine, 10 µCi/ml, for 6 hours . After labelling, cells were harvested and DNA was extracted according to Kidson (1966), except that sodium triisopropylnaphthalene sulphonate, 1 per cent, was used as detergent, and residual phenol was removed
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by ether extraction, followed by dialysis . DNA was analysed by alkaline CsCl isopycnic gradients by adjusting the initial density of the solution to 1 . 76 g/ml . Centrifugation was for 60 hours at 35 000 r.p .m . in a 50Ti head in a Beckman L2-65B centrifuge . Gradients were dripped and samples taken to determine density from measurement of the refractive index, and radioactivity by scintillation spectrometry . 2 .6 . Autoradiography Cultures on cover-slips were irradiated and labelled with 3 [H]thymidine, 10 µCi/ml, for 4 hours . The cells were then rinsed with Hanks' solution and grown for a further 1 hour in medium supplemented with thymidine, 2 . 5 µg/ml, and deoxycytidine, 2 . 5 µg/ml, before fixation in methanol for 5 min . Fixed cultures were dipped in Ilford K2 liquid emulsion for 50 sec and exposed for 21 days before development . Autoradiographs were developed with Kodak D19, fixed, and stained through the emulsion with Giemsa before counting . 2.7 . Degradation o f B UdR-substituted DNA In experiments utilizing BUdR to fill in gaps after U .V.-irradiation, monolayer cultures were rinsed on plates with isotonic EDTA solution . Culture plates containing 5 ml of ice-cold saline EDTA solution were then exposed to a long-wave-length U .V .-source (Ultra-Violet Products, Inc ., San Gabriel, California) at an exposure rate of 300 ergs/mm 2 /sec for 30 min. 3. Results 3 .1 . Synthesis of DNA after U . V. -irradiation When cultures of HeLa and MM96 were U .V .-irradiated with a dose of 250 ergs/mm 2 and 3 [H]thymidine incorporation in the subsequent 4 hours measured by autoradiography, the number of heavily-labelled HeLa nuclei dropped to 16 per cent of the unirradiated value while the number of heavilylabelled nuclei for MM96 remained essentially unchanged at this U .V .-dose . Unscheduled synthesis was apparent in both cases as evidenced by the presence of lightly-labelled nuclei . These data suggested that MM96 had the ability to continue semi-conservative DNA synthesis after a U .V .-dose of 250 ergs/mm 2 at a substantial rate, whereas DNA synthesis in HeLa cells was severely restricted. Accordingly, we used the method of Pettijohn and Hanawalt (1964), using BUdR-labelled DNA as a means of distinguishing semi-conservative DNA synthesis and repair replication . Figure 1 shows that semi-conservative replication continued in MM96 immediately after a U .V.-dose of 250 ergs/mm2 ; repair replication was also apparent . However, in the case of HeLa-QB1, most of the label incorporated into DNA was as repair replication . These findings are consistent with the autoradiographic results, which show a considerable decrease in the number of cells carrying out semi-conservative synthesis at this U .V .-dose in the case of HeLa-QB1 but not of MM96 . 3 .2 . Size o f newly-synthesized DNA after U . V. -irradiation The molecular weight of pulse-labelled DNA from U .V.-irradiated human cells has been shown to be lower than that from unirradiated cells (Buhl et al. 1972) . Figure 2 shows that this also is the case with U .V .-resistant human
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FRACTION NUMBER Figure 1 . Nature of DNA synthesis after U .V .-irradiation (250 ergs/mm 2 ) . Melanoma and HeLa cells were incubated in the presence of BUdR (5 µg/ml) and [ 3H]thymidine (10 µCi/ml) for 6 hours as described in the text, before extraction and density analysis of DNA on alkaline CsCl gradients .
melanoma cells, a decrease in the molecular weight of newly-synthesized DNA being obtained after U .V .-irradiation . The decrease in molecular weight was dose-dependent over the range tested, 50-200 ergs/mm 2 (figure 2) . This decrease in molecular weight with increasing U .V .-dose is due either to the presence of discontinuities in the newly-synthesized DNA, because of the presence of pyrimidine dimers in, or other damage to the parental template, or it may arise from a slower rate of synthesis in irradiated cells . To distinguish between these possibilities cells were prelabelled with [14 C]thymidine for 24 hours and pulsed with [ 3 H]thymidine after U .V .-irradiation . The relative rates of synthesis in irradiated and unirradiated cells are represented in figure 3 . The rate of DNA synthesis in cells irradiated with 100 ergs/mm 2 was approximately one-fifth of that in unirradiated cells . The molecular weight of DNA from irradiated and unirradiated cells, having the same 3 H/ 14 C ratio, that is the same amount of DNA synthesized, was determined by sedimentation analysis (figure 4) . Under these conditions, the weight-average molecular weight of DNA from irradiated cells was 1 . 1 x 10', while that from unirradiated cells was 2 . 3 x 10' . This suggests that after U .V.-irradiation, in the extra period of time required to synthesize the same amount of DNA as in unirradiated cells, synthesis is initiated at many more sites giving rise to short pieces . When the irradiated cells were incubated for a further 1 . 5 hours, the molecular weight of the DNA (2 . 1 x 10') had reached approximately that of DNA from the pulse-labelled unirradiated cells . Six hours after U .V .-irradiation, cells which had received a dose of 100 ergs/mm 2
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Figure 2 . Sedimentation profile of DNA synthesized in MM96 immediately after U .V.irradiation . Cells were pulse-labelled with [ 3 H]thymidine (50 µCi/ml) for 40 min immediately after U .V . -irradiation, lysed on top of alkaline sucrose gradients in 1 m NaOH containing 0.01 M EDTA for 30 min, and centrifuged for 3 hours at 30 000 r .p .m . in an SW56 rotor . Fractions were collected and analysed for radioactivity : (A) unirradiated cells, Mw=4 .0 x 10' ; (B) cells irradiated with 50 ergs/mm2, Mw=1 . 4 x 10' ; (C) 100 ergs/mm 2 , Mw=1 . 1 x 10' ; (D) 200 ergs/mm2, Mw= 9 . 5 x 10 6 .
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Figure 3 . Effect of U .V .-irradiation on DNA synthesis immediately after irradiation . Cells pre-labelled with [ 14 C]thymidine for 24 hours as described in the text and pulsed with [3H]thymidine for the times indicated after U .V .-irradiation . Unirradiated cells (.), cells irradiated with 100 ergs/mm 2 (o) .
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were capable of synthesizing DNA of molecular weight equal to that of unirradiated cells when the same pulse times were used (figure 5) . When HeLa-QB1 cells were treated under the same conditions (figure 5) DNA of molecular weight
of the same size as that obtained in unirradiated cells was found, but a high proportion of material of lower molecular weight was still present .
3 .3 . Gap filling To examine the post-replication repair process, cells were U .V .-irradiated, then pulse labelled with [ 3H]thymidine so that the same amount of DNA synthesis occurred in U .V .-irradiated and unirradiated cells . Under these conditions, gaps existed in the DNA of U .V .-irradiated cells (figure 4) . Label was washed out and the cells were further incubated in BUdR . In the case of
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Figure 5 . Sedimentation profile of DNA synthesized in MM96 and HeLa cells 6 hours after U .V .-irradiation . Cells were irradiated (100 ergs/mm 2) and pulse-labelled with [ 3H]thymidine (50 µCi/ml) : (A) unirradiated MM96 pulsed for 20 min, Mw=3 . 3 x 101 ; ( B) irradiated MM96 pulsed for 20 min, Mw=3 . 0 x 10' ; (C) unirradiated HeLa cells pulsed for 20 min, Mw=2 . 4 x 10' ; (D) irradiated HeLa cells pulsed for 20 min, Mw =1 . 0 x 10 1 ; lysis and centrifugation were as described in figure 2 .
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Figure 6 . Sedimentation profile of DNA from U .V .-irradiated cells exposed to longwave-length, high-intensity U .V.-light. Cells were irradiated (100 ergs/mm 2 ), pulse-labelled with [ 3H]thymidine (50 µCi/ml) for 40 min, chased with BUdR (5µg/ml) and exposed to long-wave-length U .V.-light (5 x 10 1, ergs/mm 2 ) . Cells were lysed as described in figure 2 and sedimentation was for 3 hours at 30 000 r .p .m . in a Beckman SW56 rotor : (A) irradiated cells, 40 min pulse, Mw=1 . 1 x 10' ; (B) irradiated cells, 40 min pulse, 5 hour chase ; (C) irradiated cells pulsed, chased and exposed to long-wave-length U .V ., Mw=1 . 6 x 10' ; (D) unirradiated cells 8 min pulse, Mw=3 . 3 x 10' ; (E) unirradiated cells 8 min pulse, 2-hour chase ; (F) unirradiated cells pulsed, chased and exposed to long-wave-length U .V., Mw=3 . 9 x 10' .
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the U .V .-irradiated cells, the process of elongation of the shorter pieces of DNA involved filling in of the existing gaps with MR . Since bromouracil containing sections of DNA are selectively photolysed by longwave length U .V .-light (Hutchinson and Hales 1970), this provides a means of determining whether gap-filling which involves de novo synthesis plays an important part in the elongation process involved in post-replication repair . Figure 6 shows that the DNA fragments of low molecular weight produced after U .V . -irradiation increased in molecular weight when the cells were further incubated in the presence of BUdR . Exposure of these cells to long-wave length U .V .-light led to a reversion to approximately the initial low molecular weight DNA . A reduction in molecular weight was also observed in unirradiated, BUdR-substituted cells but only to that obtained in unirradiated cells pulsed with [ 3H]thymidine (figure 6) . A control using cells in which DNA was not substituted with BUdR but was exposed to long-wave-length U .V .-light showed no reduction in molecular weight (data not shown) . This suggests that the gaps produced after the synthesis of DNA in U .V .-irradiated cells are filled in by a mechanism involving de novo synthesis .
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Figure 7 . The effect of theophylline on DNA synthesis in irradiated cells . Cells were U .V .-irradiated with 100 ergs/mm2 , pulsed with [3H]thymidine (50 µCi/ml) for 50 min in the presence or absence of theophylline (0 . 3 mg/ml) and chased for 2 hours in cold medium containing thymidine (2. 5 pg/ml) . Sedimentation and lysis as in figure 2 : (A) unirradiated cells pulsed for 10 min, chased for 60 min in absence of theophylline, Mw=B •0 x 107 ; ( B) in presence of theophylline (0 . 3 mg/ml), Mw=7 . 8 x 10 7 ; (C) irradiated cells, in absence of theophylline, Mw=3 . 5 x 10 7 ; (D) in presence of theophylline (0 . 3 mg/ml), Mw= 3-4 x 107 .
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3 .4 . Effect o f theophylline Theophylline inhibits the filling-in of gaps in mouse L5178Y cells (Lehmann and Kirk-Bell 1972) . When MM96 cells were exposed to U .V.-light and allowed to synthesize DNA in the presence and absence of theophylline, no difference in the rate of elongation was observed (figure 7) . The results indicate that theophylline does not interfere with the process of gap-filling opposite U .V .-damage in human melanoma cells, in keeping with data on some human cells (Lehmann, Kirk-Bell, Arlett, Paterson, Lohman, de Weerd-Kastelein and Bootsma 1975), although it has been reported that caffeine, a structural analogue, can bring about post- U . V .-sensitization in other human cells (Schroy and Todd 1976) . 4.
Discussion The results presented have shown that, although there was an initial decrease in the rate of DNA synthesis in U .V .-resistant human melanoma cells after U .V. -irradiation (figure 3), these cells continued to carry out semi-conservative replication after a U .V.-dose of 250 ergs/mm 2 . In contrast, HeLa-QB1, a U .V .-sensitive cell-line, showed a considerable decrease in capacity to carry out semi-conservative synthesis at this dose although it retained the capacity to do so at 100 ergs/mm 2 . When the same amount of radioactive nucleotide had been incorporated into the DNA, the molecular weight of the DNA initially synthesized in U .V .irradiated cells was smaller than that in unirradiated cells . Elongation of these smaller pieces of DNA occurred on further incubation of the irradiated cells . This indicates that gaps exist in the newly-synthesized material of U .V.-irradiated cells and that these gaps are subsequently filled by a post-replication repair process (Buhl et al. 1972) . When bromodeoxyuridine was present during gapfilling, and the DNA was subsequently photo-degraded, a reversion to the initial molecular-weight distribution occurred . This reversibility of gap-filling by photo-degradation suggests that de novo DNA synthesis is required . These data would be consistent with either de novo synthesis alone (Lehmann 1972), or a recombination repair mechanism, which has been reported to occur in mammalian cells (Buhl and Regan 1973, Meneghini and Hanawalt 1976) . If a small amount of de novo synthesis is required in the recombination process, BUdR would be incorporated at the ends of the recombinant parental segment, which would then be excised by photo-degradation . Calculations based on the methods of Lehmann (1972) show that, at short times after irradiation (100 ergs/mm 2), the size of the DNA fragments is approximately equal to the interdimer distance . This calculation takes into account the low level of dimer removal in this time (Chalmers et al. 1976) . The numberaverage molecular weight of the DNA pieces obtained at short times after U .V.irradiation was of the order of 3 x l0 6 daltons (figure 4), and the distance between dimers on single strands at this dose was of the same order (calculated from the data of Chalmers et al. 1976) . It has been demonstrated in mouse L5178Y cells (Lehmann 1972) and some other human cells (Buhl, Setlow and Regan 1973) that the size of the DNA pieces formed after U . V .-irradiation is approximately equal to the interdimer distance . On the other hand, fragments of a size greater than the interdimer distance were obtained in Chinese hamster cells (Meyn and Humphrey 1971) and mouse L cells (Chiu and Rauth 1972) .
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The elongation rate of newly-synthesized DNA after U .V .-irradiation in MM96 is somewhat more rapid than that of the bulk of newly-synthesized DNA in HeLa-QB1 (figure 5). Although it is possible, on this basis and on the basis of DNA degradation in the latter line (Chalmers et al . 1976), to suggest reasons why HeLa-QB1 is more U .V .-sensitive than MM96, the extreme U .V .-resistance of MM96 and other human melanoma lines is not explained fully by the present data, since the DNA elongation rate in MM96 is similar to those reported in some other cell-lines that are less sensitive than HeLa-QB 1 but much more sensitive than MM96 (Buhl et al . 1972, Lehmann 1972, Buhl et al. 1973) . The rate of DNA elongation does not comment on the fidelity of this process in melanoma cells ; a qualitative assessment of the accuracy of replication and post-replication repair after U .V .-irradiation may well contribute further to the understanding of the basis of the resistance . This is presently being examined by measurement of mutation frequency . ACKNOWLEDGMENTS
This work was supported in part by the Queensland Cancer Fund, the Cancer Research Fund of the University of Queensland and the National Health and Medical Research Council, Australia . The authors wish to thank Dr . Alan R . Lehmann for his critical reading of the manuscript and his helpful suggestions . We also thank P . Chen, L . Holder and K . Baker for technical assistance .
On a etudie l'effet du rayonnement ultraviolet (U .V .) sur la replication de dADN de cellules de melanome humain, U .V.-resistantes (lignee MM96) . Par une irradiation de 100 ergs/mm2 , on diminue d'environ cinq fois la synthese semi-conservatrice de dADN . Les fragments d'ADN synthetises par les cellules peu apres l'irradiation U .V . sont plus courts que ceux synthetises par des cellules non sujettes au rayonnement U .V . Il y a elongation de ces fragments en fonction du temps et, 6 heures apres irradiation, les cellules synthetisent des fragments d'ADN de la meme taille que ceux obtenus avec des cellules non irradiees . Dans ce systeme de reparation post-replicatrice, it apparait que le mode d'elongation a besoin d'une synthese de novo et n'est pas inhibe par la theophylline .
Die Wirkung ultravioletter (U .V .) Strahlung auf die DNS-Replikation in einer U .V .resistenten menschlichen Melanom-Zellinie (MM96) wurde untersucht . Die semikonservative DNS-Synthese wurde durch eine U .V .-Dosis von 100 erg/mm2 etwa filnffach reduziert . Die GroBe der DNS-Bruchstucke, die in bestrahlten Zellen in kurzen Abstanden nach der U .V .-Bestrahlung synthetisiert wurden, war kleiner als jene, die in unbestrahlten Zellen gebildet wurden . Diese Bruchstucke wurden mit der Zeit verlangert ; sechs Stunden nach Bestrahlung bauten die Zellen DNS-Bruchstucke derselben GrS13e auf wie jene, die von unbestrahlten Zellen erreicht wurden . Es zeigte sich in diesem Post-ReplikationsreparaturprozeB, dad die Verlangerung de novo Synthese erfordert and nicht durch Theophyllin gehemmt werden kann .
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