somatic mosaicism for dna repair capacity in fibroblasts ... · somatic mosaicism for dna repair...

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Somatic Mosaicism for DNA Repair Capacity in Fibroblasts Derived from a Group A Xeroderma Pigmentosum Patient Hae Ryong Chang, M.Sci., Kanji Ishizaki, Ph.D., Masao S. Sasaki, Ph.D., Junya Toguchida, M.D., Mitsuo Kato, M.Pharm., Yusuke Nakamura, M.D., Susumu Kawamura, M.D., Takahiko Moriguchi, M.D., and Mimo Ikenaga, Ph.D. R3di:nion Biology Ccnter. Kyoto Universiry (He . KI . MS. JT. MK. MI ), Kyoto. J;lp:m; Howard Hu ghes Mcdicallnsritutc, University of Utah (YN). Salt Lake Ciry, Utah; and Departmcnt of Plastic Surgery, Kawasaki Medic:!! School (SK. TM), Kurashi ki , Jap an. A fema le Japanese xeroderma pigmentosum (XP) patient with severe skin lesions and various neurologic abnormalities was assigned to complClnentation group A by conventional cell fu sion studies. Ultraviolet (UV)-irradiated skin fibro- blasts showed a biphasic survival curve, as measured by col- ony-f orming ability. The surviving fraction decreased rap- idly up to 2 J /m' ofUV , with a steep slope ofD. (mean lethal dose) = 0.95 J/ m'. At much higher doses it decreased more slowly, with D. = 3.5 J/ m'. To elucidate the cause of this uniqu e s urvival response. we isolated a large number of in de- pendent clones from single colonies and measured their sponses to UV. Of 81 clones anal yzed, ten showed. marked resistance to killing by UV, which was only slightly more T he neredit3ry disease xeroderm.a pigmentosurn (X P), transmitted as an autosomal recessive trait, is charac- terized by hypersensitivi.ry of the skin ro sunlig ht and a high frequency of skin cancers [1,2]' I.n addition, some XP patienrs have progressive neurologic abnor- malities l2.31. The disorder comprises at least [line genetic comple- mentation groups, A- I. and a variant form [3.41. Cells from patients belonging to groups A - I arc more or less defective i.n excisjon repair of ultraviolt"t (UV)-induced pyrimidine dimers in DNA, whereas cells from the variant form are proficient in excision repair. All [h e XP ceU strains of groups A - I. as well as XP variant strains, are hypersensitive to various degrees to [he killing effect ofUV [3,4J . In l aran . nearly halfof the XP patients so far reported belong to group A 5J. Group A patients, except for a few cases: 13.6J. have severe and progressive cutaneous and neurologic abnormal.ities. Fibroblasts de- rived from these patients show the least DNA repair capacity. usu- ally Jess than a few percent of normal cells as measured by unsched- Manuscript' received February 7. 1989; accepted for publication May 11 . 1989. Thi s work. was supporred in part by a Grant-in-Aid for Ca ncer Research from the Mini .. try of EduCOIrion, Science and Culture. Japan . Reprint requeStS to : Dr. Mituo Ik e nag2 . Biology Center, KyOto Universiry. Yo.shidako IlO(-cho. Sakyo-ku. KyotO 606. Japan. Abbreviation s: Do: mean It' t hal dose RFLP; rt'srricrion fngment length polymorphism uos: unscheduled DNA UV: ulrnviolet XI': xeroderma pi gmenrosum sensi ti ve than normal cells, and the se clones had a rate of un sc heduled DNA syn th es is (UDS) tha, was .bout 45% of normal cells. By contrast. th e remaining 71 clones were ex- tremely sensiti ve to UV, typical ofXP group A strai n s, and had a UDS level 1 % - 3% of normals. An aly siS of restriction fragment length polymorphism using seven polymorphic DNA probes indicated that the UV-resistant clones were derived from the same individual as th e UV-sensitive clones. These results clearl y demonstrate that this patient's fibroblast cel Is consis t of twO types with differing responses [0 UV , and provide direct evidence of soma tic mosaicism for DN A repair capacity in an XP patient. J [,Wtsl Dermalol 93:460 - 465, 1989 ulcd DNA synthesis (UDS). and arc most se nsitivr to UV among the different groups of strains P,4] . In general. affected members within a given family show si milar skin and neurologic symptoms; their cells show rhe same race of UDS and belong to rhe same complemenration group [1.2,7.8j. This provides further support for the notion tbt the level of DNA repair capacity is inherited as a distinct genetic rrait. Although the def ecrive repair in XP cells has been studied exclusivdy in dermal fibroblasts. ocher types of cells examincd also exhibited a similar deft'c [ in UDS. These include epidermal cells {3.91 . peripherallym- phocyt es [2.3] . conjunctival and corneal cells [3J . smooch muscle cel ls 13j. and liver cells {IOj, indicating that probably all the cells in any XP patient have a similar defective capacity for DNA repair. Th e presence of a mixed cell population (mosaicism) has been recognized in culrured lymphocytes and fibroblasts derived from individuals with certain congenital anomalies [11]. ln fact. chromo- some mosaicism has long been known in lympho cytes fcom patients with Down' s sy ndrome [12J. Somatic mosaicism may r es ult from chromosomal Or gene mutacions during the course of embryonic, fetal . and postnatal life. JIl theory. such mosaicism could also occur in XP. hecausl': XP cdls arc hypermuublc no r o nl y by UV but also bY:1 certain class of environmen tal mutagens 113]. However, thus far there has been 110 conclusive evidence: for somatic mosaicism in this disease. Here we report a J apanese XP pa tient with mosaicism for DNA repair activity in tbe fibroblasts. The fibroblasts from this patient were assigned to complt'mentation group A. Th ey showed a unique biphasic UV survival curve, as measured by colony-forming abiliry. Measurements of UV sensitiviry (measured as dono genic survival) and UV-induccd UDS with independently isolated clonal cells 0022-202X!89!S03.50 Copy ri ght © 1989 by The Society for Ill vl':Srigative Dcrrmtology.lnc. 460

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Page 1: Somatic Mosaicism for DNA Repair Capacity in Fibroblasts ... · Somatic Mosaicism for DNA Repair Capacity in Fibroblasts Derived from a Group A Xeroderma Pigmentosum Patient Hae Ryong

Somatic Mosaicism for DNA Repair Capacity in Fibroblasts Derived from a Group A Xeroderma Pigmentosum Patient

Hae Ryong Chang, M.Sci., Kanji Ishizaki, Ph.D. , Masao S. Sasaki, Ph.D., Junya Toguchida, M.D. , Mitsuo Kato, M.Pharm., Yusuke Nakamura, M.D., Susumu Kawamura, M.D., Takahiko Moriguchi, M.D., and Mimo Ikenaga, Ph.D. R3di:nion Biology Ccnter. Kyoto Universiry (He. KI . MS. JT. MK. MI ), Kyoto. J;lp:m; Howard Hughes Mcdicallnsritutc, University of Utah (YN). Salt Lake Ciry, Utah; and Departmcnt of Plastic Surgery, Kawasaki Medic:!! School (SK. TM), Kurashi ki , Japan.

A female Japanese xeroderma pigmentosum (XP) patient wi th severe skin lesions and various neurologic abnormalities was assigned to complClnentation group A by conventional cell fusion studies. Ultraviolet (UV)-irradiated skin fibro­blasts showed a biphasic survival curve, as measured by col­ony-forming ability. The surviving fraction decreased rap­idly up to 2 J /m' ofUV, with a steep slope ofD. (mean lethal dose) = 0.95 J/ m'. At much higher doses it decreased more slowly, with D. = 3.5 J / m'. To elucidate the cause of this unique survival response. we isolated a large number of in de­pendent clones from single colonies and measured their re~ sponses to UV. Of 81 clones anal yzed, ten showed. marked resistance to killing by UV, which was only sligh tly more

T he neredit3ry disease xeroderm.a pigmentosurn (X P), transmitted as an autosomal recessive trait, is charac­terized by hypersensitivi.ry of the skin ro sunlight and a high frequency of skin cancers [1,2]' I.n addition, some XP patienrs have progressive neurologic abnor­

malities l2.31. The disorder comprises at least [line genetic comple­mentation groups, A - I. and a variant form [3.41. Cells from patients belonging to groups A - I arc more or less defective i.n excisjon repair of ultraviolt"t (UV)-induced pyrimidine dimers in DNA, whereas cells from the variant form are proficient in excision repair. All [he XP ceU strains of groups A - I. as well as XP variant strains, are hypersensitive to various degrees to [he killing effect ofUV [3,4J. In l aran. nearly halfof the XP patients so far reported belong to group A 5J. Group A patients, except for a few cases: 13.6J. have severe and progressive cutaneous and neurologic abnormal.ities. Fibroblasts de­rived from these patients show the least DNA repair capacity. usu­ally Jess than a few percent of normal cells as measured by unsched-

Manuscript' received February 7. 1989; accepted for publication May 11 . 1989.

This work. was supporred in part by a Grant-in-Aid for Cancer Research from the Mini .. try of EduCOIrion, Science and Culture. Japan.

Reprint requeStS to: Dr. Mituo Ikenag2 . ~diation Biology Center, KyOto Universiry. Yo.shidakoIlO(-cho. Sakyo-ku. KyotO 606. Japan.

Abbreviations: Do: mean It' thal dose RFLP; rt'srricrion fngment length polymorphism uos: unscheduled DNA syn{hesi~ UV: ulrnviolet XI': xeroderma pig menrosum

sensi ti ve than normal cells, and these clones had a rate of unscheduled DNA synthesis (UDS) tha, was .bout 45% of normal cells. By contrast. the remaining 7 1 clones w ere ex­tremely sensi ti ve to UV, typical ofXP group A strains, and had a UDS level 1 %- 3% of normals. AnalysiS of restriction fragment length polymorphism using seven polymorphic DNA probes indicated that the UV-resistant clones were derived from the same individual as the UV-sensitive clones. These results clearl y demonstrate that this patient's fibroblast cel Is consist of twO types with differing responses [0 UV , and provide direct evidence of somatic mosaicism for DN A repair capacity in an XP patient. J [,Wtsl Dermalol 93:460 - 465, 1989

ulcd DNA synthesis (UDS). and arc most sensitivr to UV among the different groups of strains P,4].

In general. affected members within a given family show similar skin and neurologic symptoms; thei r cells show rhe same race of UDS and belong to rhe same complemenration group [1.2,7.8j. This provides further support for the notion tbt the level of DNA repair capacity is inherited as a distinct genetic rrait. Although the defecrive repair in XP cells has been studied exclusivdy in dermal fibroblasts . ocher types of cells examincd also exhibited a similar deft'c[ in UDS. These include epidermal cells {3.91. peripherallym­phocytes [2.3]. conjunctival and corneal cells [3J. smooch muscle cel ls 13j. and liver cells {IOj, indicating that probably all the cells in any XP patient have a similar defective capacity for DNA repair.

The presence of a mixed cell population (mosaicism) has been recognized in culrured lymphocytes and fibroblasts derived from individuals with certain congenital anomalies [11]. ln fact. chromo­some mosaicism has long been known in lymphocytes fcom patients with Down's syndrome [12J. Somatic mosaic ism may result from chromosomal Or gene mutacions during the course of embryonic, fetal . and postnatal life. JIl theory. such mosaicism could also occur in XP. hecausl': XP cdls arc hypermuublc nor only by UV but also bY:1 certain class of environmental mutagens 113]. However, thus far there has been 110 conclusive evidence: for somatic mosaicism in this disease.

Here we report a J apanese XP patient with mosaicism for DNA repair activity in tbe fibroblasts. The fibroblasts from this patient were assigned to complt'mentation group A. They showed a unique biphasic UV survival curve, as measured by colony-forming abiliry. Measurements of UV sensitiviry (measured as dono genic survival) and UV-induccd UDS with independently isolated clonal cel ls re~

0022-202X!89!S03.50 Copyright © 1989 by The Society for Ill vl':Srigative Dcrrmtology.lnc.

460

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VOL. 93. NO. 4 OCTOBER 1989

vealed that the 6broblast strain consists of twO distinct types of cells. one with a rate of UDS about 45% of normal cdls and resistant to UV, and the other with 1%-3% UDS and extreme sensi ti vity to UV.

MATERIALS AND METHODS

Cell Strains and Culture Conditions A biopsy specimen of the patient XPIKU was taken from dernutologically unaffected skin of the right lateral thorax. Fibroblast cultures were prepared from the biopsy as described [6.14]. The following fibroblast strains served as references for the complementation analysis. Normal cells: N 170S [6j ' NIK.A [15J, C HFU [16J: complementation ~roup A: XP30S [8, XP330S, XP350S [6] , Xl'390S 16]: group C: GM3176(XPlAA) [1 7J: group D: GM5424(XPIPO) [171; group F: XPIOIOS [18]: group G: XP2BI [1 91; and group I: XP20MA [4J . The strains GM31 76 and GM5424 were kindly supplied by Dr. K. H. Kraemer, XPIOIOS by Dr. C. Nishigori , XP2BI by Dr. Y. Fujiwara, XP20MA by Dr. E. G. Jung (through Dt. S. Arase), and N1KA by Dr. M. Inoue. The rest (two normal and fou r group A XP strains) were initi ated in our laboratory.

All cells were grown in Dulbecco's modi6ed minimum essential medium supplemenred with 10% fetal calf serum (Hyclone Lab .. Logan. UT) and antibioti cs as described [6, 16}. C ultures were main­tained at 37°C in an atmosphere of 10% CO2 in air.

Complementation Analysis and Measurement of Unsched­uled DNA Synthesis The complementation group assignment of XPIKU cells followt'd conventional techniques as described [6.14.18J. Briefly, 5 X 10' cdls each of XPIKU and referencecells were mixed and seeded on an 18 X 18· mm coverslip which was placed in a 28-mm petri dish. The next day the cells were fused with 45% polyethylene glycol (#6000, Nakarai T esque, Kyoto. Japan). About 24 h after rhe treatment, the cells were irradiated with 20 J/ m2 0f254-nm UV, incubated with culture medium containing 10 /Lei/ml of[mcth yPHJthymidine (25 Ci/m.M: Amersham Interna­tional, Buckinghamshirc. UK) at 37°C for 3 h. and fu rther i_ncu­bated for 1 h in medium containing 5 /Lg/ ml of nonradioactive thymidine. Autoradiography was performed using Konica NR-M2 emulsion (Konica Co .. T okyo, Japan), and the samples were ex­posed for 2 wk in the dark at 4°C f6.9J . Finally. cells were stained with Giernsa solution and the numbers o f grains per nucleus were counted in lightly labeled binuclear cells. Unless Otherwise speci­fied. the mean grain count was determined by scoring 100 nucb per specimen.

Survival Assay of U1traviolet~lrradjated Cells Appropriate numbers of cel ls were inoculated into 6-cm dishes with 5 011 each of culture medium. After 14 - 16 h incubation, cd Is were w:l:shed once with pbosphate-buffered sa line and irradi:l:ted with UV ar a dose rate of 0.05,0.3. or 1.2 Jim' Is 16, 15J. The cells were then incubated for 2 wk, with medium changed twice a week, and colonies were counted after staining to determine surviving fractions [15.16J . Plating efficiency of unirradiared cells ranged from 8% - 30%. de· pending on the strains.

Isolation of Clonal Cells and Their Response to Ultraviolet Light A culture ofXPlKU cells that was frozen at passage 4 and stored in liquid nitrogen was thawed and grown co a logarithmic growth phase. The cells then were seeded sparsely into lO-em dishes. so that only a few colonies could emerge in a dish after about ten days of incubation. More than 100 independem clones were isolated from the arisen single colonies with use of a stainless steel cloning ring (6-0101 inner diameter) and grown into a mass. Ultravi­olet survival and the rate ofUV -induced unscheduled DNA synthe­sis (UDS) were analyzed with these clones as described.

Analysis of Re5triction Fragment Length Polymorphism High molecular weigh t DNA was isolated from about 10' fibrobla. .. t cells as described [20.21] . T he merhods for restriction enzyme di­gestion of DNA samples, agarose gel electrophoresis, Southern hy­bridization. labeling of probes by nick translation. and autoradiog­raphy were essentially the same as those described [20.2 1 J. Briefly. 5

SOMATI C MOSAI CISM IN XP .61

Jlg DNA was digested by restriction enzymes. subjected to electro­phoresis on a 0.8% or 1.2% agarose gel and transferred onto a nylon 6lter. Filters were hybridized with 32P_labeled DNA probes: after washing, they were exposed to X-ray 61m in the dark at -75°C with an inrensifying screen.

T he probes used were those homologous [0 various polymorphic human chromosomal DNA segments. Probes pTHH 59(0 17S4), pYNZ22(DI7S30), and cMCOC46(D I3S54) contain var1.bl< numbers of tandem repears and are thus highly polymorph.ic 122). Ptobcs of pIE8(DI3S4) and p7FI2(DI3SI) were generous gifts from Dr. W . K. Cavenee {23]' Probes for human c-Ha-ras(pHS49) and L-m)'{ (#6929) were obtained from Dr. Y. Yuasa and from the Japauese Cancer Research Resources Bank, respectively.

RESULTS

Clinical Features A 9-year~0Id Japanese girl (patient identi6ca­tion number XPl KU) exhibited severe photosensitivity. with ery­thema and blistering on minimum sun exposure, about 1 mo after birth. She gradually developed freckle-like pigmentation on sun­exposed areas. At the age of 4 yr, she was diagnosed as XP at another hospi tal. She was 6rst seen ar Kawasaki Medical School Hospita.l at age 7; at rhat time a basal cell carcinoma had already developed on her cheC'k. At [he age of 9 yr she developed several basal cell carci­nomas on the face; these were surgically excised. followed by graft­ing with skin that had received less sun exposure. BasaJ cell carci­nomas were also noted on the pal pebral conjunctiva of both eyes.

When we fim saw the patient at age 7, she al ready displayed a variety of neurologic abnormal.i ties. typical of De Sanctis-Cac· chione syndrome [2.3)' These included microcephaly. severe men­tal reta rdation. dumbness. spastic paralysis . ata,xia, areflexia of the patel lar lendons. Ach illes tendon shortening. and short stature . All th ese neurologic symptoms were more or less progressive. and phys­ical examination at 9 years of age revealed a height of 11 2 em and a weight" of 16.5 kg (both below the fi rsr percentile). Discussions with the patient's parents indicated that they had noticed several neurologic abnonnaliries beginning in the first years of her life, such as mjcrocephaly with mental deterioration and impaired motor development. She has never spoken any meaningful word from infancy. She- could walk alone. though with totrering gai t. at the age of 3: at tha r time shortening of righ t Achilles tendon became appar­ent. She has been attending a school for men tally retarded children.

The parents were not consanguineous. and there is no indication of XP within the family. induding her 16-yr-old elder brother and 14-yr-o ld elder sister.

Unscheduled DNA Synthesis and Complementation Analy­sis The amount of UDS in XPI KU cells irradiated with 20 J / m2 UV was about 8% of that in normal cells (Table I). The ra te ofU DS in this strain was slightly bur consistently greater than those of rypical group A XP strains observed in our rourine assay of UDS (usually 1%- 4% of nonnals; Table I). Furthermore, the XP1KU cells were much more resistant to killing by UV than other group A strains. as described later. Therefort". instead of using a single group A XP strain for complementation analysis. we fused XP1 KU cells with four differem reference group A strains.

Figure 1 shows the distribution of tht" number of grains per nu­cleus in fused binuclear cells. No complementation occurred in any combination berween XP I KU cells and any of the four XP strains belonging to group A. By contrast, when XP1KU cells were fused with cel ls of a representative strain from groups C. D, F. G, and I. .aboul half of the binuclear cells showed UDS levels in the range of normal cells (Fig 2). Although fusion with representarive strains of groups H. E. and H was not performed. the XP 1 KU was un.ambigu­ously assigned to complementarion group A.

UV Survival Figure 3 shows the surviv.al of UV -irradiated XPlKU cells,:I:S measured by colony-forming abil ity, in compari­son with those of the reference XP strains belonging to groups A. C , D, and F. The surv ival of ali XP and normal strains shown in this fi gure were determined in our laboratory. The XPtKU cells exhib­ited:l bipha. .. ic survival curve with rwo distinct slopes; the surviving

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462 CHANG lIT AL THE JOURNAL OF INVESTIGA"lVE DERMATOLOGY

Table L Cellular Characteristics of XP and Nonnal Strains Used in This Study

Complementation UDS Level· UV Killing Cell Strain Group (% of nortn:ll cells) D,O/m') Reference~

XP cells XPIKU A Exp. l 8.2 slope I ' 0.95 This paper

Exp.2 7.3 slope 2' 3.5 XP30S A 1.1 DAD T,k<be er aI [8J XP330S A 2.7 DAD Ikenav er a.1 [unpobll XP350S A 1.0 0.37 Sara er al 16j XP390S A 2.3 0.54 Sara er a.1 [6 GM3176 (XPtAA) C 21 0.76 Cleaver et 31 [17J GM5424 (XPt rO) D 51 0.32 Cleaver et al [J7J XPIOIOS F 20 2.1 Nishigori et :l11181 XP2BI G 4.8 Keijzer et a.l f 191 XP20MA I 19 Fischer er al 41

Nonm.l. cells NI70S 100 3.9 Sato et al [6] NIKA 100 4.5 lkenaga et al [15J CHFU 100 4.3 Tsujimura et al {16]

• Number of grains W~ measured over 100 nuclei for each str.lin. exct'pr for XPIKU strain where 200 nuclei wt're scored in t'ach ex~ri ment . ~ Referenct' for complemt'ntation group usign mem ofxp nroru. For normal cells. these refer 10 original descriptions. All the UDS levels and Do values listed in this Table were

determined by the presenT authono. , Slope I :,md slope 2 are those of UV·serniti.vt': ",nd UV-resistmt compone/l! in the survival curve. (Figure 3). respectively. Xl'; xerodenna pigmentosum: UDS; unscheduled DNA syntltesis: UV: ultraviol~t .

frac tion decreased rapidly wi th increase in UV dose to 2 J/ m2. and then decreased much more slowly beyond chis dose . At higher UV doses XPl KU cells were surprisingl y more resistant to UV than four other group A XP strains. The slope of the UV -resistant com­ponen t (Do = 3.5 11m2) was comparable with those of normal cells (Do = 3.9-4.5 J/m 2). If we extrapolate thLoi slope to the ordinate. the intercept on the axis gives a value of 15% -20%, suggesting that roughly one fifth of the cell population in the XP1 rev culture may have the UV -resistant phenotype.

Response of Clonal XP1KU Cells to UV We isolated more than 100 independent clones from different single colonies. Among them. 81 clones re tained enough growth capacity that their UV sensitivities could be measured. Figure 4 shows the UV survival

" " "

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,. ( a ) KPII(U 10) l PIKU • XJl'30S

,. " (ell llPl,\U . l(P390S I f ) JlPIIO,I • lCPJJOS

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NUMBER OF CRAINS PER NUCLEUS

(el XPIKU • XPJ~OS

I I ) XPJJOS, lCPJOS

,. • I

",.

Figure 1. Lack of complemen[3tion between XPI KU and reference grollp A XP str.ains. The test (X PIKU) and th~ reference cells wer~ hybridi2ed by treatment with polyethylene glycol and incubated for 24 h. Then rhe cells were irndi2ted with 20 J/m2 ofUV • labeled with [lHJthyruidine for 3 h. and proc(:sscd for .autoradiognphy with usc of KoniQ NR-M2 emulsion. as described in [he text. The numbers of gnins per nucleus in fused binuclear cells were 5Cor~d with 100 nuclei per S2mple. except for XP 1 K U mononu­cle<lr cells (a) in which grains were counted with 200 nuclei. For the sake of comp2rison, dat2 showing the distribution of numbers of gnins in hybrid cells Ix-tween two reference strains (XP30S <lnd XP330S) are 21se included (f) . Arroul shows the mean number of grains per nucleus of normal cells. measured on the nIne day.

curves obtained with some represenrarjve clones. The results of such survival assay demonstr.ued that aU these clones can be classified into two distinct gtOUps in terms o f UV sensitivity; ten clones ex­hibited a remarkable UV resistance which was only slightly more sensitive than normal cell s, whereas the remaining 71 clones showed the same high sensi tivity as rypical group A XP strains . The Do of these numerous sensiriveclones ranged from 0.33 - 0.51 J/m2,

although the values were not accurately determined with some clones because of their low plating efficiency. It should be nored that these UV-sensiti ve dones did not have any UV-resisranr com­ponent seen in the origi.nal XPIKU cuJture.

The amounts ofu V -induced UDS were measured with four each of [he UV-resistam and UV-sensitive clones; typical resuhs arC' shown in Figure 5. A UV -resistant clone, cl-2, had an UDS level of about 38% of normal cells, and a similar increased rate of UDS was also observed with other resistant clones (ranging from 37% - 55% of normals). By contrast, UDS levels of the UV-sensitivc clones wert: less than 3% of nonnal cel ls (1.0% -2.8%). similar to the reference group A XP strains. It is therefore clear thar the reduced

" " " ! ;o j ~PIKU. Q'PuP C (bl XPIK U ~ g.ouP 0 !cl .... P!IIU ~ !I'ou p F

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~ ~ g z ~ oc ~ • (d) lP1K\J • g.auo G If ) l PlllU. !I'pup I ~ z ()(P28ll (KPlOloU.l

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". NUMBER OF GRAINS PER NUCLEUS

Figure 2. Complementation analysis ofXP1KU stram. The XPIKU and refmnee [GM3176 (gmup C), GM5424 (D) , XPIOIOS (F) , XP2BI (G), XP20MA (1)] cells were hybridized. UV-irradiated . labeled with IJH1thy­rnidine. and sobjecred to autoradjographic an21ysis of UDS. in the same m.anner 2S described in the legend to Figure 1. The histogram of normal cells (f) represents the data with the CHFU smiD (mean. 76.6 gnins per nu· deus).

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VOL. 93. NO. 4 OCTOBER 1989

XPJOS <) .

Gt.l5424 )(PJ505

NIKA

o CHF U 0_____.... NI70 5

XPIKU

o XPI0105

0.' L-____ ~ ____ ~ ____ ~ ____ _L ____ _L ____ _L __ _

o 4" a 10 12

Figure 3. UV survival curves of XP 1 KU. XP reference strains and normal cells. Appropriate numbers of cells were inocul2ted into dishes. incubated for 14 - 16 h. and lcf2di2ted with diH"erent UV doses. The cells were further incubated for 2 wk. :md colonies were counted to detennine survivaJs. Each symbol denotes the geometric.al me2n of two to four independent experi­ments.

UV sensitivity of clones such as those shown in Figure 4 is due to their high DNA repair capacity.

Restriction Fragment Length Polym orphism Analysis in Isolated Clones To prove that the clones showing different UV sensitiv ity we.re derived from the same individual (XPl KU) , we analyzed restriction fragment length polymorphism (RFLP) in a UV -resistant clone, cl-2, and two sensitive clones. cl-3 and cl-14, by use of seven polymorphic DNA probes. As shown in T able II . RFLP analysis in these three clones revealed exactly the same genotype with all probes used . Based on the &equency of each genotype in the J apanese population (unpubLished data), the possibility thata norue­lated individual other than XP1KU might have this particular genotype is 1.5 X 10-4. This strongly suggests that the UV-resist­am clones in the XVl KU culture must not be caused by contamina­tion of some UV-resistant cells derived from a differenr individual .

DISCUSSION

A Japanese XP patient. XP1KU. who exhibits severe skin lesions and a150 various neurologic abnormalities, was assigned to comple­mentation group A by assaying UV -induced UDS in cultured skin fibroblasts. However, unlike typical group A XP strains. the XV I KU cel ls showed a markedly reduced sensitivity co ki.lling by UV, es pecially at higher UV doses. To account for this. we isolated morc than 100 independent clones from the original culture of XV1 KU and measured their UV sensitivities. The tesults revealed that the original XPl KU culture is made up of twO types of ceUs with distinct DNA repair phenotypes: one group of cells that are almost as resistant to UV as normal cells and with UDS levels about 45% of normal cells, and a second group of cells that are extremely UV sensitive and show only a small percentage afUDS, similar co representative group A XP srrains. The frequency of UV-resistant clones obtained, 1.2% (ten of 81 clones), agreed with the relative ratio of UV-resistant cells in the original culture (15%-20%), which was estimated from the survival curve by extrapolating the slope of the resistant component to the ordinate . The presence of a mixed cell population (mosaicism) has been recognized in cultured lymphocytes and fibroblasts derived from individuals with certain congenital anomal ies [11, 12,24]. To the hestof our knowledge, the present observation provides the first evidence for mo~~c~sm of fibroblasts in a single XP patient, although such a poSSibility has been suggested fo, gcoup A strains XP I LO [25J and XP6PR [26J on

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SOMATIC MOSAICISM IN XP 463

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~ "fZ.:',~::: -..... \ \

\ CI-3~CI_14

CI-2 3 ~CI-300

\

..... ~

0 .1 L-__ -L __ -L ____ L-__ --'-__ -L __ ~ __

a 4 a 12 UV DOSE (J/ m2)

Figure 4. UV survival curves of c1on21 cells. Eighry-one clones i$Obted from independent single colonies were analyzed for UV sensitivity by mea­suring colony-forming abi.lity. The resu lts of four represent:lrive UV-rCSls-r­:tnt clones (cl -2. c1-30S. c1~317. c1-333) and four UV-sensirive clones (c1-3. cl-14, cl-23. d-300) 2re showtL ElIch symbol represents rhe mean of at least two independent experiments. except for those of c1-23 . c1-30S, 2nd c1-317. which were determi.ned by single me.a.surement. Dort~d lil1t, Straight litle, and dash t:d line indicate surviv2l curves of norrmJ cdls. XP1KU. and XP3QS (grollp A), respectively, which were t2ken from those shown in Figure 3.

the basis of a biphasic UV surv ival curve and a heterogeneous distri­burion of UDS. respectivel y.

Four group A XP patients have been described whose skin fibro­hlasts exhibited decreased UV sensitiviry compared with the major­iry of gcoup A XP mains. These ace XPI2BE [2,27.28]' XPILO [27,29J, XP8LO [27,30J , and XP390s 16J. Among these "",ins, [he most UV-rcslstant strain. XP8L01. had a sensitivity similar to group F XP cells [3 ,27J, and showed a rate ofUDS rhatwas 36% of normal cell s [30]. The st:rai ru XPt2BE and XPl LO were about two to three rimes less sensitive to UV [han typical group A strains [27]. although their UDS levds were less than 2% of normaJ cells (29]. The XP390S strain also showed similar characteristics {Fig 3.

40

CI-2

20

N v u

A ~ c 0 ~ 0

~ 70 v ~

E ~ CI- 3

Z 40

N

! B

Number of grains per nucleus

Figure S. UDS in UV-resistant 2nd UV-scnsitive clones. Samples for 2utO­radiography were prepared in the ~me way as described in the lege~d to Figllrc I. except th2( the [Te2crnen! with polyethylene glycol was omitted. (ll) c1-2 (UV-resistantclone) and (6) c1-3 (UV-sensitiveclone). Arrowshow$ the me.1n numbl':.r of gr2ins in normal cells.

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·6. CHANG ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Table D. Analysis of Restriction Fragment Length Polymorphism in XPl KU Clones

Pro~

pTHH59 pYNZ22 cMCOC46 plE8 p7F12 pHS49 ~6929

• UV-rtsiJunt clone. l UV-sC'.nsltlvC' clonts.

Restriction Enzyme

TDq I TDq I Msp l M' p l Mspl M,p I/ HJ'" 11 Eto RI

Table I). Interestingly, all these exceptional group A patients showed mild skin lesions and had no (XP1 LO {3.27J and XP8LO [27.30]} or only mild (XP 12BE 12.28J and XP390S [6]) neurologic abnormalities. On the basis of these findings , Andrews et al [27] sugges ted that the. degree of neurologic abnorrnaliry in XP panehes is correlated with the UV sensitiviry of their fibroblases in culNre. T he case' ofXP lKU described herein is also in agreement with this observation, as this patient has severe neurologic symptoms and about 90% of the clones derived from her fibroblasts were as highly UV sensi tive as are most group A XP strains .

The real question is whether or not the heterogeneity in the fibroblast culture reflects somatic mosaicism in the patient. Very recently , a second biopsy specimen was taken &om skin of the left lareraJ thorax . an area amipod.11 [0 the first biopsy. UV survival of fibroblasts grown from this biopsy specimen again exhibited a simj­lar hiphasic curve (data not shown), indicating the presence o f UV­resistant cell s in this second XPI KU culture. This strongly suggests that somatic cdl mosaicism for UV sensitivity may exist in this patient's cells. at least in connective tissue of the derrn.is around her upper trunk . W e do not know whether or not these UV -resistant cells may also exist in other tissues. such as sun-exposed areas of skin and the central nervous system. The presence of mosaicism for a chromosomal abnormality in fibroblascs. but its absence in other tissues, has been reported for several cases of phenotypically abnor­mal individuals [11.24J.

Somatic mosaicism may result either from chro moso mal abnor­malities or from gene mutat io ns during pre- or postnatal life. One plausible explanatio n for the observed mosaicism in the XPl KU may be that a kind o f back mutadon to a UV -resistant phenotype occurred at a cenain stage, possibly during embryonic o r fetal devel­opment. C hromosome analysis revealed no structural anomalies de­tectable by the' Giemsa banding method , such as translocatio ns or deletions. in isolated clones of both UV-sensitive and UV-resistam cells. It is possible that the UV -resistant clones resulted from a gene mutation at the XP-A locus, leading to direct reversal of che original mutation in XPl KU . Alternatively , a mutation at another locus may have suppressed the repair defects caused by the original muta­tion . Cleaver et aJ [31J have isolated from a SV 40-transformed XP group A strain, XP 12RO(SV). a UV -resistanr revenant th:n showed normal resistance to killing by UV and about 50% of normal DNA repair capacity, as measured by repair replication . The unique DNA repair properties of this revertant were that it repaired UV -induced (6 - 4) photo products normally bur not pyrimidine dime rs. 1t will be interesting to measure the repair of (6-4) photoproducts. as well as of pyrimidine dimers, in the UV-resistant XPIKU clones.

Wt wish to tlltmlt Drs. K. H. Kratmt!f (Notionol Conetr InSlitlltt, NIH) , C. N ishigo'" (Kyoto Unillmiry) . Y. Fujiu10ra (KOM UniI1wiry), and E. C. Jung (Mo'mh~jm Mtaitlll School) jar thtir gtlltfOUS supply oj tht riftrtrtu XP SUllins. Wt Il/JO (Iltlllk DN. W. K. CQlltrlft (U"i'lfrsiry ojCincin"D(i ColftgtoJMtdilint). CHId

Y. YU/1S0 (Tokyo Medilaf o,ld Den/Ill V,I iI/miry) jor prollidi"g polymorphic DNA probes.

cl-2"

4.2/ 4.0 3.0/ 2.7 2.7/ 2.5

10.5/ 7.6 4.8/ 3.9 1.0/ 1.0 6.0/ 6.0

Genotype (fragment size in kilobases)

c1-3~

4.2/4.0 3.0/ 2.7 2.7/ 2.5

10.5/ 7.6 4.8/ 3.9 1.0/ 1.0 6.0/ 6.0

REFEREN C ES

4.2/ 4.0 3.0/2.7 2.7/ 2.5

10.5/ 7.6 4.8/3.9 1.0/ 1.0 6.0/ 6.0

1. Cle:aver JE. Bootsma 0 : Xeroderma pigmentosUln: Diochemjca l and genetic chanllcteristics. Annu Rev Ge net 9:19-38. 1975

2. Robbins JH. Knemer RH. Lutl.ner MA. PeHolf BW, Coon HG: Xerodenna pigmentosum: An inheritt'd disease with sun sensitiviry. multiple cuuntous neoplasms. and abnormal DNA repair. Ann In­tern Med 80:221 -248.1974

3, Kraemer KH. Sior H: Xeroderma pigmcntosum. Clin Dermarol 3,33-69. 1985

4. Fi scher E. Keijzcr W. Th ielmann HW . Popanda O. Bohnen E. Edler L. Jung EG. BOOfsma D: A ninth {'Qmplementation group in xero­derma plgmenl'osum. XPI. Mum Res 145:2 17 - 225, 1985

5. Taktbe H. Nishigori C. Satoh Y: GenClics and skin cancer of xern­derm:a pigmcnrosum in Japan . Jpn J Cancer Res 78: 11 35 - 1143. 1987

6, Sato K. W:a[:at:alli M. lkena ga M, Kozuk..:I T . Kit::mo Y. Yoshibw:a K. Mim:aki T. Abe J . Sugita T : Sensitivity to UV r:ldi.:ltion offibroblasts from aJ:apanese group A xeroderma pigmentosllln pacicni with mild neurologicalabnonnaliries. fir J DermatoI116:101 - 108. 1987

7. Kraemer KH . Coon HG , Petinga RA . .Barrett SF. Rahc: AE. Robbins JH: Genetic heterogeneity in xeroderm.:l pigmenrosum: Comple­mentation groups and thtir rdarionship to DNA repair rates. Proc Nat! Acad Sci USA 72:59 - 63. 1975

8. Takebe H, Miki Y. Kozub T . Furu )'.:anu J. Tanak.:a K. Sasaki MS. FujiwOlr:a Y, Akiba H: DNA repair chancteris t ic~ and skin canct'rs of xerodenna ptgmentosum p.:atients in Japan . Cancer Res 37:490 -495. 1977

9. Kondo S. Fukuro S. Mamad.:a A. Kawada A. Saloh Y. Fujiw.:Ir:I Y: A.~sigllmen[ of three patienrs with xeroderm:a pigmentosum to corn­plcment:nion group E and their characteristic!;. J Invest Dennatol 90:152- 157.1 988

10. Dupuy JM . Lafforet D. Rach l11 .:an F: Xeroderma pigmentosum with liver involvement. Hdv Paediatr Act:! 29:2 13-219. 1974

II . HaJl JG : Review and hypotheses: Somatic mosaicism: Observations related to clinical genetics. Am J Hum Genet 43:355 - 363. 1988

12. Nielsen KG. Poulsen H. Mikkelsen M. Steuber E: Multiple recurrC'.nce of trisomy 21 Down syndrome. Hum Genet 78:103-105. 1988

13. Maher VM. McCormick JJ: Effeci of DNA repair on the cytotoxicity .:and mutagen icity of UV irradiation and of chemical carcinogens in Ilormaland xeroderm.a pigmcntosum cells. ln .JM Yuhas. R W Ten­nant. J D Regan (cds). Biology of IOdiation Carcinogenesis. Raven Press. New York. 1976, pp 129-145

14. Sato K, !I.:.enaga M. $ano 5: Kinetic analysis of polyethy lene glycol-in­duced cell fusion in cultured human fibroblasts : Its appl ication to genetic complementation analysis of xerodemta pigmentosum. Med J Osaka Univ 33: 19-28.1982

15. Ike-naga M. Midorikawa M. Abt J . Mimaki T: The sensitivities to radiations and radiomimetic chel1lic ls of cells from patients with ataxia telangiectasia. Jpn J Hum Genet 28:1 - 10. 1983

16. Tsujimun T. Zhang Y. Fujio C. Chang HR. Watatani M. Ishizak.i K. Kit:amura H. Ikenaga M: O'-Methylguani ne methyltr:msferase ac­tivity and sensitivity ofJapancse tumor cell smins to 1-(4-amino-2-methyl-S-pyrimidinyl)methyl-3-(2-chlotoethyl)-3-nitrosouro hy­drochloride. Jpn J Cancer Res 78: 1207 - 1215. 1987

17. Cleaver JE: Rapid complementation method for chssifying excision repair-defective xeroderma pigment05um cell strains. Somatic Cell Genet 8:801-810. 1982

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VOL. 93. NO. 4 OCTOBER 1989

l B. Nish lgori C.lshizaki K, Takebe H , Imamura S. H ayakawa M : A case of xe roderma pigmentosum group F with late onset of clinical symp­tOms. Arch Dermatol 122;5 10 - 511, 1986

19. Keijzer W .J aspers NGJ, Abrahams PJ , T aylo r AMR, Arlett C F. Zelle S, Takebe H , Kinmom f'DS, Boorsma 0 : A se vemh complementa,. tion group in excisio n-deficient xerodcrm.a pigmentosum. Murat Res 62; 183- 190. 1979

20 . Ma niatis T , Fritsch EF. Sambrook J: Molecuhr C loning: A Laboratory Manual. New Yo rk. Cold Spring H arbor Lab. 1982

2 1. Honda K.lshizaki K. lkenaga M, ToguchidaJ, In.amotOT. T illl.;lka K. Ozawa K: Incrc.;lSed frequency of specific alleles of c- Ha-raJ gene in Japanese cancer patients. Hum Genet 79:297 - 300. 1988

22. Nakamura Y , Leppert M , O'Connell P, W ol ffR. Ho lm T. Culver M, Martin C, Fuj imoto E. HoffM, Kurnlin E, White R: Variable num­ber of tandem repeat (VNTR) markers for human gene mapping. Science 235;1616 - 1622.1987

23. C.avcnee W . ~ach R. Mohandas T . Pearson P. Wh ite R: Isolation and regiornlilocaliz.ation of DNA segments rf" veali ng polymorphic loci from human ch romosome 13. AmJ Hum Gener 36:10-24, 1984

24. Paga n RA. Hall JG. Davenport SLH. Aase J . Norwood TH. Hoehn HW; Abnormal skin fibroblast cytoge netics in (our dysmorphic pa[iclHs with normal lymphocyte chromosomes. Am J H um Genet 3 1:54-61. 1979

25. Andrews AD, Barrett SF. Robbins jH: ReI.ation of DNA repair pra-.

SOMATIC MOSAICISM IN XV 465

ccsses to pathological ageing of the nen'ous system in xeroderm.a pigmentosum. Lancet i;1318 - 1320. 1976

26. Stefanini M. Keijzcr W. Dalpra L. Elli R. Porro MN. Nicoletti B, N uzzo F: D ifferences in the levels of UV repair and in clinical symptoms in twO sibs affected by xeroderma pigmentosum. Hum Genet 54: 177 - 182. '1 980

27. Andrews AD. Barrett SF, Robbins JH; Xerodemu pigmenrosum neu­rological abnormalities correlate with colony-forming ability .after ultr:l,violer radiation. Proc N at! Ac.ad Sci USA 75:1984- 1988. 1978

28 . Robbins JH. Pol insky Rj , Moshell AN: Evidence rhar lack of deoxyri­bonucleic acid repair causes death of neurons in xeroderm.a pigmen. tosum. Ann NeuroI13:682 -684, 1983

29. Pctinga RA. Andrew$ AD. T arone RE. Robbins JH: Typical xero­derma pigmelllosu m complemell t;l, tion group A fibroblasts h:lVe de· tee.cable ultraviolet light-induced unscheduled DNA synthesis . Bic­chim .Biophy' Act:;! 479:400_ 41 0. ]977

30. de Weerd~K.astelein EA. Keijzer W . Sabour M. Parrington jM. Bootsm.a D: A xeroderma pigmencosum paric.1lI having a high resid­ual .acti ,,· iry of unscheduled DNA synthesis after U V is assigned to compit'll1c.ntation group A. Mutat Res 37:307 - 312. 1976

31. Cleaver JE. Cortes F, Lutu LH, Morgan WF, Pla yer AN, Mitchdl D L; Uniqut' DNA repair properties of ~ xcrodernu pigmenrosum r('vcm.nt. Mol Cdl Bioi 7:3353-3357, 1987