Clonal chromosome rearrangements in a fibroblast strain from a patient affected by xeroderma pigmentosum (complementation group C)

Mutation Research,

219 (1989) 209-215

209

Elsevier MUTAGI 09018

Clonal chromosome rearrangements in a fibroblast strain from a patient affected by xeroderma pigmentosum (complementation group C) F. Nuzzo, P. Lagomarsini, A. Casati, R. Giorgi 1, E. Berardesca 2 and M. Stefanini lstituto di Genetica Biochimica ed Evoluzionistica del C.N.R., I Dipartimento di Genetica e Microbiologia, Unioersitd di Pavia and 2 Clinica Dermatologica, Policlinico S. Matteo, Pavia (Italy)

(Received 5 April 1988) (Revision received 25 November 1988) (Accepted 29 November 1988)

Keywords:

DNA repair; Clonal chromosome rearrangements; Xeroderma pigmentosum

Summary We report the results of D N A repair studies and cytogenetic investigations in a patient presenting acute phothosensitivity and cancerous skin lesions. In lymphocytes and fibroblasts a reduced level of unscheduled D N A synthesis after UV irradiation was found and the presence of xeroderma pigmentosum, complementation group C, mutation was demonstrated by complementation analysis. In lymphocyte and fibroblast cultures the frequency of spontaneous chromosome gaps and breaks was normal, whereas the frequency of chromosome rearrangements was higher than expected. In fibroblasts from the 4th to the 18th passage of the culture, 4 reciprocal translocations with a clonal distribution were identified. The rearranged chromosomes were Nos. 2, 13, 14 and 15, Nos. 2 and 13 being both involved in 3 different translocations with breakpoints at 2q21, 2q31, 2p23 and 13q31, 13q12 or 3. The biological significance of this finding is discussed in view of a possible correlation with the D N A repair defect and a possible relevance in tumor development of specific chromosome rearrangements.

Xeroderma pigrnentosum (XP) is a hereditary disorder in which hypersensitivity to UV light is correlated with tumor development in the sun-exposed skin (Kraemer et al., 1987). Clinical, cellular and genetic heterogeneity is well documented in XP; cells derived from affected individuals are characterized by a varying degree of impairment in their capacity to repair the UV-induced D N A damage and may be classified into 10 groups

Correspondence: Dr. F. Nuzzo, Istituto di Genetica Biochimica ed Evoluzionistica del C..N.R., Via Abbiategrasso 207, Pavia (Italy).

according to complementation studies (Cleaver, 1983, 1985). Chromosome instability, clearly demonstrated in the ' c h r o m o s o m e breakage syndrome's that may be somehow defective in processing D N A damage, seems not to be present in XP (Ray and German, 1983; Hanawalt and Sarasin, 1986). In these cells the frequency of chromosome aberrations and sister-chromatid exchanges appears to be increased after UV irradiation and exposure to a range of chemicals (Parrington et al., 1971; Sasaki, 1973). In this paper we describe the presence of spontaneous structural chromosome rearrangements with clonal distribution in cultured skin fibrob-

0921-8734/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

210

lasts obtained from an XP patient assigned to complementation group C.

Case report The patient, a male born in 1953, was the second of 4 sibs. The parents were first cousins. An older brother was healthy; 2 younger sisters with clinical symptoms of XP died at a young age. The patient had been showing acute photosensitivity of the skin since childhood (age 4). Characteristic freckling and hypopigmented skin areas developed soon afterward. Multiple neoplastic lesions (basal cell carcinomas) appeared on the face when the patient was 15-16 years old. At 20 years a large nodular basalioma was surgically removed from the lower eyelid. Despite plastic reconstruction, light exposure induced chronic conjunctivitis, keratitis and, more recently, a highly invasive spinalioma. Actinic keratoses on the scalp and on the neck are present. The patient is neurologically normal. He does not show any specific alteration of the immunologic system; the helper-suppressor lymphocyte ratio is normal.

Materials and methods

Cells and culture conditions Leukocytes were separated by spontaneous sedimentation from 20 ml of blood obtained from the patient and healthy donors and resuspended at a concentration of 106 mononucleated cells per ml minimal essential medium (MEM, Gibco). Phytohemoagglutinin (PHA-M, Difco) at a final concentration of 1 : 5 0 and pokeweed (PWM, Difco) at a final concentration of 1:100 were used as mitogens. Fibroblasts were derived from a biopsy of unaffected skin of the patient (XP9 PV strain). Fibroblast strains from 3 healthy individuals and from XP patients belonging to complementation groups (e.g.) D (TTD3 PV (Stefanini et al., 1986)) and C (XP21 RO) were used in this study. The cells were routinely grown in Eagle or F10 medium supplemented with 15% calf serum and subcultured by trypsinization. All cell strains were examined according to established procedures and found to be mycoplasma-free (Chen, 1977).

UV irradiation The cells were exposed to UV-C radiation (254 nm) using a Philips T U V 15-W lamp, giving a dose rate of 2 j / m 2 / s , as previously described (Stefanini et al., 1980). Sensitivity to UV irradiation in stimulated lymphocytes UV-irradiated cultures of 2 x 105 cells in medium supplemented with 20% calf serum were set up in microtiter wells, and incubated at 37 ° C for 68 h. 3H-thymidine (3H-TdR, Amersham, spec. actir. 2 C i / m m o l e ) was then added at a final concentration of 2.5/~Ci/ml and the incorporated radioactivity was measured 24 h later (Stefanini et al., 1986). Sensitivity to UV irradiation in non-dividing cells 1.5 x 105 fibroblasts were seeded in a 6-cm dish in medium containing 0.5% serum and UVirradiated 7 days later. After incubation at 3 7 ° C for 14 days, the adhering cells were fixed and counted (Mayne and Lehmann, 1982). Analysis of unscheduled DNA synthesis (UDS) U D S was studied in G O lymphocytes by measuring the UV-stimulated 3H-TdR incorporation in the presence of hydroxyurea (Stefanini et al., 1980). The cells were irradiated with a UV dose of 7 or 14 J / m z and then incubated at 3 7 ° C in the presence of 2.4 m M hydroxyurea (Sigma). After 15 min, 10 t t C i / m l 3H-TdR (spec. activ. 25 C i / m m o l e ) was added and the incubation continued for 3 h. Incorporated radioactivity was measured as described elsewhere (Stefanini et al., 1979). In fibroblast cultures, repair synthesis was analyzed by the autoradiographic technique. After UV irradiation the cultures were incubated in medium containing 10 t t C i / m l 3H-TdR (spec. activ. 2 C i / m m o l e ) for 3 h at 37 o C. Autoradiography was performed with Ilford emulsion. After 11 days at 4 o C, the slides were developed and stained with M a y - G r u n w a l d and Giemsa solution. U D S was measured by counting the number of grains on at least 50 non-S-phase nuclei. Complementation analysis The fibroblasts used as parentals in fusion experiments were labeled with latex beads of dif-

211

ferent size (0.75 and 1.72 # m in diameter) by growing the cells for 3 days in beads containing medium. The cells were then trypsinized, mixed in a 1 : 1 ratio, centrifuged, and suspended in medium containing 45% PEG-4000 (Merck). After 2 rain the cells were diluted in medium supplemented with 3% calf serum and seeded in dishes provided with a coverslip. After 48 h incubation at 37 o C, cells were UV-irradiated (20 j / m 2 ) , incubated for 3 h in the presence of 3H-TdR (spec. activ. 25 Ci/mmole), and processed for autoradiography 2 days later as described before. The grains over nuclei in 40 mononuclear parental cells and 20 heterokaryons, identified as binuclear cells containing beads of different size, were counted (Stefanini et al., 1986).

Chromosome analysis Chromosome preparations were established from whole-blood and fibroblast cultures. Cell samples from the patient fibroblast strain (XP9 PV) at the 4th and 5th passages were harvested either immediately or after various freezing periods (maximum 15 months). Slides were processed for conventional karyotyping and for G banding according to standard methods (Benn and Perle, 1986). Results

DNA repair study The sensitivity to UV irradiation was studied by measuring the inhibition of D N A synthesis both in PHA- and in PWM-stimulated lymphocytes after exposure of G O cells to UV light. The rnitogen response in unirradiated patient cells was similar to that in normal cells; as expected, the 3H-TdR uptake in PWM samples was 50% of that in PHA samples (data not shown). The incorporation values decreased with increasing UV doses and were lower in the patient cells compared with cells from the healthy individual (Fig. 1). The D N A synthesis rate after exposure to 14 J / m 2 UV light was reduced to about 6% and 35% in patient and control cells respectively. Cell survival was measured after UV irradiation of non-dividing fibroblasts. Again we found that patient cells were more sensitive to the killing action of UV than were normal cells (Fig. 2).

u

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UV dose ( J i m ~) Fig. 1. D N A synthesis rate in PHA- (open symbols) and PWM- (closed symbols) stimulated lymphocytes after exposure of G O ceils to UV light. 0 o normal subject, zx zx patients.

Therefore we investigated in lymphocytes and fibroblasts the capacity to perform D N A repair synthesis. The UV-induced 3H-TdR uptake in the presence of hydroxyurea was measured in G O and in 72-h PHA-stimulated lymphocytes. In both cases after UV irradiated a marked increment of incorporation was observed in normal cells whereas a slight variation was detectable in the patient's cells (Fig. 3). The unscheduled D N A synthesis (UDS) was analyzed on autoradiographic preparations of irradiated fibroblasts (Fig. 4). The mean numbers of autoradiographic grains 100

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UV dose (Jim ") Fig. 2. Survival of non-dividing fibroblasts irradiated with UV light, zx zx normal subject; • • patient.

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7'

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Fig. 3. UV-induced repair synthesis (3H-TdR incorporation in the presence of hydroxyurea) in G O (left panel) and PHAstimulated lymphocytes (right panel), o o normal subject; zx ,', patients. The SEM within triplicate samples was less than 10%.

0

30 60 0 30 60 grain number / nucteus

Fig. 4. UDS in fibroblasts from the patient (upper panel) mad from the healthy individual (lower panel). The frequencydistribution of nuclei with different grain number is shown (7 J/m 2 at the left, 14 J/m 2 at the right).

per nucleus ( + s t a n d a r d error) in normal cells were 32.7 -t- 0.7 and 44.6 -t- 1.1 after 7 and 14 J / m 2 respectively. The corresponding values in the patient were 3 . 4 _ 0.4 and 4.3 + 0.3. These results clearly indicate that in cells from the patient D N A repair synthesis was not stimulated by irradiation as in normal cells.

Cytogenetie study Chromosome analysis on lymphocyte cultures from the patient showed a normal karyotype. Chromosome aberrations were obtained in 7 out of 148 mitoses analyzed; 2 mitoses showed a chromatid gap whereas structural c h r o m o s o m e anomalies were present in 5 mitoses (the anomalies were: a reciprocal translocation involving a No. 3 and a C-group chromosome; a dicentric involving 3p and 17q; 2 deletions in 2 different C-group chromosomes; 2 acentric fragments). Chromosome analysis was performed in fibroblast cultures starting from the 4th passage, and was repeated at subsequent passages since chromosome anomalies were observed. They consisted mainly of structural aberrations, such as dicentric chromosomes, translocations and deletions; at the 4th and 5th passages few triradial and quadriradial figures were observed.

Complementation study Genetic analysis of the D N A repair defect was performed by measuring UDS in heterokaryons obtained by fusion of the patient's cells (XP9 PV) with XP cells belonging to e.g. D and C. Table 1 shows the mean values of grain number per nucleus both in parental cells and in the heterokaryons. Normal levels of U D S were restored in XP9 PV by fusion with X P D fibroblasts. By contrast, complementation was not observed in heterokaryons between the patient's cells and XPC cells. Thus the patient's cells were assigned to group C. TABLE 1

C O M P L E M E N T A T I O N ANALYSIS IN H E T E R O K A R Y O N S O B T A I N E D BY F U S I O N O F XP9 PV CELLS W I T H N O R M A L FIBROBLASTS OR XP FIBROBLASTS F R O M e.g. C A N D D Cross A x B

Grain number/nucleus + SEM Parental cells

Normal × XP9 PV XPD × XP9 PV XPC × XP9 PV

Heterokaryons

A

B

51.48 + 2.67 10.16 + 0.77 9.48 + 0.53

9.00 + 0.40 9.16 + 0.73 10.68 + 0.67

41.17 + 2.92 43.37 + 1.40 13.22 + 0.79

213 a

b

--o,| 2

13

2

14

c

2

d

13

15

Fig. 5. Partial karyotypes showing the 4 clonal chromosome rearrangements identified in cultured fibroblasts. (a) t(2;13) (q(21;q31); (b) t(2;14)(q31;111); (c) t(2;13)(p23;q12 or 3); (d) t(13;15)(q12 or 3;q13). Arrows indicate the breakpoints on the rearranged chromosomes. M o r p h o l o g i c analysis o f b a n d e d c h r o m o s o m e s r e v e a l e d the p r e s e n c e of s o m e s p o r a d i c s t r u c t u r a l c h a n g e s with b r e a k p o i n t s r a n d o m l y d i s t r i b u t e d over all the c h r o m o s o m e s . T h e m o s t significant f i n d i n g was the p r e s e n c e of 4 different c l o n a l t r a n s l o c a t i o n s , all r e c o g n i z e d at s u b s e q u e n t p a s sages of the c u l t u r e (Fig. 5). T a b l e 2 s u m m a r i z e s the results of cytogenetic analysis. T h e overall

f r e q u e n c y of m e t a p h a s e s w i t h c h r o m o s o m e rea r r a n g e m e n t s was a b o u t 9% at the first p a s s a g e s a n a l y z e d a n d d e c r e a s e d thereafter. T h e specific c l o n a l c h r o m o s o m a l changes i d e n t i f i e d at each p a s s a g e are r e p o r t e d . T h e r e a r r a n g e d c h r o m o s o m e s were N o s . 2, 13, 14 a n d 15, N o s . 2 a n d 13 b e i n g b o t h i n v o l v e d in 3 different t r a n s l o c a t i o n s with d i f f e r e n t b r e a k p o i n t s , n a m e l y : 2q21, 2q31, 2p23 a n d 13q31, 13q12 o r 3. T h e t r a n s l o c a t i o n t(2;13)(q21;q31) o c c u r r e d m o r e f r e q u e n t l y a n d was also f o u n d at late passages. C y t o g e n e t i c investigations o n f i b r o b l a s t s f r o m a c o n t r o l i n d i v i d u a l were c a r r i e d o u t in parallel. In a p o o l e d s a m p l e of 206 m i t o s e s a n a l y z e d at the 4th a n d 5th passages the f r e q u e n c y of c h r o m o s o m e b r e a k s was 3.9% whereas n o c h r o m o s o m e r e a r r a n g e m e n t s were detected. This f i n d i n g allows us to exclude the effect of c u l t u r e c o n d i t i o n s on the o c c u r r e n c e of the c h r o m o s o m e r e a r r a n g e m e n t s in the p a t i e n t cells.

Discussion T h e results of D N A r e p a i r studies in cells f r o m the p a t i e n t u n d e r e x a m i n a t i o n c o n f i r m e d the diagnosis of X P a n d d e m o n s t r a t e d t h a t the r e d u c e d c a p a c i t y to p e r f o r m U D S was d u e to the p r e s e n c e o f c.g. C m u t a t i o n . B o t h clinical s y m p t o m s (absence of n e u r o l o g i c a b n o r m a l i t i e s a n d p r e s e n c e of severe skin lesions) a n d low level o f excision r e p a i r ( 1 0 - 2 0 % of n o r m a l ) are in a c c o r d w i t h those of

TABLE 2 FREQUENCIES AND TYPES OF CHROMOSOME ABNORMALITIES IN CULTURED SKIN FIBROBLASTS Passage

No. of analyzed mitoses

No. of mitoses with rearrangements (%)

Clonal abnormality

No. of mitoses with the clonal abnormality

4th

216

19 (8.8)

5th

644

61 (9.2)

6th

100

6 (6.0)

10th 18th

646 390

7 (1.1) 6 (1.5)

t(2;13Xq21;q31) t(2;14Xq31;qll) t(2;13)(p23;q12 or 3) t(2;13Xq21;q31) t(2;14Xq31;qll) t(13;15)(q12 or 3;q13) 6(2;14Xq31;qll) t(2;13Xp23;q12 or 3) t913;15Xq12 or 3;q13) t(2;13Xq21;q31) t(2;13)(q21;q31)

7a 4b 5c 39 a 1b 4d 1b 1c 1a 3a 1a

a, b, c, d identify mitoses with the same rearrangement (Fig. 5).

214

other XP patients belonging to this c.g. (Cleaver, 1983). On the other hand an unexpected finding emerged from the cytogenetic analysis. In lymphocyte culture a low frequency of gaps and breaks ( 1 . 3 % ) was observed whereas the frequency of chromosome rearrangements (3.4%) was higher than that commonly found in normal individuals. A high frequency of such anomalies was also present in fibroblasts in which a systematic cytogenetic analysis allowed the identification of 4 mutant clones, each characterized by a presumably balanced translocation. Thus, although a tendency to chromosome breakage was not evident, the occurrence of clonal chromosome rearrangements can be demonstrated. Data from the literature indicate that cytogenetically marked clones are occasionally found in fibroblast cultures from apparently normal healthy persons (Littlefield and Mailhes, 1975; Hoehn et al., 1975; Harnden et al., 1976). The occurrence of such mutant clones has been related to culture conditions, as well as to physiological or pathological changes. According to Harnden et al. (1976) the donor age may be one of the responsible factors. The donor genotype certainly plays an important role as has been demonstrated by the observations of site-directed chromosome rearrangements in norfiaal skin fibroblasts from persons (patients and obligate carriers) carrying genes for hereditary neoplasia (Sasaki et al., 1980). Examples are also the chromosome rearrangements found in fibroblasts from the recessively transmitted cancer-predisposing diseases Bloom syndrome (German, 1972), ataxia telangiectasia (Cohen et al., 1975; Oxford et al. 1975), Nijmegen syndrome (Maraschio et al., 1986), Werner syndrome (Salk et al., 1981; Scappaticci et al., 1982) and Fanconi anemia (Auerbach et al., 1980). In these cases a predisposition to chromosome rearrangements is justified by the constitutional chromosome fragility of the affected individuals (Ray and German, 1983). As previously mentioned, in XP the frequency of spontaneous chromosome breakage is reported to be normal. However, it is worth noticing that chromosome investigations in XP fibroblasts derived either from normal skin or from tumors are scarce. An observation similar to that described in this paper was made by German et al. (1973).

They found 9 abnormal metaphases out of 211 analyzed at passage 4 of an XP fibroblast strain, and identified 2 clones each composed of cells with the same chromosome rearrangement. Data reported by Thielmann et al. (1983) document a different situation. An extreme genomic imbalance was observed at the 5th passage in an XP (c.g. F or G) fibroblast strain of normal origin. In this case multiple numerical and structural chromosome alterations were present, accompanied by malignant transformation and increased repair capacity. In our case the cell phenotype of cultured cells did not change and the frequency of abnormal metaphases decreased at late passages. This means that the mutant clones do not have any selective advantage in the culture conditions. Recently Aledo et al. (1988) reported the first cytogenetic study in primary culture of a skin squamous cell carcinoma from an XP patient. They found that virtually all mitoses had chromosome rearrangements; in particular they observed a recurrent involvement of chromosome 14 in translocations with various other chromosomes. It is tempting to attribute a biological significance to the chromosome anomalies observed in XP9 PV fibroblasts, from 2 viewpoints. One concerns a possible correlation with the D N A repair defect present in the patient cells, a second concerns a possible relevance of specific chromosome recombinations for tumor development. With the exception of 15q13, all breakpoints involved in the 4 clonal translocations we found (2q21, 2q31, 2p23, 13q31, 13q12 or 3, 14qll), are at or near the same band at which a chromosome fragile site has been localized (Yunis et al., 1987). This circumstance may be remarkable, in view of the known relationship between fragile sites, cancer chromosome breakpoints, and oncogenes (Yunis, 1987).

Acknowledgements P. Lagomarsini and A. Casati were supported by fellowships from the Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.). This work was partially supported by grants from Regione Lombardia (Ricerca Finalizzata Area Prevenz-

215 ione) and from the A.I.R.C. The authors thank M r s . C. B a g a r o t t i f o r t y p i n g t h e m a n u s c r i p t .

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L. Gargantini, L. Minoli and O. Zuffardi (1986) A new chromosome instability disorder, Clin. Genet., 30, 353-365. Mayne, L.V., and A.R. Lehman (1982) Failure of RNA synthesis to recover after UV-irradiation: an early defect in cells from individuals with Cockayne syndrome and xeroderma pigrnentosum, Cancer Res., 42, 1473-1478. Oxford, J.M., D.G. Harnden, J.M. Parrington and J.D.A. Delhanty (1975) Specific chromosome aberrations in ataxia telangiectasia, J. Med. Genet., 12, 251-262. Parrington, J.M., J.D.A. Delhanty and H.P. Baden (1971) Unscheduled D N A synthesis, UV-induced chromosome aberrations and SV40 transformations in cultured cells from xeroderma pigrnentosum, Ann. Hum. Genet., 35, 149-160. Ray, J.H., and J. German (1983) The cytogenetics of the 'chromosome-breakage syndromes', in: "Chromosome Mutation and Neoplasia", A.R. Liss, New York, pp. 135-168. Salk, D., K. Au, H. Hoehn, M.R. Stenchever and G.M. Martin (1981) Evidence of clonal attenuation, clonal succession, and clonal expansion in mass cultures of aging Werner's syndrome skin fibroblasts, Cytogenet. Cell Genet., 30, 108-117. Sasaki, M.S. (1973) DNA repair capacity and susceptibility to chromosome breakage in xeroderma pigrnentosum cells, Mutation Res., 20, 291-293. Sasaki, M.S., Y. Tsunematsu, J. Utsunorniya and J. Utsum (1980) Site-directed chromosome rearrangements in skin fibroblasts from persons carrying genes for hereditary neoplasms, Cancer Res., 40, 4796-4803. Scappaticci, S., D. Cerimele and M. Fraccaro (1982) Clonal structural rearrangements in primary fibroblast cultures and in lymphocytes of patients with Werner's syndrome, Hum. Genet., 62, 16-24. Stefanini, M., E. Ascari and F. Nuzzo (1979) UV-induced repair in hairy cell leukaemia patients, Cancer Left., 7, 235-241. Stefanini, M., W. Keijzer, L. Dalprh, R. Elli, M. Nazzaro Porro, B. Nicoletti and F. Nuzzo (1980) Differences in the level of UV repair and in clinical symptoms in two sibs affected by xeroderma pigrnentosum, Hum. Genet., 54, 177-182. Stefanini, M., G. Orecchia, G. Rabbiosi and F. Nuzzo (1986a) Altered cellular response to UV irradiation in a patient affected by premature ageing, Hum. Genet., 73, 189-192. Stefanini, M., P. Lagomarsini, C.F, Arlett, S. Marinoni, C. Borrone, F. Crovato, G. Trevisan, G. Cordone and F. Nuzzo (1986b) Xeroderma pigmentosum (complementation group D) mutation is present in patients affected by trichothiodystrophy with photosensitivity, Hum. Genet., 74, 107-112. Thielmarm, H.W., E. Fischer, R.T. Dzarlieva, D. Komitowski, O. Popanda and L. Edler (1983) Spontaneous in vitro malignant transformation in a xeroderma pigmentosurn fibroblast line, Int. J. Cancer, 31, 687-700. Yunis, J.J. (1987) Multiple recurrent genomic rearrangements and fragile sites in human cancer, Somat. Cell Mol. Genet., 4, 397-403. Yurtis, J.J., A.L. Soreng and A.E. Bowe (1987) Fragile sites are targets of diverse mutagens and carcinogens, Oncogene, 4, 59-69.