Tendency to high levels of UVR-induced unscheduled DNA synthesis in Bloom syndrome

transcript

Mutation Research, 81 (1981) 229--241 © Elsevier/North-Holland Biomedical Press

TENDENCY TO HIGH LEVELS OF UVR-INDUCED UNSCHEDULED DNA SYNTHESIS IN BLOOM SYNDROME

F. GIANNELLI, S.A. PAWSEY and P.K. BOTCHERBY

Paediatric Research Unit, The Prince Philip Research Laboratories, Guy's Hospital Medical School, London SE1 9RT (Great Britain)

(Received 7 July 1980) (Revision received 15 October 1980) (Accepted 16 October 1980)

Summary

The unscheduled DNA synthesis (UDS)induced b y ultraviolet radiations (UVR) in fibroblasts from 5 patients with Bloom Syndrome (BS)has been studied. Since often a high proportion of BS cells has large, probably polyploid nuclei, care was taken to select cells of normal size. Furthermore, UDS was usually measured over constant nuclear areas by a photometric method and each cell strain was tested on several different occasions. This showed that each BS cell strain had levels of UDS exceeding control values, on average, by 19--29%. The difference between the UDS of BS and control cells did not change with UVR doses from 5 to 100 J . m -2 but, unirradiated BS cells showed no evidence of "spontaneous" UDS. Also erythemal UVR (>295 nm)elicited excessive UDS in the BS cell strain which was exposed to such radiations. When BS and control fibroblasts were incubated at low (32.5°C) and high temperature (40.5°C) before and after UVR doses of 5, 25 and 100 J • m -2, it was found that at the lowest dose the temperature of incubation did not modify the difference in UDS between normal and BS cells while, at the highest dose, BS cells incubated at 32.5°C did not show more UDS than the controls. Finally, BS and control fibroblasts were fused with xeroderma pigmentosum (XP) cells of complementation groups C and D, which are complemented slowly by their part- nets, and it was then found that BS cells may transfer their tendency to high levels of UVR-induced UDS to their fusion partners. In keeping with these findings, UV-irradiated fibroblasts from BS heterozygotes seemed to show more UDS than normal cells. The anomalous behaviour of BS cells exposed to UVR is unexplained but trivial factors such as differences in cell geometry do not seem to account for our findings. Therefore, since an abnormally small endo- genous pool of thymidine and "spontaneous" UDS in BS were not observed, it seems possible that the greater UDS performed during the first 1.5 h of repair by BS cells may be due to: greater numbers of damaged sites repaired; greater

incorporation of thymidine per site repaired; or finally, both. In fact, the cell- fusion experiments suggest that BS cells may contain a diffusible factor which influences at least the initial rate of UDS. If such a factor were the product of the BS allele it could be argued that the BS mutations are not amorph and that the BS gene product may compete with that of the normal allele and modify the initial rate of UDS induced by UVR. It is hoped that our observations and their many possible interpretations will stimulate further work on BS.

Bloom syndrome (BS) is a rare autosomal recessive condition of marked pre- and post-natal growth retardation, sensitivity to sunlight, immunodeficiency and marked predisposition to cancer (German, 1979; Polani, 1979). The pri- mary metabolic defect(s)is(are)still unknown as many investigations have given negative results. Thus it seems that BS cells are able to excise pyrimidine dimers, perform daughter-strand repair and rejoin most of the single strand breaks induced by large doses of X-rays (Regan et al., 1973; Giannelli et al., 1977; Vincent et al., 1978). They also do not seem grossly defective in the ability to reactivate virus damaged by ultraviolet radiations (UVR), although, one strain appeared slightly defective under some experimental conditions (Selsky et al., 1979). DNA polymerase activities have also been reported to be within the rather broad normal limits (Parker and Lieberman, 1977) and the slow rate o f progression of DNA growing forks observed by Hand and German (1975, 1977) in both BS lymphocytes and fibroblasts has not been observed by Ockey (1979) in BS fibroblasts except at low density growth. Modest reductions in sur- vival after exposure to UVR or ethyl methanesulphonate have been reported in some strains but whether this response is a direct consequence of the metabolic defect in BS is unclear (Giannelli et al., 1977; Arlett and Lehmann, 1978). The principal cellular feature of BS is still, therefore, the high incidence of "spontaneous" chromosomal aberrations. These often involve exchanges between the chromatids of homologous chromosomes at homologous sites but the X chromosomes, which are known to replicate asynchronously are usually exempt (C.A. Bourgeois, personal communication, 1976; Kuhn, 1976). Such exchanges, together with the characteristic high incidence of sister-chromatid exchanges (Chaganti et al., 1974)are perhaps still the best (though circum- stantial) evidence that the BS defect(s)may express itself during DNA synthesis.

In the course of our first observations on BS in 1976 we noted that 2 BS fibroblast strains exposed to 254-nm UVR had higher levels of unscheduled DNA synthesis (UDS) than control cells. In some cultures, this seemed to be partly due to the relatively high number of BS fibroblasts with large nuclei. However, the BS fibroblasts showed levels of unscheduled DNA synthesis slightly above normal even after allowance for nuclear size. Since this may suggest anomalies in excision repair we have collected further data on the UDS of 5 of our own BS flbroblast cell strains and such data are the object of this communication.

Materials and methods

Fibroblasts from 5 patients with BS (BS 1 LO or H11325/PRU 8638; BS 2 LO or H12572/PRU 10003; BS 3 LO or H13106/PRU 10624; BS 4 LO or H15887/PRU 13671; BS 5 LO or H17549/PRU 15247), 3 with xeroderma pigmentosum (XP 106 LO of complementation group C and XP 104 and 110 LO both of group D), 9 normal volunteers (9172; 9366; 9527; 11677; 11696; 14815; 15161; 15526; 18800) and 6 heterozygotes (BSH 1 L O d or Hl1326/ PRU 8638; BSH 2 LO ~ or H12574/PRU 10003; BSH 3 LO 9 H13107/PRU 10624; BSH 4 LO ~ or H15888/PRU 13671; BSH 5 LO 9 or H17789/PRU 15247; BSH 5 LO ~ H17790/PRU 15247)were established from small skin biopsies and propogated in Ham's F10 deficient in thymidine (Tdr) and hypo- xanthine or Dulbecco MEM with 20% serum and antibiotics. Cells were irradiated as described previously (Pawsey et al., 1979) using a germicidal UV-light source emitting mainly {95%)radiations of 254 nm or a sunlamp emitting essentially UV-B radiations with a peak at 310 nm. Cells were exposed to the latter source through the lids of plastic dishes transparent only to wavelengths longer than 295 nm. UDS, taking place during the first 90 min after UVR was detected by autoradiography as previously described (Giannelli et al., 1973) and autoradiographs were analysed either by eye or photometrically by measuring the light reflected by the silver grains in the autoradiographs with a Zeiss microphotometer using incident illumination and polarised light in order to ob- tain optimum dark background as discussed by Rogers (1973). The measuring area of the photometer was chosen so that its diameter (12.6 #) was slightly smaller than the shorter axis of the average fibroblast nucleus. Nuclei t h a t would fill the measuring area were chosen for measurement, but those exces- sively large were excluded. Autoradiographic slides were processed in batches so as to secure uniform conditions of autoradiographic exposure and develop- ment and comparisons were made only within batches. Slides were coded prior to microscopy and analysed blind.

Cells were fused with ~-propiolactone inactivated Sendal virus (Harris and Watkins, 1965) and incubated either in normal medium or in medium containing cycloheximide (5 pg/ml) so as to inhibit protein synthesis. After harvesting, heterokaryons were identified by the X and Y sex markers as previously described (Giannelli et al., 19q3).

In order to estimate the intracellular Tdr pool 2 X l0 s cells were placed in 60-mm plastic petri dishes and left to settle overnight. The next day the medium was replaced by one containing 0.001 pCi/ml (37 Bq/ml) of ~4C-Tdr (spec. act. 59.6 mCi/mmole) (2.2 GBq/mmole) and the cells were labelled for 3 days. Then they were washed twice with phosphate buffered saline (PBS) and chased for 1--2 days in fresh medium. The confluent cells were washed again with PBS and covered with 0.2 ml of the same solution prior to irradiation with germicidal UV light (100 J . m-2). Pairs of irradiated monolayers were then incubated in medium containing 5 pCi/ml (185 kBq/ml) of 3H-Tdr (spec. act. 20 Ci/ mmole) (740 GBq/mmole)and either 10 -6 or 10 -s M Tdr for 3 h. The cells, washed twice with ice-cold PBS, were harvested by scraping in an ice-cold solution of 0.25% trypsin in 0.02% versene and centrifuged. The pellet was then resuspended in ice cold PBS, 5 × l0 s cells were lysed in 1% Sarkosyl (Ciba-

Geigy) and the lysate precipitated with an equal volume of ice cold 10% trichlomcetic acid (TCA). The precipitate was then filtered through Whatman GF/C filters and washed with 5% TCA and with absolute ethanol. The filters were then solubilised with NCS tissue solubiliser (Amersham) and counted in a toluene-based scintillation fluid containing PPO and POPOP. The ratio of 14C/ SH dpm was obtained and used to calculate the pool size by the procedure described by Clarkson (1978).

Results

Visual automdiographic grain counts over whole nuclei or identical nuclear areas of randomly selected BS and control cells are shown in Table 1. This shows that, at least in some cultures, differences in mean nuclear size may con- tribute to the discrepancy between the UDS of normal and BS cells since such discrepancy is greater when whole nuclei are considered. However, differences in nuclear area do not seem totally to account for the higher UDS of BS cells because these show higher grain counts even when identical areas are considered. This is amply confirmed by experiments, analysed photometrically by measuring the density of aut0radiographic grains over identical areas of nuclei of similar size. Out of 51 comparisons the BS cells had higher grain densities in 49 and the difference was statistically significant, at least, at the 5% level, in

T A B L E I

U V R - I N D U C E D U D S IN BS F I B R O B L A S T S R E L A T I V E T O C O N T R O L S ( V I S U A L M E A S U R E - M E N T S ) a

E x p t . Cell strain Cell R a n d o m l y s e l e c t e d ce l l s N o . s a m p l e

s ize Wh ole nuc le i C o n s t a n t nuc lear area

Grain BS as % Grain BS as % c o u n t o f c o n t r o l c o u n t o f co n tro l

1 9 1 7 2 3 1 3 9 . 5 8 1 8 . 3 9

BS 1 L O 3 6 6 7 . 5 1 7 0 b 1 8 7 b 3 4 1 1 . 5

2 9 1 7 2 4 0 8 0 . 1 4 0 2 6 . 3

9 3 6 6 8 0 9 9 . 8 8 0 2 8 . 5

BS 2 L O 3 2 1 1 1 . 7 1 3 9 b 1 0 6 4 0 1 1 2 2 7 . 8 9 7 . 5

3 9 1 7 2 8 0 6 8 . 5 8 0 23.8

9866 80 90.9 8 0 2 4 . 5

BS 2 L O 8 8 1 3 3 . 6 1 9 5 b 1 2 5 b 8 0 1 4 7 b 2 9 . 7 1 2 1 b

a U V R d o s e , I 0 0 J • m - 2 . b D i f f e r e n c e f r o m c o n t r o l s igni f icant at the 0 .1% level .

S tat i s t ica l s ign i f i cance s h o w n in this and s u b s e q u e n t tables w a s t e s t e d b y the t w o - s a m p l e s t t e s t and refers t o t he d i f f e r e n c e b e t w e e n th e o b s e r v e d m e a n s o f autoradiographie grain c o u n t s .

T A B L E 2

U V R o I N D U C E D U D S IN BS F I B R O B L A S T S R E L A T I V E , T O C O N T R O L S

A v e r a g e o f d i f f e r e n t E x p t s . a n a l y s e d p h o t o m e t r i c a l l y .

BS cell U V R d o s e BS U D S as N u m b e r o f S i g n i f i c a n t s t r a i n (J • m -2 ) % o f c o n t r o l s s a m p l e s c o m p a r e d a d i f f e r e n c e s b

BS 1 L O 1 0 1 2 1 2 2 1 0 0 1 2 2 7 6

BS 2 L O 1 0 1 2 5 1 1 1 0 0 1 1 8 5 2

BS 3 LO 5 125 3 2 2 5 1 2 8 2 2 5 0 1 1 1 1 1

1 0 0 1 3 4 6 5

BS 4 L O 5 1 2 9 3 3 1 0 1 2 0 1 1 2 5 1 0 5 2 0 5 0 9 5 . 5 1 0

1 0 0 12"/ 1 0 7

BS 5 L O 5 1 3 8 1 1 2 0 1 6 0 1 1 5 0 1 2 5 1 1

1 0 0 1 0 8 4 1

a T h e s a m p l e size v a r i e d f r o m 2 0 t o 1 7 0 cel ls a n d m o s t f r e q u e n t l y w a s > 5 0 . b D i f f e r e n c e s b e t w ^ e n o b s e r v e d m e a n g r a i n c o u n t s s i gn i f i c an t , a t l eas t , a t t h e 5% level .

= 150 o

A o •

• z , o

" o o z~ o

5O i i

i e r i i I

25 50 100

UVR dose (J. m -2)

Fig. 1. The UDS in samples o f BS cells has been expxessed as % o f the UDS in the appropriate ~umples o f c o n t r o l cells. These p e r c e n t a g e s h a v e t h e n b e e n p l o t t e d a g a i n s t U V R d o s e s ( 2 5 4 n m ) a n d t h e y d o n o t s h o w a n y r e g r e s s i o n o n s u c h doses , e , BS 1 L O ; o , BS 2 L O . A, BS 3 L O ; 4 BS 4 L O ; o, BS 5 L O .

36. Only on one occasion have BS cells shown lower grain densities and the difference was not statistically significant. Marked differences between the 5 BS cell strains were not observed but BS 2 LO showed the lowest and BS 3 LO the highest levels of UDS. These were, respectively, 19 and 29% higher than normal (see Table 2).

If the BS autoradiographic grain counts are expressed as percent of control counts and plotted against the dose of UVR, no regression on dose is observed (Fig. 1). This suggests that in the conditions of our experiments the difference between the response to UVR of BS and control cells is not a function of dose. It must, however, be noted that doses of 25 J . m -2 elicit maximal UDS responses in normal fibroblasts labelled for the first 1.5 h after irradiation. UVR of wavelengths shorter than 290 nm, such as those used in the previous experiments, are absorbed by the atmosphere and are not present in natural sunlight at the surface of the Earth. Therefore, BS 1 LO and control fibroblasts were exposed to UV-B radiations (peak band 310 nm) filtered through plastic transparent only to wavelengths greater than 295 nm and UDS measurements (Table 3) showed that even such UVR induce more UDS in BS than in control cells. Since BS cells show spontaneous chromosomal aberrations it seemed relevant to test whether the high levels of UDS in BS cells could be explained by "spontaneous" UDS. BS 1,4,5 LO and control fibroblasts were, therefore, tested for UDS in the absence of UVR. Labelling and autoradiographic exposure were chosen so that if the difference in UDS observed between control and BS fibroblasts exposed to UVR were due to a background of "spontaneous" UDS in the BS cells such background would have given photometric measurements of approx. 20 units. The values observed were, however, 0.4 for the control and 0.35, 0.17 and 0.25 for the 3 BS cell strains.

Because of reported effects of incubation temperature on the excision repair of mammalian cells (Goss and Parsons, 1976) experiments were performed to check if the difference between the levels of UDS in BS and control cells could be influenced by changing the incubation temperature.

TABLE 3

UDS INDUCED IN BS 1 LO AND CONTROL FIBROBLASTS BY A UVR SOURCE WITH PEAK EMIS-

SION AT 310 nm

Photometric measurements.

Dose Cell s ample size Con t ro l s t ra in BS UDS as % of contro l (mln )

5 60 9527 110 5 60 15161 116 b

10 a 60 9527 88 b 10 60 15161 100

15 60 9 5 2 7 128 c 15 60 15161 130 c

20 60 9527 96 20 60 15161 124 c

a U D S reached plateau o f m a x i m u m response at this dose . b ,c Di f ference from contro l s i~nlf icant at the 1% and 0.1% level .

o 180-

N 160-

N 140-

~+20 -

E-20 - 3

31i" A

[ 37'. 5 40. 5 32. 5

~ A _ _ _ _ . ~ . . . . % _ _ _

A j a ~ A

37'.5 4d.5 ~5 3/.5 I 40. 5 32. 5 32. 5

Incubation temperature ( °c )

Fig. 2. Effect o f incubat ion temperature and U V R dose o n the U D S o f BS and contro l f lbroblasts. A .A' ; B,B' and C,C' resp. s h o w data from cells e x p o s e d to 5, 25 and 100 J • m -2 o f U V R ( 2 5 4 nm) . In A--C the U D S o f BS cel ls is expressed as % o f the U D S o f contro l f ibroblasts: 9 5 2 7 and 1 5 1 6 1 ( s y m b o l s as in Fig. 1). A t the l o w e s t U V R dose (A) incubat ion temperature has n o e f fec t o n the dif ference b e t w e e n the U D S of BS and contro l ceils but at the highest U V R dose (C) l o w incubat ion temperature suppresses such a difference. A'---C' s h o w the temperature- induced changes in U D S levels o f contro l f lbzoblasts relative to the U D S o f cells incubated at 37 .5°C.

In 3 Expts. the cells were incubated at 32.5, 37.5 and 40.5°C for a total o f 7.5 h (6 preceeding the 1.5 fol lowing UVR treatment). A UVR dose of 100 J • m -2 was used in all the experiments while, in two, doses of 5 and 25 J • m -2 were also used. The results o f all 4 Expts. are summarized in Fig. 2. This figure shows that the uptake of SH-Tdr after saturating doses of UVR ( > 2 5 J • m -2) tends to be lower at 32.5°C than at the higher temperatures, and that the temperature of incubation appears to modi fy the difference in the levels o f UDS in BS and normal cells only after the highest UVR dose since, aRer such a dose, BS cells incubated at 32.5°C do not perform more U D S than, control cells. Finally, it seemed particularly relevant to see if normal and B S'cells after fusion to excision<lefective XP fibroblasts would differ i n their:ability to influence the levels o f UDS o f their defective partners. ,,

XP cells o f complementat ion group C and D which 'hze cSm~lemented slowly

~ 4o- O

"~ 30-

o ~ 20-

-~ I0-

N o r m a l leve l of UDS

CY 6h 24h

24h CY*

B ' N o r m a l l eve l of UDS

CY 24h

C' N o r m a l l eve l of UDS

6h* 24h*

Fig . 3. U D S i n XP n u c l e i o f h e t e r o d t k a , ~ o n s o r u n f u s e d m o n o k a r y o n s e x p o s e d t o 1 0 0 J • m -2 o f UVR ( 2 5 4 n m ) a t v a r i o u s t i m e s a f t e r f u s o g e n i e t r e a t m e n t . XP f l h r o h l a s t s we re f u s e d w i t h BS 1 o r BS 2 L O o r c o n t r o l s t r a in s (9172, 9366, 9527, 11677). In e a c h e x p e x t m e n t t h e a u t o r a d i o g r a p h t e g r a i n c o u n t s we re s t a n d a r d l s e d b y m u l t i p l y i n g e a c h va lue b y t h e c o r r e c t i o n f a c t o r 7 0 / X , w h e r e X is t h e m e a n gzaln c o u n t ove r c o n t r o l m o n o k a r y o u s . D a t a f r o m d i f f e r e n t E x p t s . o n t h e s a m e XP s tza ln a re p o o l e d t o g e t h e r . A - - C , UDS o f XP n u c l e i o f h e t e r o d i k a r y o n s i r r a d i a t e d : 6 h a f t e r f u s i o n a n d i n c u b a t i o n in m e d i u m c o n t a i n i n g 5 / ~ g / m l o f c y e l o h e x i m t d e (CY) ; 6 h a f t e r f u s i o n (6 h ) a n d 2 4 h a f t e r f u s i o n ( 2 4 h ) . A ' - - C ' , UDS o f XP m o n o k a r y o n s f o u n d i n t h e s a m e m i c r o s c o p i c f ie ld o f t h e h e t e r o d i k a r y o n s . S t i p p l e d a n d e m p t y c o l u m n s h o w m e a n U D S in f u s i o n e x p e r i m e n t s i nvo lv ing reep . BS o r c o n t r o l cell s t r a ins . T h e e r r o r b a r (+1 SE) is s h o w n a t t he t o p o f e a c h c o l u m n a n d s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s b e t w e e n m e a n s are m a r k e d b y *. A , A ' , XP 1 0 6 L O p lus BS I L O o r c o n t r o l s ; B, B ' a n d C , C ' , r e sp . X P 1 0 4 a n d I I I L O p lus BS 2 L O o r c o n t r o l s . T h e f igure s h o w s t h a t XP n u c l e i t e n d t o r e a c h h i g h e r levels o f U D S w h e n t h e y a re in h e t e r o k a r y - one eon tAin lng a BS n u c l e u s w h i l e XP m o n o k a r y o u s a re n o t i n f l u e n c e d b y t h e t y p e o f cel l t h e y a re eul- t u z e d w i t h .

B S Heterozygous Cell Strain (B S H L O)

1 2 4 (~

d 9 1901 •

160 1 •

150 1 • • e

1201 • • ~ H 0 o" _ = . _

o o 1001 ~o e o

oO o o o ,

Fig. 4. UDS of BS he te rozygous cell s trains (BSH) expressed as % of controls. The cells were exposed to 5, 10, 50 and 100 J • m -2 of UVR (254 rim) bu t since the difference in UDS between controis and BSH showed no regression on UVR doses the data f rom samples exposed to different UVR doses are shown together. Each point represents the mean UDS of a sample of 20--80 cells (most f requent ly ~ 40 cells). Each hor izontal line is a mean of means. Solid points and emp ty ctreles are values that , resp., do or do not differ significantly f rom control means.

and show low levels of UDS in young heterokaryons (Pawsey et al., 1979) were thought particularly suitable to compare the effect of normal and BS cells on their XP partner and were, therefore, fused with normal and BS fibroblasts prior to UVR treatment. The results of such experiments are shown in Fig. 3. They indicate that the XP cells fused with BS fibroblasts reach higher levels of UDS than those fused with control fibroblasts. By contrast, the XP monokary- ons found in the mixed cell populations subjected to the fusogenic treatment have similar levels of UDS whether they are cultured together with BS or normal fibroblasts. It seems, therefore, that through fusion (but not co-cultivation) the BS cells may transfer their tendency to high levels of UVR-induced UDS to their XP partners. Such heterokaryon data raised the question of whether BS heterozygotes might also show high values of UDS after UVR. The observations made so far and summarised in Fig. 4 would seem to indicate that this is so.

Discussion

Since BS patients show clinical evidence of photosensitivity the UDS induced in their cells by far UVR has been studied in a number of other laboratories. Cleaver (1970) lists one case of BS among other light-sensitive patients he examined, but does not comment on it in detail. More recently, Evans et al.

(1978) observed a statistically significant excess of UDS in the lymphocytes of one BS patient bu t only on one of the two occasions in which such lymphocytes were tested. Tice et al. (1978a) have examined a single cell sample from one normal and two BS cell strains exposed to 254-nm UVR and have found twice as many grains over the BS than over the control cells. However, these authors do no t ment ion differences in the mean nuclear size of BS and normal cell samples and the differences they report may have been inflated by larger mean nuclear sizes in the BS cell populations examined. Ahmed and Setlow (1978) who have performed a detailed s tudy of excision repair in one BS fibroblast strain have no t observed high levels of UDS in such cells. However, UDS was tested on cells treated with hydroxyurea (HU), which not only inhibits semi-conservative DNA synthesis but also alters DNA-repair synthesis (Ben-Hur and Ben-Ishai, 1971; Collins, 1977; Clarkson, 1978; Johnson and Collins, 1978; Andrae and Greim, 1979; Francis et al., 1979). We have tested UDS in our 5 BS strains on several occasions and have observed small differences between BS and control cells. Such differences, in individual experiments, could be explained by interstrain or interculture variations bu t our results, as a whole, indicate a clear tendency to excessive UDS in BS cells and suggest a true, though small, difference in the response of BS and control cells to UVR. The cause of such a difference is not clear but certain simple explanations may be rejected. For example, systematic differences in mean nuclear area do not affect our photometr ic measurements because they were taken over a constant area, smaller than the whole nucleus. Differences in nuclear thickness are unlikely to contr ibute because of the poor penetrat ion of the ~ radiations from tri t ium (Rogers, 1973). In 1 Expt. measurements were taken over areas of 12.6 and 8 g diameter and very similar relative differences between control and BS cells were obtained (i.e. 44% between BS 3 LO and controls over both areas and 31 and 30.5% between BS 4 LO and controls over the 2 areas). Finally, systematic differences in cell geometry fail to explain why only BS cells exposed

T A B L E 4

E S T I M A T E O F T H E T d r P O O L S I Z E OF BS A N D C O N T R O L F I B R O B L A S T S

Ceil s t ra in Mean poo l size N u m b e r o f d e t e r m i n a t i o n s (M)

BS 1 LO 9.4 × 10 -7 3 BS 3 LO 3.3 × 10 -7 4 BS 4 LO 7.5 X 10 -7 3 BS 5 LO 6.1 × 10 -7 2 G M 1 4 9 2 a 3.6 × 10 -7 1

To ta l BS b 6.0 X 10 -7 13

9 1 7 2 3.8 X 10 -7 1 9527 2.1 × 10 -7 3

1 5 1 6 1 8.6 X 10 -7 2 1 8 8 0 0 3.3 X 10 -7 1

To t a l con t ro l s b 4 .5 X 10 -7 7

a BS ceil s tain f r o m the Ins t i tu te o f Medica l Re se ar c h , C a m d e n , NJ (U.S.A.) . b T h e d i f f e r e n c e b e t w e e n th e t o t a l BS and c o n t r o l m e a n s is n o t stat is t ical ly s ignif icant .

to 100 J • m -2 UVR and incubated at 32.5°C do not show excessive UDS nor why differences are observed between the UDS of XP nuclei in heterokaryons formed by fusion of the same XP cell strain with either a BS or a normal strain. These 2 observations and the data shown in Table 4 are also inconsistent with the possibility that different intracellular Tdrpools (i.e. smaller pools in BS) may be responsible for the excess of UDS in the BS fibroblasts by allowing synthesis of repair patches of higher specific activity.

Tice et al. (1978b) have claimed that co~ultivation of BS and normal cells or the incubation of normal cells in medium conditioned by BS cultures increased the frequency of SCE in normal cells and they have suggested that BS cells pro- duce and release into the medium substances capable of damaging DNA. How- ever, the co~ultivation experiments of Van Buul et al. (1978), Bartram et al. (1979) and especially those of Schonberg and German (1980), who studied the effect of BS cells on HGPRT- fibroblasts coupled to BS cells in metabolic co- operation, are at variance with those of Tice et al. (1978b). Furthermore, the suggestion of Tice et al. (1978b) could have explained our findings only if BS cells had shown "spontaneous" UDS but, in keeping with Evans et al. (1978), we have failed to detect such UDS.

What explanations can then be offered for our results? Obviously an excess of UDS may be caused either by the repair of a greater number of damaged sites or by the incorporation of greater amounts of aH-Tdr per site repaired. Ahmed and Setlow (1978)compared the disappearance of UV~ndonuclease- sensitive sites and the average size of repair patches in normal and BS cells and found no difference. However, by the UV~ndonuclease assay, these authors measured essentially the number of pyrimidine dimers removed over a period of 24 h whilst we obtained a measure of the DNA synthesised for the repair of pyrimidine dimers as well as other lesions during the first 1.5 h of repair. Simi- larly, the estimates of patch size made by Ahmed and Setlow (1978) refer to patches synthesised over a period of 12 h. Moreover, the measurements of patch size were performed on cells exposed to HU, a metabolic inhibitor which may interfere with repair. We feel, therefore, that the results of Ahmed and Setlow (1978) do not exclude the possibility that our results are due to small differences in the number and/or the size or repair patches synthesised by BS and control cells during the first 1.5 h of DNA repair.

The comparison of UDS in XP nuclei fused with BS or control cells is inter- esting because it suggests that the factor causing higher than normal rates of UDS in BS cells can be transferred from nucleus to nucleus upon cytoplasmic fusion. The XP cells used in our experiments, belonging to complementation groups C and D are complemented very slowly by either normal or XP partners. Polykaryon studies have also shown that the complementation of XP cells of group D during the first few hours after fusion is not strongly dependent on the dose of wild-type alleles at the relevant XP locus (Giannelli, Pawsey and Avery in preparation). It seems, therefore, unlikely that the greater UDS of XP cells fused with BS fibroblasts is due to a greater than normal transfer of the factor defective in the XP cells. Our heterokaryon data, therefore, suggest that BS cells contain an intra~ellularly diffusible factor which influences the initial rate of UDS independently of the factors involved in the incision defect of XP.

If such a factor were the product of the BS allele it could be argued that the

BS mutations are not amorph and that the BS gene product may compete with the product of the normal allele and modify the initial rate of the UDS induced by UVR.

Our observations do not provide a direct clue to the metabolic defect of BS but we hope that they will provoke speculation and new working hypotheses to further the investigation of the cellular defect in BS.

Acknowledgements

We are very grateful to Professor J.M. Tanner for referring his patients to us and to Professor G. Belyavin for the gift of Sendal virus. Professor P.E. Polani has given us valuable advice and constant support. We acknowledge the techni- cal help of Miss F. Moir and the secretarial assistance of Miss M. Thomas. This work was supported by the Spastics Society, The Cancer Research Campaign and the National Fund for Research into Crippling Diseases.

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Tendency to high levels of UVR-induced unscheduled DNA synthesis in Bloom syndrome

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