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Biochimica et Biophysica Acta, 4 3 5 ( 1 9 7 6 ) 9 5 - - 1 0 3 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 98596

DNA-PROTEIN CROSS-LINKING BY U L T R A V I O L E T R A D I A T I O N IN N O R M A L H U M A N AND X E R O D E R M A PIGMENTOSUM FIBROBLASTS

A L B E R T J. F O R N A C E Jr. * a n d K U R T W. K O H N

Laboratory of Molecular Pharmacology, Division o f Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, Md. 20014 (U.S.A.) (Received November 18th, 1975)

Summary DNA-protein cross-linking b y ultraviolet radiation was measured in h u m a n fibroblasts by an adaptation o f the m e t h o d of D N A alkaline elution. To measure cross-linking, a controlled frequency of D N A single-strand breaks was introduced by exposing the cells to a low dose o f X-ray at 0°C prior to analysis by alkaline elution. The effect of prior exposure of the cells to ultraviolet radiation was to reduce the rate and/or extent of DNA elution from X-irradiated cells. This effect was attributed to DNA-protein cross-linking, since the effect was reversed by treatment of the cell lysates with proteinase-K. Cross-linking in normal h u m a n fibroblasts occurred immediately after ultraviolet irradiation, prior to the appearance of D N A single-strand breaks due to excision repair. U p o n incubation of normal cells after exposure, to ultraviolet radiation, the cross-linking was partially repaired. In xeroderma pigmentosum cells, cross-links appeared as in normal cells, b u t there was no repair. Instead, the extent of cross-linking appeared to increase u p o n incubation after ultraviolet irradiation.

Introduction Although the most extensively studied photochemical effect of ultraviolet radiation on cellular D N A has been the formation o f pyrmidine dimers, this reaction m a y n o t fully account for the deleterious effect of ultraviolet radiation on cells [1,2]. It has been proposed that the lethal effect o f ultraviolet radiation may depend also on the formation of cross-links b e t w e e n D N A and protein [1,3--9]. The formation of DNA-protein cross-links has been inferred from a reduction in extractability o f DNA from cell lysates, and from the * Present address: D e p a r t m e n t o f P a t h o l o g y , 721 H u n t i n g t o n A v e n u e , Boston, Mass. 02115, U.S.A.

96 finding that extractability is restored by treatment of the lysates with trypsin [10,11,7]. Model photochemical reactions have been demonstrated that may be the basis for linkage between DNA and protein [ 1 2 - - 2 2 ] . The evidence relating cross-linking with viability is weakened b y the limited sensitivity of the extractability assays, which have required ultraviolet exposures above the range in which the major changes in viability occur. In the current work, we show the applicability of a more sensitive assay, based on the alkaline elution of DNA from cells lysed on filters [23,24]. We have employed this m e t h o d to examine the formation and possible repair of DNA-protein crosslinks in normal and xeroderma pigmentosum fibroblasts. Materials and Methods Cell lines and cell labeling. H u m a n fibroblast cell lines were obtained from the American Type Culture Collection, Rockville, Md. Lines CRL 1119, CRL 1220, CRL 1229 were from normal adult donors; line XP12BE (CRL 1223) was from a patient with xeroderma pigmentosum, of complementation group A [25]. The cells were grown in Dulbecco's medium with 10% fetal calf serum plus penicillin and streptomycin at 37°C under 5% CO2. 6 days prior to the experiments, 2 - l 0 s fibroblasts were plated on 50 cm 2 plastic petri dishes (Falcon no. 3003) with [2-14C]thymidine (0.02 pCi/ml, 57 Ci/mol); 3 days later, the medium was replaced with non-radioactive medium. On the day of experiments the cells were in a confluent monolayer. L1210 mouse leukemia cells were grown in suspension culture in RPMI 1630 medium supplemented with 20% fetal calf serum plus penicillin and streptomycin [ 2 6 ] . Exponentially growing L1210 cells were labeled with [3H]thymidine (0.1 pCi/ml, 10 -6 M thymidine) for 20 h. Irradiation. Confluent fibroblast monolayers were irradiated with a GE 254 nm germicidial lamp calibrated with a short wave ultraviolet intensity meter (J-225 UV meter, UV Products, Inc., San Gabriel, Calif.} Prior to irradiation, the cells were washed with warm phosphate-buffered saline (0.15 M NaC1/ 0.7 mM KH2PO4/4.3 mM K2HPO4, pH 7.4). After irradiation, fresh non-radioactive medium, buffered with 0.01 M HEPES (N-2-hydroxyethylpiperazine-N'2-ethane sulfonate), was quickly added, and the cells were incubated at 37°C for various times ("repair" time). At the end of this incubation, the cells were washed with warm saline fo~ 40 s, incubated with 0.25% trypsin at 37°C for 60 s, and suspended in cold saline. When there was no incubation after ultraviolet exposure, the cells were trypsinized for 60 s, gently rinsed with warm saline, ultraviolet irradiated at 37°C, and then immediately suspended in cold saline. X-irradiation o f fibroblasts was carried o u t on cells suspended in saline at 0°C. X-ray was delivered by vertically opposed dual Westinghouse Quadrocondex units operated at 200 kV, 15 mA; 0.25 mm copper plus 0.55 m m aluminum filter. The dose rate was 140 rad/min. Alkaline elution. The procedure used is a modification of that described by Kohn et al. [ 2 3 ] . Cells were diluted in cold saline and filtered onto a 25 mm diameter, 2 pm pore size polyvinylchloride filter (Millipore Corp., Bedford, Mass.) and washed several times with cold saline. The cells were then lysed at

97 r o o m temperature with 5 ml of 2 M NaC1, 0.02 M Na3EDTA, and 0.2% Sarkosyl (pH 10.2), which was allowed to flow slowly through the filter without suction. The filter was then washed with 3 ml of 0.02 M Na3EDTA (pH 10.3) and eluted in the dark with 0.10 M t e t r a p r o p y l a m m o n i u m hydroxide/0.02 M EDTA (acid form) (pH 12.2) at a p u m p speed o f 0.04 ml/min. Fraction were collected at 90-min intervals for 15 h. The fractions were mixed with 10 ml Aquasol (New England Nuclear, Boston, Mass.) containing 0.3% acetic acid, and counted in a liquid scintillation counter. Radioactivity remaining on the filter was determined b y treating the filter with 0.4 ml of I M HC1 at 70°C for 1 h, followed by 2.5 ml of 0.4 M NaOH at r o o m temperature for 30 min and 10 ml of Aquasol. Radioactivity remaining in the funnel was recovered by five washes with 3 ml of 0.4 M NaOH and the counts were added to those remaining on the filter. The procedure was carried o u t using 2 • l 0 s 14C-labeled fibroblasts. In order to provide for an internal standard, 4" l 0 s 3H-labeled L1210 cells that had received 150 rad at 0°C were included in each assay. The fraction of a sample DNA which is retained on the filter when 50% of the internal standard DNA has eluted is termed "relative retention." Proteinase-K treatment. Cells were lysed on filters for alkaline elution. After the pH 10.2 lysis solution had run o u t by gravity, the filters were washed once with 3 ml of 0.01 M Tris/0.01 M EDTA/0.01 M NaC1/0.5% sodium dodecyl sulfate, pH 8.2. 3 ml of 0.5 mg/ml proteinase-K (E. Merck, Darmstadt, Germany) in the same buffer was then added. After a b o u t 1 ml of this solution had passed through t h e filter, the exit tube from the funnel was clamped o f f and incubation was continued to 30 min at 23°C. The solution was then allowed to drip o u t and alkaline elution was carried o u t at pH 12.2. Results To elicit the effects of DNA cross-linking in the alkaline elution method, D N A single-strand breaks were introduced in a controlled manner b y X-irradiation o f the cells. Fig. 1 illustrates typical elution curves, and shows the increase in elution rate produced b y exposure of normal human fibroblasts to 220 tad o f X-ray. In order to prevent repair of X-ray-induced DNA breaks, cells were k e p t at 0°C during and following X-irradiation. Fig. 1 also shows that the DNA elution from the 150-rad irradiated L1210 reference cells is nearly linear in the semi-logarithmic plot. In subsequent figures, the elution of D N A from sample cells is plotted against the elution o f D N A from the internal reference cells, since this m e t h o d o f presentation improves quantitation. This normalization procedure caused little change in the shape of the elution curves, because o f the linearity of the elution curve of the internal reference cells. The dependence of elution on X-ray dose is shown in Fig. 2. The measure of elution used was the fraction of the sample DNA retained on the filter when 50% o f the internal standard DNA has eluted. We call this quantity "relative retention." Relative retention is seen to decrease approximately according to a first-order relation with X-ray dose up to at least 200 tad. The Do dose was 165 rad. This dose corresponds to one break per 2.6 • 109 daltons, assuming an efficiency for DNA single-strand breakage of 2.3 • 10 -12 per dalton per tad

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[ 2 7 ] . The X-ray sensitivities of D N A in the different cell lines were the same. The effect o f a cross-linking agent would be to decrease the apparent level of strand breakage induced by X-ray [ 2 8 ] . In order to test this expectation, normal human fibroblasts were treated with nitrogen mustard (bis-2-chloroethylmethylamine), a known D N A cross-linking agent [ 2 9 ] , and then X-irradiated at 0°C and analysed by alkaline elution. Fig. 3 shows that nitrogen mustard markedly inhibits the ability of X-ray to make the D N A elutable. The effect of ultraviolet radiation on D N A in normal human fibroblasts is shown in Fig. 4. When cells were harvested immediately after ultraviolet exposure, there was little evidence of D N A single-strand breakage, (upper curve in Fig. 4B). When cells were incubated after ultraviolet exposure, single-strand breaks attributable to excision repair appeared (upper curve in Fig. 4C) [ 2 4 ] . In both types of experiments, however, the ability o f X-ray to increase elution

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