327
Mutation Research, 33 (1975) 3 2 7 - - 3 4 0 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - Printed in The Netherlands
FIVE COMPLEMENTATION GROUPS IN XERODERMA PIGMENTOSUM
K.H. K R A E M E R l, E.A. DE W E E R D - K A S T E L E I N 2, J.H. R O B B I N S l, W. K E I J Z E R 2, S.F. B A R R E T T l, R.A. P E T I N G A 1 A N D D B O O T S M A 2
1 Dermatology Branch, National Cancer Institute, National Institutes o f Health, Bethesda, Maryland 20014 (U.S.A.) and 2 Department o f Cell Biology and Genetics, Erasmus University, P.O. Box 1 738, Rotterdam (The Netherlands) (Received July 24th, 1975) ( A c c e p t e d July 28th, 1975)
Summary A collaborative study was undertaken to determine the relationship between the three DNA repair complementation groups in xeroderma pigmentosum found at Erasmus University, Rotterdam, and the four groups found at the National Institutes of Health, Bethesda. The results of this study reveal that there are five currently known complementation groups in xeroderma pigmentosum.
Introduction Patients with xeroderma pigmentosum (XP) develop malignancies and pigmentation abnormalities on areas of skin exposed to sunlight [10]. Skin fibroblasts from most patients with this autosomal recessive disease are unable to perform excision repair of UV-induced pyrimidine dimers in their DNA as rapidly as normal fibroblasts [2,3,10]. This defective repair can be manifest as a decreased rate of UV-induced unscheduled DNA synthesis (UDS) [2,3,10]. In 1972 investigators at Erasmus University, Rotterdam, found two complementation groups in XP by showing that nuclei in heterokaryons formed by fusing fibroblasts from certain pairs of such repair-defective XP patients performed UV-induced UDS at a normal rate [11 ]. Subsequently a third complementation group was found among the Rotterdam XP strains [12]. The demonstration of genetic heterogeneity for DNA repair among the Rotterdam XP strains prompted investigators at the National Institutes of Health Abbreviations: XP, xeroderma pigmentosum; UDS, unscheduled DNA synthesis; 3HTdR, tritiated thymidine; NIH, National Institutes of Health.
328 (NIH), Bethesda, to search for complementation groups among their strains. Four complementation groups were found among the ten kindred tested from the NIH XP patient series [8,10]. The Rotterdam and NIH investigators considered it useful to perform a collaborative study to determine the relationship between the R o t t e r d a m complementation groups and the NIH groups. In this paper we present the results of this collaborative project which indicate that there are five currently known complementation groups in XP. Materials and methods
Representative XP complementation group fibroblast strains The representative Rotterdam complementation group strains were grown from skin biopsies in Rotterdam and then sent to The American Type Culture Collection, Rockville, Maryland, for further growth and dissemination to the NIH. The NIH group strains were grown from biopsies by the American Type Culture Collection. Table I identifies the representative complementation group strains used in our collaborative study.
Rotterdam experimental procedure Details of the procedure have been published elsewhere [11]. Trypsinized cells of 2 different strains were mixed in a 1 : 1 ratio and incubated in the presence of ~-propiolactone-inactivated Sendai virus. One million cells were incubated with 125 hemagglutinating units of Sendai virus in a final volume of 1 ml. After 4 to 6 min at 4°C followed by 20 min at 37°C, the cells were seeded on coverslips. Two days after fusion the fusedcell population was labelled 1 h before and 2 h after UV-irradiation in medium containing [ 3H ] thymidine (3 HTdR; 10 pCi/ml; spec. act., 2 Ci/mmol). Before irradiation the culture was washed with a balanced salt solution, drained and exposed to 10 J/m 2 UV light (254 nm). After the 2 h post-irradiation incubation, the cells were fixed in Bouins fixative. Cells not treated with virus were seeded on coverslips and served as TABLE 1 REPRESENTATIVE ROTTERDAM AND NIH COMPLEMENTATION GROUP FIBROBLAST STRAINS USED IN C O L L A B O R A T I V E STUDY a
C o m p l e m e n t a t i o n group
Strains t e s t e d
Rotterdam nomenclature De Sanctis C a c e h i o n e Classical XP, severe Classical XP, light to m o d e r a t e l y severe
XP25RO XP4RO XP2RO
NIH nomenclature A B C D
XPILO, XP12BE XPllBE XP1BE, XP2BE XP5BE, XP6BE
a T h e p r o p o s e d s t a n d a r d i z a t i o n o f n o m e n c l a t u r e of XP strains [7] is used t h r o u g h o u t ; R O , R o t t e r d a m LO, L o n d o n ; BE, B e t h e s d a .
329
controls. The control coverslip cultures were handled like the fused cultures. Autoradiographs were prepared with Kodak AR 10 stripping film. Exposure time was 1 week. This exposure time resulted in a mean grain c o u n t of 20 grains per nucleus in a control strain. After development, the preparations were stained with hematoxyline and eosin. By evaluating the frequency distribution of grain counts over normal nuclei under these labelling conditions, nuclei with less than 50 grains were considered to be G1- or G2-phase nuclei. The grain numbers 8 and 50 were adopted as the limits of UDS, and nuclei showing between 8 and 50 grains per nucleus are referred to as lightly-labelled. In each experiment 50 binuclear cells with non-S-phase nuclei were included in the countings. Binuclear cells showing between 8 and 50 grains over each nucleus were considered as lightly-labelled. In experiments with XP2RO cells the UV dose was lowered to 5 J/m 2 and the number of binuclear cells used to estimate the number of grains over their nuclei was extended to 100. The autoradiographic exposure time was 15 days. This exposure time resulted in a mean grain c o u n t of 26 grains per nucleus in a control strain.
NIH experimen tal procedure Details of the NIH experimental procedure have been published [8]. Variations in that procedure are presented in the appropriate legends of figures and table legends herein. As in the R o t t e r d a m procedure,/~-propiolactone-inactivated Sendai virus was used to produce cell fusion, and autoradiograms were used to determine the 3HTdR incorporation representing UV-induced UDS. In contrast to the Rotterdam procedure, the strains to be fused were n o t mixed in suspension, for fusion was accomplished by placing drops of a suspension of one strain on another strain which had already been growing on coverslips. Also in contrast to the Rotterdam procedure, at the NIH the fused cells were irradiated approximately 16 h after the two strains to be fused were placed in contact with each other. Since there was a very low frequency of synchronized S-phase synthesis in the nuclei of the binuclear cells after only 16 h following the initiation of cell fusion, preirradiation incubation of the cells with 3HTdR to label S-phase nuclei was n o t necessary and was n o t performed. Results
(I) Rotterdam studies (A) Confirmation of four complementation groups among the NIH groups" strains The cell strains XP1LO, X P l l B E , XP2BE and XP5BE were investigated as representatives of the complementation groups A, B, C and D respectively. A small fraction of the cells in these parental cell populations were lightly-labelled following UV exposure (0--18%, Table IIa). In contrast 96% of the nuclei in a control strain (C5) were labelled when treated under the same conditions. The six possible pairwise fusions between the XP strains resulted in formation of binuclear cells showing complementation as evidenced by the fact t h a t from 28 to 40% of the binuclear cells were lightly labelled (Table IIb). These percent-
330 T A B L E II COMPLEMENTATION
ANALYSIS PERFORMED
BY R O T T E R D A M
GROUP
a. U n s c h e d u l e d D N A s y n t h e s i s in p a r e n t a l s t r a i n s Cell s t r a i n
C5 XP1 L O XPllBE XP2BE XP5BE XP25RO XP4RO
Complementation group
Control A B C D De S a n c t i s - C a c c h i o n e Classic XP, s e v e r e
% L a b e l l e d cells f o l l o w i n g a d o s e o f 0 J/m 2
10 J / m 2
2 2 2 0 0 2 2
96 0 2 18 2 0 8
b. C o n f i r m a t i o n of t h e N I H c o m p l e m e n t a t i o n g r o u p s Fusion
% L a b e l l e d b i n u c l e a r cells f o l l o w i n g a d o s e o f
Complementation group
Cell s t r a i n s
0 J/m 2
10 J / m 2
A/B A/C A/D B/C B/D C/D
XPILO/XPllBE XP1 L O / X P 2 B E XPILO/XP5BE XPllBE/XP2BE XPllBE/XP5BE XP2BE/XP5BE
0 0 0 0 0 0
30 34 40 30 34 28
c. R o t t e r d a m ' s " D e S a n c t i s - C a c c h i o n e " g r o u p c o m p l e m e n t s all N I H g r o u p s e x c e p t A XP25RO fibroblasts fused with NIH group strains
% L a b e l l e d b i n u c l e a r cells f o l l o w i n g a d o s e o f
Group
Strain
0 J/m 2
10 J / m 2
A B C D
XP1LO XPllBE XP2BE XP5BE
0 0 0 0
0 30 48 42
d. R o t t e r d a m ' s " c l a s s i c XP, s e v e r e " g r o u p c o m p l e m e n t s all N I H g r o u p s e x c e p t C XP4RO fibrohlasts fused with NIH group strains
% L a b e l l e d b i n u c l e a r cells f o l l o w i n g a d o s e o f
Group
Strain
0 J/m 2
10 J / m 2
A B C D
XPILO XPIIBE XP2BE XP5BE
0 0 0 0
38 38 0 40
In t h e p a r e n t a l s t r a i n s o n l y cells w i t h 0 - - 5 0 g r a i n s o v e r t h e i r n u c l e i w e r e c o u n t e d ; cells w i t h 8 - - 5 0 g r a i n s w e r e c o u n t e d as labelled. I n t h e f u s e d cell p o p u l a t i o n s b i n u c l e a r ceils w i t h 8 - - 5 0 g r a i n s o v e r e a c h o f b o t h n u c l e i w e r e c o u n t e d as labelled cells. E a c h v a l u e is b a s e d on c o u n t i n g o f 50 cells.
ages approach the 50% level which is the expected fraction of hybrid binuclear cells in the population. The binuclear cells in the unirradiated fused cultures were unlabelled (Table IIb). These results confirm the complementation group studies previously performed at the NIH, which indicated the existence of four complementation groups among these strains [ 8 , 1 0 ] .
331
(B) Assignment o f XP25RO and XP4RO fibroblasts to complementation groups A and C respectively. The X P 2 5 R O fibroblasts belong to the complementation group which was previously indicated as the De Sanctis Cacchione group [11]. These cells were fused with each of the four representative strains of the complementation groups A, B, C and D (Table IIc). In the fused and UV exposed populations 30--48% of the binuclear cells were lightly labelled except in the fusion with XP1LO cells (Table IIc). These results indicate that complementation occurred after fusion with cells from complementation groups B, C, and D and not with cells belonging to A. Therefore the mutation resulting in the Rotterdam complementation group De Sanctis Cacchione can be assigned to the NIH group A. In the same way the representative strain of the R o t t e r d a m complementation group "severe classic X P " [ 1 1 ] , the X P 4 R O strain, could be assigned to the NIH group C. This is indicated by the absence of lightly-labelled binuclear cells following fusion with XP2BE and UV exposure (Table IId). (C) Assignment o f strain XP2RO to separate complementation group E The third complementation group f o u n d in Rotterdam, the light to moderately severe classic XP [12], is represented by the X P 2 R O strain. Since it has previously been shown that X P 2 R O fibroblasts are n o t in the complementation groups to which strains X P 2 5 R O and XP4RO belong, and since the latter strains are n o w shown to be in groups A and C respectively, it was considered unnecessary in the present study to fuse strains XP2RO with the NIH strains from groups A and C. However, strain XP2RO was fused with the NIH strains X P l l B E (group B) and XP5BE (group D). In previous experiments [7] it was found that by lowering the UV dose from 10 to 5 J/m 2 a better distinction can be made between the residual level of UDS in XP2RO cells and the level of UDS in control cells. Therefore a 5 J/m 2 UV dose was performed in these experiments and in each fusion the grain numbers over 100 binuclear cells were counted. The results of these grain countings were presented in scatter diagrams in Fig. 1. Each binuclear cell is represented by one point indicating the correlation between the labelling intensity of both nuclei. If the scatter falls outside the lines enclosing the parental XP cells complementation is indicated. As seen in Fig. l a and Fig. l b fusion with X P l l B E and with XP5BE resulted in complementation, whereas it is absent in the X P 2 R O / XP2RO population (Fig. lc). Thus, strain X P 2 R O is n o t in groups A, B, C or D and is, therefore, assigned to a group of its own, group E. (II) NIH studies (A ) Assignment o f XP4RO fibroblasts to NIH complementation group C Histographic analysis of UV-induced UDS in normal fibroblasts is presented in Fig. 2. The histograms show the mean grain counts (arrows) and the distribution of the number of nuclei in various autoradiographic grain classes for unirradiated (Fig. 2a) and irradiated (Fig. 2b) mononuclear fibroblasts from control donor K. Each of the unirradiated cells (Fig. 2a) had less than 5 grains per nucleus, and their mean grain c o u n t (i.e., their nuclear background) Was 1.2
332
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Fig. 1. C o m p l e m e n t a t i o n a n a l y s i s w i t h t h e X P 2 R O s t r a i n r e p r e s e n t i n g t h e R o t t e r d a m " l i g h t to m o d e r a t e l y s e v e r e " X P c o m p l e m e n t a t i o n g r o u p . E a c h p o i n t r e p r e s e n t s o n e b i n u c l e a x cell, d e t e r m i n e d b y t h e n u m b e r o f g r a i n s o v e r e a c h o f b o t h n u c l e i ( o n e n u c l e u s on t h e o r d i n a t e a n d o n e o n the a b s c i s s a ) . T h e s q u a r e s ind i c a t e t h e a r e a s e n c l o s i n g t h e g r a i n n u m b e r s f o u n d o v e r p a r e n t a l n u c l e i a n d n u c l e i o f c o n t r o l cells ( C 5 ) , w i t h 9 5 % t o l e r a n c e l i m i t s . (a) t h e f u s i o n X P 2 R O / X P l l B E ; ( b ) X P 2 R O / X P S B E ; (c) X P 2 R O / X P 2 R O .
grains per nucleus (arrow). The control donor's irradiated cells all had more than 35 grains per nucleus, and their mean grain c o u n t (arrow) was 62 grains per nucleus. Strain XP4RO was fused with a representative strain from each of the four NIH c o m p l e m e n t a t i o n groups. The autoradiographic data for some of these fusions are presented in the experiment of Fig. 3. When XP4RO cells were
333 1.2 too
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GRAINS PER NUCLEUS Fig. 2. h i s t o g r a m s o f a u t o r a d i o g r a p h i c d a t a s h o w i n g U V - i n d u c e d U D S d u r i n g t h e f i r s t 3 h r a f t e r U V irrad i a t i o n w i t h 30 J / m 2 in m o n o n u c l e a r cells o f a f i b r o b l a s t s t r a i n f r o m a c o n t r o l d o n o r . (a) U n i r r a d i a t e d cells; (b) i r r a d i a t e d cells. T h e cells w e r e n o t t r e a t e d w i t h S e n d a i virus. A f t e r t h e tLme o f i r r a d i a t i o n , t h e u n i r r a d i a t e d a n d i r r a d i a t e d cells w e r e i n c u b a t e d w i t h 3 H T d R (spec. act., 1 5 . 7 C i / m m o l e ) at a c o n c e n t r a t i o n o f 10 p C i / m l o f m e d i u m 1 9 9 c o n t a i n i n g 2 0 % h u m a n p l a s m a . C o n t r o l d o n o r K h a s t h e A m e r i c a n Type Culture Collection culture designation CRK 1295KD. Autoradiograms were exposed for 7 days. The g r a i n class o f 0 t h r o u g h 5 g r a i n s p e r n u c l e u s i n c l u d e d n u c l e i w i t h n o g r a i n s as well as t h o s e w i t h as m a n y as 5 g r a i n s ; g r a i n classes t h e n r u n s u c c e s s i v e l y as 6 t h r o u g h 10, 11 t h r o u g h 1 5 , etc. W h e n m o r e t h a n 4 0 n u c l e i are in a s i n g l e g r a i n class, t h e b a r r e p r e s e n t i n g t h e m is t r u n c a t e d , a n d t h e n u m b e r o f cells in t h a t class is p r i n t e d n e a r t h e t o p o f t h e bar. A r r o w s i n d i c a t e t h e m e a n g r a i n c o u n t f o r t h e c o n s e c u t i v e l y evalua t e d n u c l e i ( 1 0 0 in a, 50 in b).
mixed with XPIBE (group C) cells and treated with Sendai virus, 99% of the unfused irradiated mononuclear cells which escape fusion (Fig. 3a) had less than 40 grains per nucleus and a mean grain count of 12.4 grains per nucleus (arrow). Of the nuclei in the irradiated binuclear cells in this same virus treated mixture (Fig. 3b), 97% had less than 40 grains per nucleus and had a mean grain c o u n t of 14.8 grains per nucleus (arrow), a value not significantly different from that for the mononuclear cells on the same coverslip (arrow, Fig. 3a). Thus, fusing strain XP4RO fibroblasts with XP1BE fibroblasts did n o t result in a population of binuclear cells capable of more UV-induced UDS than these strain's unfused fibroblasts. Therefore, strains XP4RO and XP1BE are in the same complementation group. When XP4RO fibroblasts were fused with X P l l B E (group B) (Figs. 3c,f) or with XP5BE (group D) (Fig. 3e,f) fibroblasts and irradiated , a population of nuclei appeared in the binuclear cells (Figs. 3d,f) which had more than 40
334
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GRAINS PER NUCLEUS Fig. 3. R e l a t i o n s h i p o f X P 4 R O f i b r o b l a s t s to N I H c o m p l e m e n t a t i o n g r o u p s C, B, and D. H i s t o g r a m s are of a u t o r a d i o g r a p h i c d a t a for S e n d a l v i r u s - t r e a t e d , i r r a d i a t e d f i b r o b l a s t s s h o w i n g lack o f c o m p l e m e n t a t i o n w h e n X P 4 R O f i b r o b l a s t s are f u s e d w i t h X P I B E cells ( g r o u p C) (a), (b) b u t s h o w i n g c o m p l e m e n t a t i o n w h e n f u s e d w i t h X P l l B E cells ( g r o u p B) (c), (d) or w i t h X P 5 B E cells ( g r o u p D) (e), (f). H i s t o g r a m s (a), (c) a n d (e) are o f i r r a d i a t e d , u n f u s e d m o n o n u c l e a r cells w h i c h e s c a p e d f u s i o n on c o v e r s l i p s c o n t a i n i n g S e n d a i v i r u s - t r e a t e d cells o f t h e strains i n d i c a t e d . H i s t o g r a m s (b), (d) a n d (f) are of i r r a d i a t e d b i n u c l e a r cells o n t h e s a m e coverslips used in (a), (c) a n d (e) r e s p e c t i v e l y . A r r o w s in (a) a n d (b) i n d i c a t e t h e m e a n grain c o u n t s f o r n u c l e i (99 in a; 97 in b) w i t h f e w e r t h a n 40 grains p e r n u c l e u s . Of t h e 30 a n d 19 n u c l e i w h i c h h a d m o r e t h a n 4 0 grains p e r n u c l e u s in t h e binucleax cells of (d) a n d (f), r e s p e c t i v e l y , 9 3 a n d 9 8 % w e r e acc o m p a n i e d by a n o t h e r n u c l e u s w h i c h h a d m o r e t h a n 4 0 grains p e r n u c l e u s . E x p e r i m e n t a l details a n d pres e n t a t i o n of d a t a as in Fig. 2.
grains per nucleus and a grain class distribution similar to that of the irradiated, normal fibroblasts of Fig. 2b. More than 90% of these nuclei were accompanied in their binuclear cells by another nucleus with more than 40 grains, indicating that a mutual, complementary correction of the impaired UDS in both nuclei had occurred. The binuclear cells containing these nuclei were considered to be heterokaryons showing complementation [8,10] and the binuclear cells containing nuclei with less than 40 grains per nucleus were considered to be homo-
335 4.1
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Group A x A
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XP25RO x XPI2BE Binucleor Cells UV
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0
i , i i i i I0 20 30 40 50 60 70 80 90 I00110 120]30 140 150
GRAINS PER NUCLEUS Fig. 4. R e l a t i o n s h i p o f X P 2 5 R O f i b r o b l a s t s to N I H c o m p l e m e n t a t i o n g r o u p s A, B, a n d D. H i s t o g r a m s axe of a u t o r a d i o g r a p h i c d a t a for S e n d a l v i r u s - t r e a t e d , i r r a d i a t e d f i b r o b l a s t s s h o w i n g l a c k o f c o m p l e m e n t a t i o n w h e n X P 2 5 R O f i b r o b l a s t s are f u s e d w i t h X P 1 2 B E ( g r o u p A ) (a), (b) b u t s h o w i n g c o m p l e m e n t a t i o n w h e n fused w i t h X P l l B E cells ( g r o u p B) (c), (d) or w i t h X P 5 B E cells ( g r o u p D) (e), (f). I m m e d i a t e l y a f t e r the t i m e o f i r r a d i a t i o n , t h e cells w e r e i n c u b a t e d f o r 3 h w i t h 3 H T d R (spec. a c t . , 15.7 C i / m m o l e ) at a c o n c e n t r a t i o n of 10 p C i ] m l of m e d i u m R P M I 1 6 4 0 c o n t a i n i n g 20% h u m a n p l a s m a . A u t o r a d i o g r a p h i c e x p o s u r e , 7 d a y s . H i s t o g r a m s (a), (e) a n d (e) are of i r r a d i a t e d , u n f u s e d m o n o n u c l e a x cells w h i c h e s c a p e d f u s i o n o n c o v e r s l i p s c o n t a i n i n g S e n d a i v i r u s - t r e a t e d cells of t h e strains i n d i c a t e d . H i s t o g r a m s (b), (d) a n d (f) are of i r r a d i a t e d b i n u c l e a x cells o n t h e s a m e c o v e r s l i p s u s e d in (a), (c) a n d (e) r e s p e c t i v e l y . A r r o w s in (a) a n d (b) i n d i c a t e t h e m e a n grain c o u n t s f o r n u c l e i ( 1 0 0 in a; 97 in b) w i t h f e w e r t h a n 4 0 grains p e r n u c l e u s . Of the 4 2 a n d 3 5 n u c l e i w h i c h h a d m o r e t h a n 4 0 grains per n u c l e u s in the binucleax cells of (d) a n d (f) r e s p e c tively, 1 0 0 a n d 94% w e r e a c c o m p a n i e d b y a n o t h e r n u c l e u s w h i c h h a d m o r e t h a n 4 0 grains p e r n u c l e u s . I n this e x p e r i m e n t i r r a d i a t e d m o n o n u c l e a x cells of t h e c o n t r o l d o n o r s t r a i n ( A m e r i c a n T y p e C u l t u r e Coll e c t i o n c u l t u r e d e s i g n a t i o n C R L 1 1 2 1 Pat Bru) h a d a m e a n grain c o u n t of 1 0 5 . 5 grains p e r n u c l e u s . E x p e r i m e n t a l details a n d p r e s e n t a t i o n of d a t a o t h e r w i s e as in Fig. 2.
karyons [8,10]. In another experiment (data n o t shown) it was found that the XP4RO fibroblasts c o m p l e m e n t e d XP12BE (group A) fibroblasts. Thus, X P 4 R O fibroblasts c o m p l e m e n t the representative strains from groups A, B, and D b u t n o t from group C.
336 (B) A s s i g n m e n t o f X P 2 5 R O fibroblasts to N I H c o m p l e m e n t a t i o n g r o u p A Strain XP25RO was fused with a representative strain from each of the four NIH c o m p l e m e n t a t i o n groups. Fig. 4 shows t hat X P25RO fibroblasts did n o t c o m p l e m e n t XP12BE (group A) cells (Figs. 4a,b) but did c o m p l e m e n t X P l l B E
26.7, 1
30 25
Group E x E XP2RO x XP2RO
Group E x E XP2RO x XP2RO Binucleor Cells UV
MononucleerCells UV
20 15
26.5
IO G
5 0
l
44 30
~
I
L
l
l
I
III
I
l
l
I
Group E x B
20
lkz
5
3o z5 zo
Group E x B XP2RO x XPIIBE Binucleer Cells
XP2RO x XPflBE Mononucleor Cells UV
~25 z
I
t
GroupE x D
d
Group E x D XP2RO x XP6BE Binucleor Cells UV
XP2RO x XPGBE Mononucleor Cells UV
I0
5
f
e
0 0 I0 20 30 40 50 60 , , ~70N 80 . 90 . . . .I00110 ..
I0 20 30 40 50 60 70 80 90100110120130140150
GRAINS PER NUCLEUS Fig. 5. R e l a t i o n s h i p s o f X P 2 R O f i b r o b l a s t s to N I H e o m p l e m e n t a t i o n g r o u p s B a n d D. H i s t o g r a m s are of autoradiographic data for Sendal virus-treated, irradiated fibroblasts showing lack of complementation w h e n X P 2 R O f i b r o b l a s t s are f u s e d to t h e m s e l v e s to f o r m h o m o k a r y o n s (a), (b) b u t s h o w i n g c o m p l e m e n t a t i o n w h e n f u s e d w i t h X P l l B E cells ( g r o u p B) (c), (d) or w i t h X P 6 B E cells ( g r o u p D) (e), (f). I m m e d i a t e l y a f t e r t h e t i m e of i r r a d i a t i o n w i t h 8 J / m 2 t h e cells w e r e i n c u b a t e d f o r 3 h w i t h 3 H T d R (spec. act., 2 1 . 0 C i / m m o l e ) in C o o n ' s m o d i f i c a t i o n [ 4 ] o f H a m ' s F - 1 2 m e d i u m ( b u t w i t h o u t t h y m i d i n e ) c o n t a i n i n g 5% fetal c a l f s e r u m . A u t o r a d i o g r a p h i c e x p o s u r e , 14 d a y s . H i s t o g r a m s (a), (c) a n d (e) are o f i r r a d i a t e d , unf u s e d m o n o n u c l e a r cells w h i c h e s c a p e d f u s i o n on c o v e r s l i p s c o n t a i n i n g S e n d a l v i r u s - t r e a t e d cells o f t h e s t r a i n s i n d i c a t e d . H i s t o g r a m s (b), (d) a n d (f) are o f i r r a d i a t e d b i n u c l e a r cells o n t h e s a m e c o v e r s l i p s u s e d in (a), (c) a n d (e) r e s p e c t i v e l y . A r r o w s in (a) a n d (b) i n d i c a t e t h e m e a n g r a i n c o u n t s f o r t h e n u c l e i evalua t e d ( 1 0 0 in a; 4 8 in b), all o f w h i c h h a d 50 or f e w e r g r a i n s p e r n u c l e u s . O f t h e 41 a n d 29 n u c l e i w h i c h h a d m o r e t h a n 50 g r a i n s p e r n u c l e u s in t h e b i n u c l e a r cells o f (d) a n d (f) r e s p e c t i v e l y , 9 8 a n d 9 0 % w e r e acc o m p a n i e d b y a n o t h e r n u c l e u s w h i c h h a d m o r e t h a n 50 g r a i n s p e r n u c l e u s . In t h i s e x p e r i m e n t i r r a d i a t e d m o n o n u c l e a r cells o f t h e c o n t r o l d o n o r s t r a i n ( A m e r i c a n T y p e C u l t u r e C o l l e c t i o n c u l t u r e d e s i g n a t i o n C R L 1121 Pat Bru) had a m e a n grain c o u n t of 45.9 grains per nucleus. E x p e r i m e n t a l details and presentat i o n o f d a t a o t h e r w i s e as in Fig. 2.
337
(group B) (Figs. 4c,d) and XP5BE (group D) (Figs. 4e,f) cells. In another experiment (data n o t shown) X P 2 5 R O fibroblasts were found to complement XP1BE (group C) fibroblasts. (C) Assignment of strain XP2RO to a separate complementation group E When XP2RO fibroblasts were fused to themselves to form homokaryons, the mean grain c o u n t for irradiated nuclei in the binuclear cells was the same as that for the irradiated nuclei in the mononuclear cells (arrows, Figs. 5a and 5b). Thus, the fusion process per se did n o t affect the rate of UV-induced UDS in the XP2RO homokaryons. When XP2RO fibroblasts were fused with X P l l B E {group B) fibroblasts (Fig. 5d), with XP6BE (group D) fibroblasts (Fig. 5f), or with XP2BE (group C) fibroblasts (data n o t shown}, complementation occurred in the hybrid binuclear cells. XP2RO cells were n o t fused with any group A strains at the NIH since it had already been demonstrated [12] that XP2RO fibroblasts complemented fibroblasts from strain X P 2 5 R O which latter strain is n o w shown to be in complementation group A (Table II; Fig. 4b). (D) Rates o f UV-induced UDS in the representative strains The rates of UV-induced UDS were determined for the representative Rotterdam and NIH complementation group strains (Table III). Strains X P 2 5 R O and XP12BE, which are both in group A, have less than 5 or 2% of the normal rate, if they have any UV-induced UDS at all. Significant UDS was n o t detected in these group A cells in these experiments. The rates of UDS of strains XP4RO and X P I B E (both in group C) were within 10--20% of the normal range. The UDS seen in group B cells (XP11BE) was similar to the previously reported range of 3--7% [refs. 8,10]. A discrepancy was observed between the data of the NIH and Rotterdam groups regarding the residual UDS of the group D and E strains. The values obtained by the R o t t e r d a m group were consistently lower
TABLE
III
COMPARISON OF BOTH INSTITUTES Complementation
UV-INDUCED
group
UDS
IN THE
Strain
REPRESENTATIVE
UV-induced Rotterdam
a
NIH b
Control donors XP25RO XP12BE XP1LO
B
XP11BE
C
XP4RO XPIBE XP2BE
10--15
D
XP5BE
10
27.1
E
XP2RO
40--60
60
as a percentage as a percentage
~5 4
24--27
MEASURED
U D S (% o f n o r m a l r a t e )
A
a Expressed b Expressed
100 ~5
XP STRAINS
100 ~2 ~2 ~2 4.8 12.9 19.8 13--18
o f t h e l e v e l s f o u n d i n n o r m a l c e l l s a f t e r a U V d o s e o f 1 0 J / m 2. o f t h e l e v e l s f o u n d i n n o r m a l c e l l s a f t e r a U V d o s e o f 3 0 J / m 2.
IN
338 than those obtained by the NIH group (Table III). The different UV doses used in these experiments (10 and 30 J/m e respectively) or differences in culture conditions might be responsible for these discrepancies. Discussion The R o t t e r d a m and NIH methods for complementation testing are in principle the same. In both methods the rate of UV-induced UDS is determined autoradiographically in multinucleate cells resulting from Sendai virus-induced cell fusion. The results obtained in R o t t e r d a m concerning the complementation group assignment of XP fibroblasts were identical to the results obtained at the NIH. Thus, the R o t t e r d a m investigators have confirmed (Table II) the existence of the four complementation groups, A, B, C, and D, previously reported among the NIH strains [8,10]. Furthermore, the Rotterdam results for complementation group assignment of the representative strains (Table II) were the same as the results found at the NIH (Figs. 3--5). Thus, there are currently five known UDS complementation groups in XP. That the restored rate of UV-induced UDS observed in the heterokaryons whose nuclei show complementation reflects DNA repair is supported by the following observations: 1) the rate of UDS in these complemented nuclei is resistant to h y d r o x y u r e a [8,10] and is the same as that in normal fibroblasts [11, 12] ; 2) these complemented nuclei have normal rates of thymine dimer excision [9] and repair replication [13]. Furthermore, host-cell reactivation studies of UV-irradiated adenovirus 2 reveal that heterokaryons of complementing XP strains have the same capacity as normal strains to restore biological function to the virus [ 5]. Both the Rotterdam [7,12] and the NIH investigators [8,10] have reported that strains within a complementation group have similar rates of UV-induced UDS and that each complementation group has a characteristic rate. The present studies n o w extend these findings to the five known UDS complementation groups in XP as the following indicates. The R o t t e r d a m "De SanctisCacchione" group, which does not complement NIH group A, has the same rate of UDS as group A; the R o t t e r d a m "classical XP, severe" group, which does n o t complement NIH group C, has the same UDS rate as group C; and the R o t t e r d a m "classical XP, light to moderately severe" group, which complements all four of the NIH groups, has a much greater rate of UDS than any of the NIH groups. Our collaborative study has provided additional information concerning the relationship between the clinical manifestations of XP and the complementation groups. The NIH complementation group containing the most kindreds, group C, was known [10] to be essentially free of the neurological abnormalities of XP. The R o t t e r d a m "classical XP severe" group, which does n o t complement NIH group C, contains 6 kindred none of which has any known neurological defect [7]. Thus, it appears as though the defective gene p r o d u c t in group C does n o t result in neurological abnormalities. The kindreds in the Rotterdam "De Sanctis-Cacchione" group, which does n o t complement NIH group A, all have numerous neurological abnormalities [6,7] while most of the kindreds in group A also have numerous neurological abnormalities [ 10]. However,
339 one kindred strain (XP12BE) of group A has only two neurological abnormalities known to date [10], while another kindred strain (XP1LO) in group A is n o t known to have any neurological abnormalities [10]. Therefore, for these reasons and because groups B and D have kindreds with neurological abnormalities, we propose that the term "De-Sanctis-Cacchione" no longer be used as a complementation group designation, and that the NIH complementation group designations A, B, C, and D, be appropriately applied to the Rotterdam groups as well as to the NIH groups. Since strain XP2RO is in its own complementation group, and since it has more UV-induced UDS than any of the other UDS-deficient strains, we propose that the complementation group of strain XP2RO be designated group E.
Acknowledgement The work of the Rotterdam group was supported by grants from Euratom, contract No. 123-74-1 BJOC and the Netherlands Organization for the Advancement of Pure Research (ZWO).
References 1 B o o t s m a , D., Cell f u s i o n in t h e s t u d y o f g e n e t i c h e t e r o g e n e i t y of x e r o d e r m a p i g m e n t o s u m , in R . L . D a v i d s o n a n d F. d e la C r u z (eds.), S o m a t i c Cell H y b r i d i z a t i o n , R a v e n Press, N e w Y o r k , 1 9 7 4 . 2 B o o t s m a , D., M.P. M u l d e r , F. P o t , a n d J . A . C o h e n , D i f f e r e n t i n h e r i t e d levels o f D N A r e p a i r r e p l i c a t i o n in x e r o d e r m a p i g m e n t o s u m cell s t r a i n s a f t e r e x p o s u r e t o u l t r a v i o l e t l i g h t , M u t a t i o n R e s . , 9 ( 1 9 7 0 ) 507--516. 3 Cleaver, J . E . , D e f e c t i v e r e p a i r r e p l i c a t i o n o f D N A in x e r o d e r m a p i g m e n t o s u m , N a t u r e , 2 1 8 ( 1 9 6 8 ) 652--656. 4 C o o n , H . G . a n d M.C. Weiss, A q u a n t i t a t i v e c o m p a r i s o n o f f o r m a t i o n o f s p o n t a n e o u s a n d v i r u s - p r o d u c e d viable h y b r i d s , P r o c . N a t l . A c a d . Sci., U.S., 6 2 ( 1 9 6 9 ) 8 5 2 - - 8 5 9 . 5 Day, R.S., K.Y. Kraemer, and J.H. Robbins, Complementing xeroderma pigmentosum fibroblasts restore biological activity to UV-damaged DNA, Mutation Res., 28 (1975) 251--255. 6 K a l o u s t i a n , V.M. d e r , E . A . d e W e e r d o K a s t e l e i n , W.J. Kleijer, W. K e i j z e r a n d D. B o o t s m a , T h e g e n e t i c d e f e c t i n t h e De S a n c t l s - C a c c h i o n e s y n d r o m e , J. I n v e s t . D e r m a t o l . , 6 3 ( 1 9 7 4 ) 3 9 2 - - 3 9 6 . 7 Kleijer, W.J., E . A . d e W e e r d - K a s t e l e i n , M.L. S l u y t e r , W. Keijzer, J. d e Wit a n d D. B o o t s m a , UV-ind u c e d D N A r e p a i r s y n t h e s i s in cells o f p a t i e n t s w i t h d i f f e r e n t f o r m s o f x e r o d e r m a p i g m e n t o s u m a n d of heterozygotes, Mutation Res., 20 (1973) 417--428. 8 Kraemer, K.H., H.G. Coon, R.A. Petinga, A.F. Barrett, A.E. Rahe and J.H. Robbins, Genetic heterog e n e i t y in x e r o d e r m a p i g m e n t o s u m ; c o m p l e m e n t a t i o n g r o u p s a n d t h e i r r e l a t i o n s h i p t o D N A r e p a i r r a t e s , P r o c . N a t l . A c a d . Sci., U.S., 7 2 ( 1 9 7 5 ) 5 9 - - 6 3 . 9 P a t t e r s o n , M.C., P.H.M. L o h m a n , A. W e s t e r v e l d a n d M.L. S l u y t e r , D N A r e p a i r m o n i t o r e d b y a n e n z y m a t i c a s s a y in m u l t i n u c l e a t e x e r o d e r m a p i g m e n t o s u m ceils a f t e r f u s i o n , N a t u r e , 2 4 8 ( 1 9 7 4 ) 5 0 - - 5 2 . 1 0 R o b b i n s , J . H . , K . H . K r a e m e r , M . A . L u t z n e r , B.W. F e s t o f f , a n d H . G . C o o n , X e r o d e r m a p i g m e n t o s u m : a n i n h e r i t e d disease w i t h s u n s e n s i t i v i t y , m u l t i p l e c u t a n e o u s n e o p l a s m s , a n d a b n o r m a l D N A r e p a i r , Ann. Internal Med., S0 (1974) 221--248. 11 W e e r d - K a s t e l e i n , E . A . de, W. K e i j z e r a n d D. B o o t s m a , G e n e t i c h e t e r o g e n e i t y o f x e r o d e r m a pigm e n t o s u m d e m o n s t r a t e d b y s o m a t i c cell h y b r i d i z a t i o n , N a t u r e , 2 3 8 ( 1 9 7 2 ) 8 0 - - 8 3 . 1 2 W e e r d - K a s t e l e i n , E . A . d e , W. Keijzer, a n d D. B o o t s m a , A t h i r d c o m p l e m e n t a t i o n g r o u p in x e r o d e r r n a pigmentosum, Mutation Res., 22 (1974) 87--91. 1 3 W e e r d - K a s t e l e i n , E . A . d e , W.J. Kleijer, M . L . S l u y t e r , a n d W. K e i j z e r , R e p a i r r e p l i c a t i o n in h e t e r o karyons derived from different repair-deficient xeroderma pigmentosum strains, Mutation Res., 19 (1973) 237--243.
Mutation Research, 33 (1975) 3 2 7 - - 3 4 0 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - Printed in The Netherlands
FIVE COMPLEMENTATION GROUPS IN XERODERMA PIGMENTOSUM
K.H. K R A E M E R l, E.A. DE W E E R D - K A S T E L E I N 2, J.H. R O B B I N S l, W. K E I J Z E R 2, S.F. B A R R E T T l, R.A. P E T I N G A 1 A N D D B O O T S M A 2
1 Dermatology Branch, National Cancer Institute, National Institutes o f Health, Bethesda, Maryland 20014 (U.S.A.) and 2 Department o f Cell Biology and Genetics, Erasmus University, P.O. Box 1 738, Rotterdam (The Netherlands) (Received July 24th, 1975) ( A c c e p t e d July 28th, 1975)
Summary A collaborative study was undertaken to determine the relationship between the three DNA repair complementation groups in xeroderma pigmentosum found at Erasmus University, Rotterdam, and the four groups found at the National Institutes of Health, Bethesda. The results of this study reveal that there are five currently known complementation groups in xeroderma pigmentosum.
Introduction Patients with xeroderma pigmentosum (XP) develop malignancies and pigmentation abnormalities on areas of skin exposed to sunlight [10]. Skin fibroblasts from most patients with this autosomal recessive disease are unable to perform excision repair of UV-induced pyrimidine dimers in their DNA as rapidly as normal fibroblasts [2,3,10]. This defective repair can be manifest as a decreased rate of UV-induced unscheduled DNA synthesis (UDS) [2,3,10]. In 1972 investigators at Erasmus University, Rotterdam, found two complementation groups in XP by showing that nuclei in heterokaryons formed by fusing fibroblasts from certain pairs of such repair-defective XP patients performed UV-induced UDS at a normal rate [11 ]. Subsequently a third complementation group was found among the Rotterdam XP strains [12]. The demonstration of genetic heterogeneity for DNA repair among the Rotterdam XP strains prompted investigators at the National Institutes of Health Abbreviations: XP, xeroderma pigmentosum; UDS, unscheduled DNA synthesis; 3HTdR, tritiated thymidine; NIH, National Institutes of Health.
328 (NIH), Bethesda, to search for complementation groups among their strains. Four complementation groups were found among the ten kindred tested from the NIH XP patient series [8,10]. The Rotterdam and NIH investigators considered it useful to perform a collaborative study to determine the relationship between the R o t t e r d a m complementation groups and the NIH groups. In this paper we present the results of this collaborative project which indicate that there are five currently known complementation groups in XP. Materials and methods
Representative XP complementation group fibroblast strains The representative Rotterdam complementation group strains were grown from skin biopsies in Rotterdam and then sent to The American Type Culture Collection, Rockville, Maryland, for further growth and dissemination to the NIH. The NIH group strains were grown from biopsies by the American Type Culture Collection. Table I identifies the representative complementation group strains used in our collaborative study.
Rotterdam experimental procedure Details of the procedure have been published elsewhere [11]. Trypsinized cells of 2 different strains were mixed in a 1 : 1 ratio and incubated in the presence of ~-propiolactone-inactivated Sendai virus. One million cells were incubated with 125 hemagglutinating units of Sendai virus in a final volume of 1 ml. After 4 to 6 min at 4°C followed by 20 min at 37°C, the cells were seeded on coverslips. Two days after fusion the fusedcell population was labelled 1 h before and 2 h after UV-irradiation in medium containing [ 3H ] thymidine (3 HTdR; 10 pCi/ml; spec. act., 2 Ci/mmol). Before irradiation the culture was washed with a balanced salt solution, drained and exposed to 10 J/m 2 UV light (254 nm). After the 2 h post-irradiation incubation, the cells were fixed in Bouins fixative. Cells not treated with virus were seeded on coverslips and served as TABLE 1 REPRESENTATIVE ROTTERDAM AND NIH COMPLEMENTATION GROUP FIBROBLAST STRAINS USED IN C O L L A B O R A T I V E STUDY a
C o m p l e m e n t a t i o n group
Strains t e s t e d
Rotterdam nomenclature De Sanctis C a c e h i o n e Classical XP, severe Classical XP, light to m o d e r a t e l y severe
XP25RO XP4RO XP2RO
NIH nomenclature A B C D
XPILO, XP12BE XPllBE XP1BE, XP2BE XP5BE, XP6BE
a T h e p r o p o s e d s t a n d a r d i z a t i o n o f n o m e n c l a t u r e of XP strains [7] is used t h r o u g h o u t ; R O , R o t t e r d a m LO, L o n d o n ; BE, B e t h e s d a .
329
controls. The control coverslip cultures were handled like the fused cultures. Autoradiographs were prepared with Kodak AR 10 stripping film. Exposure time was 1 week. This exposure time resulted in a mean grain c o u n t of 20 grains per nucleus in a control strain. After development, the preparations were stained with hematoxyline and eosin. By evaluating the frequency distribution of grain counts over normal nuclei under these labelling conditions, nuclei with less than 50 grains were considered to be G1- or G2-phase nuclei. The grain numbers 8 and 50 were adopted as the limits of UDS, and nuclei showing between 8 and 50 grains per nucleus are referred to as lightly-labelled. In each experiment 50 binuclear cells with non-S-phase nuclei were included in the countings. Binuclear cells showing between 8 and 50 grains over each nucleus were considered as lightly-labelled. In experiments with XP2RO cells the UV dose was lowered to 5 J/m 2 and the number of binuclear cells used to estimate the number of grains over their nuclei was extended to 100. The autoradiographic exposure time was 15 days. This exposure time resulted in a mean grain c o u n t of 26 grains per nucleus in a control strain.
NIH experimen tal procedure Details of the NIH experimental procedure have been published [8]. Variations in that procedure are presented in the appropriate legends of figures and table legends herein. As in the R o t t e r d a m procedure,/~-propiolactone-inactivated Sendai virus was used to produce cell fusion, and autoradiograms were used to determine the 3HTdR incorporation representing UV-induced UDS. In contrast to the Rotterdam procedure, the strains to be fused were n o t mixed in suspension, for fusion was accomplished by placing drops of a suspension of one strain on another strain which had already been growing on coverslips. Also in contrast to the Rotterdam procedure, at the NIH the fused cells were irradiated approximately 16 h after the two strains to be fused were placed in contact with each other. Since there was a very low frequency of synchronized S-phase synthesis in the nuclei of the binuclear cells after only 16 h following the initiation of cell fusion, preirradiation incubation of the cells with 3HTdR to label S-phase nuclei was n o t necessary and was n o t performed. Results
(I) Rotterdam studies (A) Confirmation of four complementation groups among the NIH groups" strains The cell strains XP1LO, X P l l B E , XP2BE and XP5BE were investigated as representatives of the complementation groups A, B, C and D respectively. A small fraction of the cells in these parental cell populations were lightly-labelled following UV exposure (0--18%, Table IIa). In contrast 96% of the nuclei in a control strain (C5) were labelled when treated under the same conditions. The six possible pairwise fusions between the XP strains resulted in formation of binuclear cells showing complementation as evidenced by the fact t h a t from 28 to 40% of the binuclear cells were lightly labelled (Table IIb). These percent-
330 T A B L E II COMPLEMENTATION
ANALYSIS PERFORMED
BY R O T T E R D A M
GROUP
a. U n s c h e d u l e d D N A s y n t h e s i s in p a r e n t a l s t r a i n s Cell s t r a i n
C5 XP1 L O XPllBE XP2BE XP5BE XP25RO XP4RO
Complementation group
Control A B C D De S a n c t i s - C a c c h i o n e Classic XP, s e v e r e
% L a b e l l e d cells f o l l o w i n g a d o s e o f 0 J/m 2
10 J / m 2
2 2 2 0 0 2 2
96 0 2 18 2 0 8
b. C o n f i r m a t i o n of t h e N I H c o m p l e m e n t a t i o n g r o u p s Fusion
% L a b e l l e d b i n u c l e a r cells f o l l o w i n g a d o s e o f
Complementation group
Cell s t r a i n s
0 J/m 2
10 J / m 2
A/B A/C A/D B/C B/D C/D
XPILO/XPllBE XP1 L O / X P 2 B E XPILO/XP5BE XPllBE/XP2BE XPllBE/XP5BE XP2BE/XP5BE
0 0 0 0 0 0
30 34 40 30 34 28
c. R o t t e r d a m ' s " D e S a n c t i s - C a c c h i o n e " g r o u p c o m p l e m e n t s all N I H g r o u p s e x c e p t A XP25RO fibroblasts fused with NIH group strains
% L a b e l l e d b i n u c l e a r cells f o l l o w i n g a d o s e o f
Group
Strain
0 J/m 2
10 J / m 2
A B C D
XP1LO XPllBE XP2BE XP5BE
0 0 0 0
0 30 48 42
d. R o t t e r d a m ' s " c l a s s i c XP, s e v e r e " g r o u p c o m p l e m e n t s all N I H g r o u p s e x c e p t C XP4RO fibrohlasts fused with NIH group strains
% L a b e l l e d b i n u c l e a r cells f o l l o w i n g a d o s e o f
Group
Strain
0 J/m 2
10 J / m 2
A B C D
XPILO XPIIBE XP2BE XP5BE
0 0 0 0
38 38 0 40
In t h e p a r e n t a l s t r a i n s o n l y cells w i t h 0 - - 5 0 g r a i n s o v e r t h e i r n u c l e i w e r e c o u n t e d ; cells w i t h 8 - - 5 0 g r a i n s w e r e c o u n t e d as labelled. I n t h e f u s e d cell p o p u l a t i o n s b i n u c l e a r ceils w i t h 8 - - 5 0 g r a i n s o v e r e a c h o f b o t h n u c l e i w e r e c o u n t e d as labelled cells. E a c h v a l u e is b a s e d on c o u n t i n g o f 50 cells.
ages approach the 50% level which is the expected fraction of hybrid binuclear cells in the population. The binuclear cells in the unirradiated fused cultures were unlabelled (Table IIb). These results confirm the complementation group studies previously performed at the NIH, which indicated the existence of four complementation groups among these strains [ 8 , 1 0 ] .
331
(B) Assignment o f XP25RO and XP4RO fibroblasts to complementation groups A and C respectively. The X P 2 5 R O fibroblasts belong to the complementation group which was previously indicated as the De Sanctis Cacchione group [11]. These cells were fused with each of the four representative strains of the complementation groups A, B, C and D (Table IIc). In the fused and UV exposed populations 30--48% of the binuclear cells were lightly labelled except in the fusion with XP1LO cells (Table IIc). These results indicate that complementation occurred after fusion with cells from complementation groups B, C, and D and not with cells belonging to A. Therefore the mutation resulting in the Rotterdam complementation group De Sanctis Cacchione can be assigned to the NIH group A. In the same way the representative strain of the R o t t e r d a m complementation group "severe classic X P " [ 1 1 ] , the X P 4 R O strain, could be assigned to the NIH group C. This is indicated by the absence of lightly-labelled binuclear cells following fusion with XP2BE and UV exposure (Table IId). (C) Assignment o f strain XP2RO to separate complementation group E The third complementation group f o u n d in Rotterdam, the light to moderately severe classic XP [12], is represented by the X P 2 R O strain. Since it has previously been shown that X P 2 R O fibroblasts are n o t in the complementation groups to which strains X P 2 5 R O and XP4RO belong, and since the latter strains are n o w shown to be in groups A and C respectively, it was considered unnecessary in the present study to fuse strains XP2RO with the NIH strains from groups A and C. However, strain XP2RO was fused with the NIH strains X P l l B E (group B) and XP5BE (group D). In previous experiments [7] it was found that by lowering the UV dose from 10 to 5 J/m 2 a better distinction can be made between the residual level of UDS in XP2RO cells and the level of UDS in control cells. Therefore a 5 J/m 2 UV dose was performed in these experiments and in each fusion the grain numbers over 100 binuclear cells were counted. The results of these grain countings were presented in scatter diagrams in Fig. 1. Each binuclear cell is represented by one point indicating the correlation between the labelling intensity of both nuclei. If the scatter falls outside the lines enclosing the parental XP cells complementation is indicated. As seen in Fig. l a and Fig. l b fusion with X P l l B E and with XP5BE resulted in complementation, whereas it is absent in the X P 2 R O / XP2RO population (Fig. lc). Thus, strain X P 2 R O is n o t in groups A, B, C or D and is, therefore, assigned to a group of its own, group E. (II) NIH studies (A ) Assignment o f XP4RO fibroblasts to NIH complementation group C Histographic analysis of UV-induced UDS in normal fibroblasts is presented in Fig. 2. The histograms show the mean grain counts (arrows) and the distribution of the number of nuclei in various autoradiographic grain classes for unirradiated (Fig. 2a) and irradiated (Fig. 2b) mononuclear fibroblasts from control donor K. Each of the unirradiated cells (Fig. 2a) had less than 5 grains per nucleus, and their mean grain c o u n t (i.e., their nuclear background) Was 1.2
332
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Fig. 1. C o m p l e m e n t a t i o n a n a l y s i s w i t h t h e X P 2 R O s t r a i n r e p r e s e n t i n g t h e R o t t e r d a m " l i g h t to m o d e r a t e l y s e v e r e " X P c o m p l e m e n t a t i o n g r o u p . E a c h p o i n t r e p r e s e n t s o n e b i n u c l e a x cell, d e t e r m i n e d b y t h e n u m b e r o f g r a i n s o v e r e a c h o f b o t h n u c l e i ( o n e n u c l e u s on t h e o r d i n a t e a n d o n e o n the a b s c i s s a ) . T h e s q u a r e s ind i c a t e t h e a r e a s e n c l o s i n g t h e g r a i n n u m b e r s f o u n d o v e r p a r e n t a l n u c l e i a n d n u c l e i o f c o n t r o l cells ( C 5 ) , w i t h 9 5 % t o l e r a n c e l i m i t s . (a) t h e f u s i o n X P 2 R O / X P l l B E ; ( b ) X P 2 R O / X P S B E ; (c) X P 2 R O / X P 2 R O .
grains per nucleus (arrow). The control donor's irradiated cells all had more than 35 grains per nucleus, and their mean grain c o u n t (arrow) was 62 grains per nucleus. Strain XP4RO was fused with a representative strain from each of the four NIH c o m p l e m e n t a t i o n groups. The autoradiographic data for some of these fusions are presented in the experiment of Fig. 3. When XP4RO cells were
333 1.2 too
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Control Donor K Mononuclear Cells No UV
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GRAINS PER NUCLEUS Fig. 2. h i s t o g r a m s o f a u t o r a d i o g r a p h i c d a t a s h o w i n g U V - i n d u c e d U D S d u r i n g t h e f i r s t 3 h r a f t e r U V irrad i a t i o n w i t h 30 J / m 2 in m o n o n u c l e a r cells o f a f i b r o b l a s t s t r a i n f r o m a c o n t r o l d o n o r . (a) U n i r r a d i a t e d cells; (b) i r r a d i a t e d cells. T h e cells w e r e n o t t r e a t e d w i t h S e n d a i virus. A f t e r t h e tLme o f i r r a d i a t i o n , t h e u n i r r a d i a t e d a n d i r r a d i a t e d cells w e r e i n c u b a t e d w i t h 3 H T d R (spec. act., 1 5 . 7 C i / m m o l e ) at a c o n c e n t r a t i o n o f 10 p C i / m l o f m e d i u m 1 9 9 c o n t a i n i n g 2 0 % h u m a n p l a s m a . C o n t r o l d o n o r K h a s t h e A m e r i c a n Type Culture Collection culture designation CRK 1295KD. Autoradiograms were exposed for 7 days. The g r a i n class o f 0 t h r o u g h 5 g r a i n s p e r n u c l e u s i n c l u d e d n u c l e i w i t h n o g r a i n s as well as t h o s e w i t h as m a n y as 5 g r a i n s ; g r a i n classes t h e n r u n s u c c e s s i v e l y as 6 t h r o u g h 10, 11 t h r o u g h 1 5 , etc. W h e n m o r e t h a n 4 0 n u c l e i are in a s i n g l e g r a i n class, t h e b a r r e p r e s e n t i n g t h e m is t r u n c a t e d , a n d t h e n u m b e r o f cells in t h a t class is p r i n t e d n e a r t h e t o p o f t h e bar. A r r o w s i n d i c a t e t h e m e a n g r a i n c o u n t f o r t h e c o n s e c u t i v e l y evalua t e d n u c l e i ( 1 0 0 in a, 50 in b).
mixed with XPIBE (group C) cells and treated with Sendai virus, 99% of the unfused irradiated mononuclear cells which escape fusion (Fig. 3a) had less than 40 grains per nucleus and a mean grain count of 12.4 grains per nucleus (arrow). Of the nuclei in the irradiated binuclear cells in this same virus treated mixture (Fig. 3b), 97% had less than 40 grains per nucleus and had a mean grain c o u n t of 14.8 grains per nucleus (arrow), a value not significantly different from that for the mononuclear cells on the same coverslip (arrow, Fig. 3a). Thus, fusing strain XP4RO fibroblasts with XP1BE fibroblasts did n o t result in a population of binuclear cells capable of more UV-induced UDS than these strain's unfused fibroblasts. Therefore, strains XP4RO and XP1BE are in the same complementation group. When XP4RO fibroblasts were fused with X P l l B E (group B) (Figs. 3c,f) or with XP5BE (group D) (Fig. 3e,f) fibroblasts and irradiated , a population of nuclei appeared in the binuclear cells (Figs. 3d,f) which had more than 40
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GRAINS PER NUCLEUS Fig. 3. R e l a t i o n s h i p o f X P 4 R O f i b r o b l a s t s to N I H c o m p l e m e n t a t i o n g r o u p s C, B, and D. H i s t o g r a m s are of a u t o r a d i o g r a p h i c d a t a for S e n d a l v i r u s - t r e a t e d , i r r a d i a t e d f i b r o b l a s t s s h o w i n g lack o f c o m p l e m e n t a t i o n w h e n X P 4 R O f i b r o b l a s t s are f u s e d w i t h X P I B E cells ( g r o u p C) (a), (b) b u t s h o w i n g c o m p l e m e n t a t i o n w h e n f u s e d w i t h X P l l B E cells ( g r o u p B) (c), (d) or w i t h X P 5 B E cells ( g r o u p D) (e), (f). H i s t o g r a m s (a), (c) a n d (e) are o f i r r a d i a t e d , u n f u s e d m o n o n u c l e a r cells w h i c h e s c a p e d f u s i o n on c o v e r s l i p s c o n t a i n i n g S e n d a i v i r u s - t r e a t e d cells o f t h e strains i n d i c a t e d . H i s t o g r a m s (b), (d) a n d (f) are of i r r a d i a t e d b i n u c l e a r cells o n t h e s a m e coverslips used in (a), (c) a n d (e) r e s p e c t i v e l y . A r r o w s in (a) a n d (b) i n d i c a t e t h e m e a n grain c o u n t s f o r n u c l e i (99 in a; 97 in b) w i t h f e w e r t h a n 40 grains p e r n u c l e u s . Of t h e 30 a n d 19 n u c l e i w h i c h h a d m o r e t h a n 4 0 grains p e r n u c l e u s in t h e binucleax cells of (d) a n d (f), r e s p e c t i v e l y , 9 3 a n d 9 8 % w e r e acc o m p a n i e d by a n o t h e r n u c l e u s w h i c h h a d m o r e t h a n 4 0 grains p e r n u c l e u s . E x p e r i m e n t a l details a n d pres e n t a t i o n of d a t a as in Fig. 2.
grains per nucleus and a grain class distribution similar to that of the irradiated, normal fibroblasts of Fig. 2b. More than 90% of these nuclei were accompanied in their binuclear cells by another nucleus with more than 40 grains, indicating that a mutual, complementary correction of the impaired UDS in both nuclei had occurred. The binuclear cells containing these nuclei were considered to be heterokaryons showing complementation [8,10] and the binuclear cells containing nuclei with less than 40 grains per nucleus were considered to be homo-
335 4.1
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GRAINS PER NUCLEUS Fig. 4. R e l a t i o n s h i p o f X P 2 5 R O f i b r o b l a s t s to N I H c o m p l e m e n t a t i o n g r o u p s A, B, a n d D. H i s t o g r a m s axe of a u t o r a d i o g r a p h i c d a t a for S e n d a l v i r u s - t r e a t e d , i r r a d i a t e d f i b r o b l a s t s s h o w i n g l a c k o f c o m p l e m e n t a t i o n w h e n X P 2 5 R O f i b r o b l a s t s are f u s e d w i t h X P 1 2 B E ( g r o u p A ) (a), (b) b u t s h o w i n g c o m p l e m e n t a t i o n w h e n fused w i t h X P l l B E cells ( g r o u p B) (c), (d) or w i t h X P 5 B E cells ( g r o u p D) (e), (f). I m m e d i a t e l y a f t e r the t i m e o f i r r a d i a t i o n , t h e cells w e r e i n c u b a t e d f o r 3 h w i t h 3 H T d R (spec. a c t . , 15.7 C i / m m o l e ) at a c o n c e n t r a t i o n of 10 p C i ] m l of m e d i u m R P M I 1 6 4 0 c o n t a i n i n g 20% h u m a n p l a s m a . A u t o r a d i o g r a p h i c e x p o s u r e , 7 d a y s . H i s t o g r a m s (a), (e) a n d (e) are of i r r a d i a t e d , u n f u s e d m o n o n u c l e a x cells w h i c h e s c a p e d f u s i o n o n c o v e r s l i p s c o n t a i n i n g S e n d a i v i r u s - t r e a t e d cells of t h e strains i n d i c a t e d . H i s t o g r a m s (b), (d) a n d (f) are of i r r a d i a t e d b i n u c l e a x cells o n t h e s a m e c o v e r s l i p s u s e d in (a), (c) a n d (e) r e s p e c t i v e l y . A r r o w s in (a) a n d (b) i n d i c a t e t h e m e a n grain c o u n t s f o r n u c l e i ( 1 0 0 in a; 97 in b) w i t h f e w e r t h a n 4 0 grains p e r n u c l e u s . Of the 4 2 a n d 3 5 n u c l e i w h i c h h a d m o r e t h a n 4 0 grains per n u c l e u s in the binucleax cells of (d) a n d (f) r e s p e c tively, 1 0 0 a n d 94% w e r e a c c o m p a n i e d b y a n o t h e r n u c l e u s w h i c h h a d m o r e t h a n 4 0 grains p e r n u c l e u s . I n this e x p e r i m e n t i r r a d i a t e d m o n o n u c l e a x cells of t h e c o n t r o l d o n o r s t r a i n ( A m e r i c a n T y p e C u l t u r e Coll e c t i o n c u l t u r e d e s i g n a t i o n C R L 1 1 2 1 Pat Bru) h a d a m e a n grain c o u n t of 1 0 5 . 5 grains p e r n u c l e u s . E x p e r i m e n t a l details a n d p r e s e n t a t i o n of d a t a o t h e r w i s e as in Fig. 2.
karyons [8,10]. In another experiment (data n o t shown) it was found that the XP4RO fibroblasts c o m p l e m e n t e d XP12BE (group A) fibroblasts. Thus, X P 4 R O fibroblasts c o m p l e m e n t the representative strains from groups A, B, and D b u t n o t from group C.
336 (B) A s s i g n m e n t o f X P 2 5 R O fibroblasts to N I H c o m p l e m e n t a t i o n g r o u p A Strain XP25RO was fused with a representative strain from each of the four NIH c o m p l e m e n t a t i o n groups. Fig. 4 shows t hat X P25RO fibroblasts did n o t c o m p l e m e n t XP12BE (group A) cells (Figs. 4a,b) but did c o m p l e m e n t X P l l B E
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0 0 I0 20 30 40 50 60 , , ~70N 80 . 90 . . . .I00110 ..
I0 20 30 40 50 60 70 80 90100110120130140150
GRAINS PER NUCLEUS Fig. 5. R e l a t i o n s h i p s o f X P 2 R O f i b r o b l a s t s to N I H e o m p l e m e n t a t i o n g r o u p s B a n d D. H i s t o g r a m s are of autoradiographic data for Sendal virus-treated, irradiated fibroblasts showing lack of complementation w h e n X P 2 R O f i b r o b l a s t s are f u s e d to t h e m s e l v e s to f o r m h o m o k a r y o n s (a), (b) b u t s h o w i n g c o m p l e m e n t a t i o n w h e n f u s e d w i t h X P l l B E cells ( g r o u p B) (c), (d) or w i t h X P 6 B E cells ( g r o u p D) (e), (f). I m m e d i a t e l y a f t e r t h e t i m e of i r r a d i a t i o n w i t h 8 J / m 2 t h e cells w e r e i n c u b a t e d f o r 3 h w i t h 3 H T d R (spec. act., 2 1 . 0 C i / m m o l e ) in C o o n ' s m o d i f i c a t i o n [ 4 ] o f H a m ' s F - 1 2 m e d i u m ( b u t w i t h o u t t h y m i d i n e ) c o n t a i n i n g 5% fetal c a l f s e r u m . A u t o r a d i o g r a p h i c e x p o s u r e , 14 d a y s . H i s t o g r a m s (a), (c) a n d (e) are o f i r r a d i a t e d , unf u s e d m o n o n u c l e a r cells w h i c h e s c a p e d f u s i o n on c o v e r s l i p s c o n t a i n i n g S e n d a l v i r u s - t r e a t e d cells o f t h e s t r a i n s i n d i c a t e d . H i s t o g r a m s (b), (d) a n d (f) are o f i r r a d i a t e d b i n u c l e a r cells o n t h e s a m e c o v e r s l i p s u s e d in (a), (c) a n d (e) r e s p e c t i v e l y . A r r o w s in (a) a n d (b) i n d i c a t e t h e m e a n g r a i n c o u n t s f o r t h e n u c l e i evalua t e d ( 1 0 0 in a; 4 8 in b), all o f w h i c h h a d 50 or f e w e r g r a i n s p e r n u c l e u s . O f t h e 41 a n d 29 n u c l e i w h i c h h a d m o r e t h a n 50 g r a i n s p e r n u c l e u s in t h e b i n u c l e a r cells o f (d) a n d (f) r e s p e c t i v e l y , 9 8 a n d 9 0 % w e r e acc o m p a n i e d b y a n o t h e r n u c l e u s w h i c h h a d m o r e t h a n 50 g r a i n s p e r n u c l e u s . In t h i s e x p e r i m e n t i r r a d i a t e d m o n o n u c l e a r cells o f t h e c o n t r o l d o n o r s t r a i n ( A m e r i c a n T y p e C u l t u r e C o l l e c t i o n c u l t u r e d e s i g n a t i o n C R L 1121 Pat Bru) had a m e a n grain c o u n t of 45.9 grains per nucleus. E x p e r i m e n t a l details and presentat i o n o f d a t a o t h e r w i s e as in Fig. 2.
337
(group B) (Figs. 4c,d) and XP5BE (group D) (Figs. 4e,f) cells. In another experiment (data n o t shown) X P 2 5 R O fibroblasts were found to complement XP1BE (group C) fibroblasts. (C) Assignment of strain XP2RO to a separate complementation group E When XP2RO fibroblasts were fused to themselves to form homokaryons, the mean grain c o u n t for irradiated nuclei in the binuclear cells was the same as that for the irradiated nuclei in the mononuclear cells (arrows, Figs. 5a and 5b). Thus, the fusion process per se did n o t affect the rate of UV-induced UDS in the XP2RO homokaryons. When XP2RO fibroblasts were fused with X P l l B E {group B) fibroblasts (Fig. 5d), with XP6BE (group D) fibroblasts (Fig. 5f), or with XP2BE (group C) fibroblasts (data n o t shown}, complementation occurred in the hybrid binuclear cells. XP2RO cells were n o t fused with any group A strains at the NIH since it had already been demonstrated [12] that XP2RO fibroblasts complemented fibroblasts from strain X P 2 5 R O which latter strain is n o w shown to be in complementation group A (Table II; Fig. 4b). (D) Rates o f UV-induced UDS in the representative strains The rates of UV-induced UDS were determined for the representative Rotterdam and NIH complementation group strains (Table III). Strains X P 2 5 R O and XP12BE, which are both in group A, have less than 5 or 2% of the normal rate, if they have any UV-induced UDS at all. Significant UDS was n o t detected in these group A cells in these experiments. The rates of UDS of strains XP4RO and X P I B E (both in group C) were within 10--20% of the normal range. The UDS seen in group B cells (XP11BE) was similar to the previously reported range of 3--7% [refs. 8,10]. A discrepancy was observed between the data of the NIH and Rotterdam groups regarding the residual UDS of the group D and E strains. The values obtained by the R o t t e r d a m group were consistently lower
TABLE
III
COMPARISON OF BOTH INSTITUTES Complementation
UV-INDUCED
group
UDS
IN THE
Strain
REPRESENTATIVE
UV-induced Rotterdam
a
NIH b
Control donors XP25RO XP12BE XP1LO
B
XP11BE
C
XP4RO XPIBE XP2BE
10--15
D
XP5BE
10
27.1
E
XP2RO
40--60
60
as a percentage as a percentage
~5 4
24--27
MEASURED
U D S (% o f n o r m a l r a t e )
A
a Expressed b Expressed
100 ~5
XP STRAINS
100 ~2 ~2 ~2 4.8 12.9 19.8 13--18
o f t h e l e v e l s f o u n d i n n o r m a l c e l l s a f t e r a U V d o s e o f 1 0 J / m 2. o f t h e l e v e l s f o u n d i n n o r m a l c e l l s a f t e r a U V d o s e o f 3 0 J / m 2.
IN
338 than those obtained by the NIH group (Table III). The different UV doses used in these experiments (10 and 30 J/m e respectively) or differences in culture conditions might be responsible for these discrepancies. Discussion The R o t t e r d a m and NIH methods for complementation testing are in principle the same. In both methods the rate of UV-induced UDS is determined autoradiographically in multinucleate cells resulting from Sendai virus-induced cell fusion. The results obtained in R o t t e r d a m concerning the complementation group assignment of XP fibroblasts were identical to the results obtained at the NIH. Thus, the R o t t e r d a m investigators have confirmed (Table II) the existence of the four complementation groups, A, B, C, and D, previously reported among the NIH strains [8,10]. Furthermore, the Rotterdam results for complementation group assignment of the representative strains (Table II) were the same as the results found at the NIH (Figs. 3--5). Thus, there are currently five known UDS complementation groups in XP. That the restored rate of UV-induced UDS observed in the heterokaryons whose nuclei show complementation reflects DNA repair is supported by the following observations: 1) the rate of UDS in these complemented nuclei is resistant to h y d r o x y u r e a [8,10] and is the same as that in normal fibroblasts [11, 12] ; 2) these complemented nuclei have normal rates of thymine dimer excision [9] and repair replication [13]. Furthermore, host-cell reactivation studies of UV-irradiated adenovirus 2 reveal that heterokaryons of complementing XP strains have the same capacity as normal strains to restore biological function to the virus [ 5]. Both the Rotterdam [7,12] and the NIH investigators [8,10] have reported that strains within a complementation group have similar rates of UV-induced UDS and that each complementation group has a characteristic rate. The present studies n o w extend these findings to the five known UDS complementation groups in XP as the following indicates. The R o t t e r d a m "De SanctisCacchione" group, which does not complement NIH group A, has the same rate of UDS as group A; the R o t t e r d a m "classical XP, severe" group, which does n o t complement NIH group C, has the same UDS rate as group C; and the R o t t e r d a m "classical XP, light to moderately severe" group, which complements all four of the NIH groups, has a much greater rate of UDS than any of the NIH groups. Our collaborative study has provided additional information concerning the relationship between the clinical manifestations of XP and the complementation groups. The NIH complementation group containing the most kindreds, group C, was known [10] to be essentially free of the neurological abnormalities of XP. The R o t t e r d a m "classical XP severe" group, which does n o t complement NIH group C, contains 6 kindred none of which has any known neurological defect [7]. Thus, it appears as though the defective gene p r o d u c t in group C does n o t result in neurological abnormalities. The kindreds in the Rotterdam "De Sanctis-Cacchione" group, which does n o t complement NIH group A, all have numerous neurological abnormalities [6,7] while most of the kindreds in group A also have numerous neurological abnormalities [ 10]. However,
339 one kindred strain (XP12BE) of group A has only two neurological abnormalities known to date [10], while another kindred strain (XP1LO) in group A is n o t known to have any neurological abnormalities [10]. Therefore, for these reasons and because groups B and D have kindreds with neurological abnormalities, we propose that the term "De-Sanctis-Cacchione" no longer be used as a complementation group designation, and that the NIH complementation group designations A, B, C, and D, be appropriately applied to the Rotterdam groups as well as to the NIH groups. Since strain XP2RO is in its own complementation group, and since it has more UV-induced UDS than any of the other UDS-deficient strains, we propose that the complementation group of strain XP2RO be designated group E.
Acknowledgement The work of the Rotterdam group was supported by grants from Euratom, contract No. 123-74-1 BJOC and the Netherlands Organization for the Advancement of Pure Research (ZWO).
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