The Journal of Dermatology Vol. 17: 465-472, 1990

Defective DNA Repair in Cultured Melanocytes from Xeroderma Pigmentosum Patients Jun Yamaguchi, Akira Mamada, Seiji Kondo and Yoshiaki Satoh* Abstract The DNA repair of ultraviolet (UV)-induced damages in primary cultured melanocytes from xeroderma pigmentosum (XP) patients and normal subjects were studied by measuring unscheduled DNA synthesis (UDS) on autoradiographs, Melanocytes were cultured in aminimum essential medium (a-MEM) supplemented with 10% fetal calf serum (FCS), 12-0tetradecanoyl-phorbol-13 acetate (TPA), and geneticin. The levels of UDS in XP melanocytes were compared with those in normal melanocytes. In both normal and XP melanocytes, post-UV-UDS increased dose-dependently at doses of 5-10 11m2. XP melanocytes exhibited various levels of defect in DNA repair, depending on the type of XP. Melanocytes from XP-A patients displayed very low levels of UDS, only 6.2-8.4% that of the normal melanocytes. However, UDS values in melanoeytes from intermediate groups, XP-D, XP-E, and XP-F, were relatively high, 37.2-53.5% of the control in XP-D, 50.0-66.5% in XP-E, and 38.2-46.7% in XP-F, respectively. Melanocytes from XP-variant patients exhibited almost normal levels ofUDS, 87.7-91.6% ofthose from normal subjects.The levels ofUDS in XP melanocytes were very similar to those in fibroblasts isolated from the same specimens. Key words: melanocyte; unscheduled DNAsynthesis; xeroderma pigmentosum

Introduction Xeroderma pigmentosum (XP) is a rare autosomal recessive disease characterized by extreme sun-sensitivity, a high incidence of cutaneous neoplasms on sun exposed areas, and frequent neurological abnormalities (1). Among malignant cutaneous neoplasms, basal cell epitheliomas and squamous cell carcinomas are most frequent and malignant melanomas are slightly less common. In XP patients under 20 years of age, rates of cutaneous melanoma are increased more than 2000 times over the general population (2). Sunlight has been suspected to playa role in the etiology of Received April 17, 1990; accepted for publication June 18,1990. Department of Dermatology, School of Medicine, Tokyo Medical and Dental University, Tokyo,Japan. *Department of Dermatology, Dai-Ni Hospital, Tokyo Women's Medical College, Tokyo,Japan. Reprint requests to: Jun Yamaguchi, M.D., Department of Dermatology, School of Medicine, Tokyo Medical and Dental University, 5-45, Yushima l-chome, Bunkyo-ku, Tokyo 113,Japan.

cutaneous melanoma. Fibroblasts from XP patients exhibit defective DNA repair of ultraviolet (UV)-induced damage (3). It is a current hypothesis that the DNA repair defect is etiologically significant in predisposing to cutaneous malignancies in XP patients (4, 5). Keratinocytes are a targetforUV carcinogenesis of the skin. We previously reported that epidermal keratinocytes from XP patients have a defective DNA repair (6, 7), and that the repair deficiency in XP keratinocytes is well correlated with the age of onset of skin tumors. In this connection, we measured post-UVunscheduled DNA synthesis (DDS) in primary cultured melanocytes from XP patients, the levels of which were compared with those from normal subjects. We found that cultured melanocytes from XP patients had various levels of post-UV-UDS defect, depending on the type of XP. Materials and Methods Cell Sources

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Human primary cultured melanocytes were obtained from the skin of 9 XP patients [XP group A: XP29TO (15-yr-old, female), XP63TO (18-yr-old, female), XP94TO (2-yr-old, male); XP group D: XP58TO (12-yr-old, male) (8), XP85TO (38-yr-old, female); XP group E: XP80TO (52-yr-old, female) (9); XP group F: XP92TO (41-yr-old, female) (10); XP variant: XP50TO (44-yr-old, female), XP91TO (36-yr-old, male)], and 16 normal subjects aged between 2 and 61 years. The frozen stocks of fibroblasts from the same XP patients in the 5th-7th passage were used for comparison. A normal fibroblast strain, SFN5 (authors), was isolated from a 25year-old woman. The types of XP were determined separately using fibroblasts by the complementation test of fused heterodikaryons. CultureofMelanocytes Melanocytes were isolated and cultured as described by Eisinger and Marko (11), with some modifications. Small skin samples (about 1 em") were obtained from the elbows ofXP patients; those of normal subjects were remainders of surgical specimens. These skin samples were cleaned of connective tissue, washed in phosphate-buffered saline (PBS), and floated overnight in 0.25% trypsin at 4°C. The epidermis was then removed from the dermis with forceps and shaken in 0.02% ethylenediamine tetraacetic acid (EDTA). Detached cells were collected by centrifugation. These cells were suspended in 2 ml of a-minimum essential medium (a-MEM) supplemented with 10% fetal calf serum (FCS, Hyclone), 10 ng/mI12-0-tetradecanoyl-phorbol-13 acetate (TPA), and 100 ,ug/ml geneticin (G5018, SIGMA). The cell suspension was divided into eight equal parts, and each (250 ,ul) was plated on a round glass cover slip (15 mm in diameter) in a 35-mm plastic dish without medium, then incubated at 37°C in 5% CO 2, After 2 days, 2 ml medium was added, and the dish was filled with medium. The medium was changed twice a week. At 1 month after cultivation, UV irradition was carried out. CultureofFibroblasts Frozen stocks of fibroblasts isolated from the same patients and SFN-5, used as a control, were thawed, and each fibroblast was seeded in a 60-mm plastic dish and grown in a-MEM supplemented with 10% FCS at 37°C in an atmosphere of 5% CO 2 in air until it reached confluence in a monolayer culture. Fibroblasts were transfered by tripsinization

and seeded on a cover slip placed in a 35-mm plastic dish at a concentration of 105 cells/dish. UV irradiation was carried out on day 2 after seeding. Measurement of UDS The cells growing on cover slips were washed once with PBS and UV irradiated at 254 nm from a 15-W germicidal lamp at room temperature in the absence of covering medium. UV doses were determined with a UVR-254 UV RADIOMETER (Tokyo Kogaku Kikai Co., Tokyo, Japan). The exposure times were 10-40 seconds at a fluence rate of 0.5 Jlm 2/sec. The UV-irradiated cells were then incubated for 3 h in medium containing 5 ,uCi/ml [methyPH] thymidine (spec. act. 25 Ci/mmol, from Amersham International, Bucks, U.K), washed 3 times with PBS and fixed with a mixture of methanol and acetic acid (3:1 v/v) for 10 min. The acid-soluble fraction was removed by treatment with 3 changes of cold 5% trichloroacetic acid; then the cover slips were mounted on slides. The labeled cells were processed for autoradiography using NR-M2 emulsion (Konica Corp., Tokyo, Japan) and exposure in darkness at 4°C for 7 days. After development, the cells were lightly stained with 2% Giemsa solution. Numbers of silver grains on 30-50 lightly labeled, non-S-phase nuclei were counted in fields selected at random under a light microscope with a x100 oil-immersion objective. The average number of grains per cell corrected for the background (0.6-1.3 grains/nuclei) was calculated for a measure ofUDS. Immunofluorescence Staining ofMelanocytes Melanocytes were identified by the presence of tyrosinase in cells, as detected by indirect immunofluorescence using TMH-2 (rat monoclonal antibody against tyrosinase) (12), and fluorescein isothiocyanate (Fl'I'Cj-conjugated second antibody directed to the monoclonal antibody.

Results

Identification ofMelanocytes Both melanocytes and keratinocytes coexisted in the early stage of cultivation. Melanocytes were distinguished from keratinocytes by their morphological characteristics: keratinocytes had a round shape, while cultured melanocytes assumed a bipolar or polydendritic shape (Fig. 1). These dendritic cells were identified

UDS in XP MeIanocytes

467

Fig. 1. Cultured melanocytes from a normal subject show dendritic shape morphologically. Rounded keratinocytes also can be seen (xlOO).

with melanocytes by stammg strongly with monoclonal antibody to tyrosinase (Fig. 2). Influence of TPA To determine any possible effect ofTPA on UDS, we first measured UDS in melanocytes grown in the presence and absence of TPA. Melanocytes were cultured under two different conditions. Although the addition of TPA was essential for growth and differentiation of melanocytes, a limited number of melanocytes were available in a short-term culture within one week, in the absence of TPA, from the beginning of the cultivation. DDS in melanocytes from 3 normal subjects was measured in the presence and absence of 10 ng/ml of TPA for one week. As seen in Table 1, post-tN-UDS increased dose-dependently, and there was no difference between the presence and absence of TPA, indicating

that TPA did not have any influence on DDS. UDS in Melanocytes from Normal Subjects Table 2 lists cultures with the ages of the donors, their sexes, and the sites from which skin samples were obtained. Post-tN-DDS increased dose-dependently in the dose range of 5-20 ]/m2 (Fig. 3). The mean grain counts and standard deviations for 16 cultures were 24.4 ± 4.5 at 5 ]/m2, 33.4 ± 5.1 at 10 ]/m2 , and 41.3 ± 4.9 at 20]/m2 • UDS in Melanocytes from XP Patients Post-tN-UDS increased in XP melanocytes dose-dependently, but its levels were found to depend greatly on the type of XP (Table 3 and Fig. 3). In XP-A, which represents a severe defect in excision repair, the levels of DDS in melanocytes were very low, only 6.2-8.4% of those in normal melanocytes; i.e., 1.4-1.8 grains/nucleus at 5 1/m2 and 1.7-2.5 at 10

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Fig. 2. Indirect immunofluorescence staining ofmelanocytes (xlOO). Table 1. Influence ofTPAon UDS in cultured melanocytes from normal subjects Subject

Sex

Age

TPAa

1.

m

2

2.

m

39

3.

m

39

(-) (+) (-) (+) (-) (+)

a b

UDS (grains/nucleus) SJlm2 b

24.9±2.8 22.7±4.9 21.3±3.6 22.1±4.4 23.7±2.9 22.8±3.1

lOJ/m 2

20Jlm2

32.9±4.5 32.0±4.3 31.3±3.7 31.0±5.9 29.8±4.0 28.7±3.4

40.8±S.O 41.6±S.1 40.3±4.2 40.S±7.4 37.0±6.2 38.9±6.3

(+): TPA was added. (-): TPA was not added. Mean ± S.D. for 30-S0 cells, corrected for the background.

Jlm 2 • On the other hand, melanocytes isolated from XP-D, XP-E, and XP-F displayed relatively high levels of residual DNA repair. In the two cases of XP-D, UDS was 8.3-13.2 grains/nucleus at 5 J/m 2 and 14.2-18.2 at 10 j/m 2 , which represented 37.2-53.5% of that of the normal melanocytes. In the case ofXP-E, UDS was 13.7 at 5 J/m 2 and 17.0 at 10 J/m 2 , which represented 50.0-66.5%. In the case ofXP-F, the data were 18.2 at 5 j/m 2 and 18.9 at 10 J/m 2 , which represented 38.2-46.7%. In the two cases ofXPvariant, DDS was 87.7-91.6% of that in normal

cells; i.e., 19.7-25.9 grainslnucleus at 5 J/m 2 and 27.1 at 10 j/m 2 • UDS in Fibroblasts from XP Patients The levels of UDS in fibroblasts were also measured for comparison with those in melanocytes. The levels of UDS in fibroblasts were measured using frozen stocks of previously isolated dermal fibroblasts. In the three cases ofXP-A, the levels ofUDS in their fibroblasts were 1.9-4.4% of those in normal controls. In the two cases ofXP-D, UDS was 30.1-35.5%. In XP-E, UDS in fibroblasts was

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Table 2. UDS in melanocytes from normal subjects Subject

Sex

Age

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

m m m m m m f f f f f m f m f f

2 39 39 20 26 31 33 8 17 18 23 29 40 60 61 21

Site thigh thigh thigh axilla thigh forearm breast foot upper arm face shoulder breast back thigh back hand

Average a

20Jlm 2

23.9±4.9a 22.3±4.0 22.0±3.6 21.0±2.2 22.3±5.6 24.9±4.1 21.5±4.1 22.7±3.1 38.9±8.1 22.1±6.9 21.2±3.3 25.0±3.3 26.l±4.6 20.6±4.4 30.7±4.9 24.9±3.5

31.3±7.4 31.8±4.8 28.1±3.3 27.5±4.0 33.4±5.5 33.3±6.8 30.9±6.3 31.8±4.6 49.5±9.9 31.7±7.2 30.1±3.9 34.0±5.8 34.6±2.8 34.0±9.0 39.6±11.0

41.8±6.6

24.4±4.5

33.4±5.l

36.8±5.5 37.8±8.5 41.5±7.4 38.3±13.0 51.4±8.7

41.3±4.9

33.2-42.0% and in XP-F, UDS was 18.0-20.0%. In the two cases of XP-variant, UDS in fibroblasts was 79.8-99.6% (Table 3). These values were very similar to those in melanocytes isolated from the same subjects. Figure 4 shows results on UDS in pairs of melanoeytes and frozen stocks of fibroblasts isolated from the same specimens after UV irradiation at 5 Jlm2• In 4 XP patients (XP50TO, XP80TO, XP85TO, XP91TO), UDS in keratinocytes was also previously reported by Kondo et al. (7). The level of UDS in XP cells was expressed as a percentage of that in normal cells.

50

!Jl

lOJlm 2

Mean ± S.D. for 30-50 cells, corrected for the background.

60

c 'iii

UDS (grains/nucleus)

5J1m 2

40

.. 15 30 ..