Rodent complementation group 8 (ERCC8) corresponds to Cockayne syndrome complementation group A

DNA Repair

ELSEVIER

Mutation Research 362 ( 1996) 167- I71

Rodent complementation group 8 ( ERCC8) corresponds to Cockayne syndrome complementation group A Toshiki Itoh a, Tadahiro Shiomi b, Naoko Shiomi b, Yoshinobu Harada b, Mitsuo Wakasugi ‘, Tsukasa Matsunaga ‘, Osamu Nikaido ‘, Errol C. Friedberg d, Masaru Y amaizumi ‘** ” Dcyutment

of Cell Gertetics. lrr.stittrte ofMolectrlar

Emhrwlog~

and Gertetics, Kumanroto

Kumumoto h Dit,isiott

qf Genetics. National

L Dil,ision ofRadiation ’ Lahoratotyv

of Molecular

Pathology.

Biology.

Institute Far@

Departmertt

of Radiological

Uniwrsity

School qf Medicirzr.

Kuttonji

4-24. I.

862, Japan Sciences. 4-V I Attagawa

of Pharmaceutical

Science. Kana:awct

of Patho1og.v. Uttirersity

Received 29 May 1995; revised 7 September

of Te.xas Southwestern 1995: accepted

Itzageku, Chiha 263, Japan

Unit,ersit_v, Kanazawa Medical

13 September

920. Japan

Center. Dullas,

7X 75235, USA

1995

Abstract US31 is a UV-sensitive mutant cell line (rodent complementation group 8) derived from a mouse T cell line L5178Y. We analyzed removal kinetics for UV-induced cyclobutane pyrimidine dimers and (6-4) photoproducts in US31 cells using monoclonal antibodies against these photoproducts. While nearly all (6-4) photoproducts were repaired within 6 h after UV-irradiation, more than 70% of cyclobutane pyrimidine dimers remained unrepaired even 24 h after UV-irradiation. These kinetics resembled those of Cockayne syndrome (CS) cells. Since US31 cells had a low efficiency of cell fusion and transfection, which hampered both complementation tests and gene cloning, we constructed fibroblastic complementation group 8 cell line 6L1030 by fusion of US3 1 cells with X-irradiated normal mouse fibroblastic LTA cells. Complementation tests by cell fusion and transfection using 6L1030 cells revealed that rodent complementation group 8 corresponded to CS complementation group A. Keyords:

Cockayne

syndrome:

Xeroderma

pigmentosum:

Thymine dimer: (6-4) photoproduct;

1. Introduction UV induces two major types of DNA lesions. cyclobutane pyrimidine dimers and (6-4) photoproducts, both of which play roles in the biological consequences of UV exposure such as cell death, mutagenesis and carcinogenesis (Cleaver and Krae-

* Corresponding 364-3554.

author.

Tel.: (81-96)

373-5343:

Fax: (81-96)

0921-8777/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 092 1-8777(95)00046-I

Complementation

test

mer, 1995). Nucleotide excision repair is the major DNA repair system which removes these lesions from damaged DNA. Cells from patients suffering from xeroderma pigmentosum (XP) or Cockayne syndrome (CS) are hypersensitive to UV-irradiation due to defects in nucleotide excision repair (Cleaver and Kraemer. 1995). Using the cell fusion techniques, excision repair-defective XP cells have been divided into seven genetic complementation groups (A through G) (Kraemer and Lee, 1987; Cleaver and Kraemer, 1995), whereas in CS two complementa-

168

T. Itoh rt (11./Mutation

bon groups (A and B) have been identified (Tanaka et al., 198 1; Lehmann, 1982). In addition to these human mutant cells, large numbers of UV-sensitive repair-deficient mutants have been isolated from established rodent (Chinese hamster or mouse) cell lines. These UV-sensitive mutants have been divided into at least 11 genetic complementation groups (Thompson et al., 1981, 1988; Shiomi et al., 1982; Stefanini et al., 1991; Riboni et al., 1992). Six human genes which complement the defects in rodent complementation groups 1, 2, 3, 4, 5 and 6 have been cloned and designated ERCC (Excision Repair Cross Complementing) 1 (Westerveld et al., 1984). ERCC2 (Weber et al., 1988), ERCC3 (Weeda et al., 1990b). ERCC4 (Thompson et al., 19941, ERCCS (Mudgett and Maclnnes, 19901 and ERCC6 (Troelstra et al.. 19901, respectively. These human genes (except ERCCl) complement defects in different complementation groups of nucleotide excision repairdeficient human disorders. ERCC2 is involved in XP complementation group D (XPD) (Flejter et al., 19921. ERCC3 in XPB (Weeda et al.. 1990a), ERCC4 in XPF (Park and Sancar, 19941, ERCCS in XPG (O’Donovan and Wood. 1993; Shiomi et al., 1994) and ERCC6 in CSB (Troelstra et al., 1992). The mouse UV-sensitive mutant US31 is the sole member of rodent complementation group 8 (Shiomi et al., 1982; Thompson et al.. 1988), and the human gene which complements the defects in US31 (ERCC8) has not been characterized. In this study, we demonstrated that US31 cells had removal kinetics of UV-induced cyclobutane pyrimidine dimers and (6-4) photoproducts which were similar to those of US46 cells (rodent complementation group 6/CSB) (Troelstra et al., 19921, and that rodent complementation group 8 corresponded to CSA demonstrated by complementation tests using cell fusion and transfection.

2. Materials

and methods

Research 362 (19961 167-I

74

son et al.. 19881, respectively, Ltkkaprt(LTA) (a thymidine kinase- and adenine phosphoribosyltransferase-deficient clone of mouse L cells), and hybrid clone 6L 1030 constructed from LTA and US3 1 cells. CS2SE, Mps 1 and Npsl cells were assigned to Cockayne syndrome complementation group A (CSA), CSA and xeroderma pigmentosum complementation group F (XPF), respectively, in our laboratory (Itoh et al., 1994). CSlMO cells (CSB) were purchased from the Japanese Cancer Research Resources Bank (Tokyo, Japan). WI38VA 13 cells were a wild-type of transfectant. All cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; ICN, USA) supplemented with 10% fetal bovine serum (Gibco BRL. USA), 100 pg/ml streptomycin and 100 units/ml penicillin (1 OF-DMEM). 2.2. Isolation of UV-sensitire

partial h_vbrid clones

6-Thioguanine-resistant (TG ‘1 clones of US3 1 were isolated by plating US3 1 cells (l-5 X 10” cells/bO-mm dish) in 0.3% soft agar medium containing 5 pg/ml of 6TG. After lo-14 days of incubation at 37°C surviving colonies were isolated and checked for their sensitivity to HAT medium (lop5 M amethopterin, 4 X lo-” M thymidine and lo-’ M hypoxanthine). To isolate partial hybrid clones by directional chromosome segregation (Pontecorvo, 19711, LTA cells cultured for 24 h at 37°C after X-irradiation (lo-25 Gy) were mixed with nonirradiated US31 (TG’) cells in a 1 : 1 ratio ( lo7 cells of each cell line). These mixed cells were treated with 50% poly(ethylene glycol) (PEG) 1500 Germany) and plated in (Boehringer-Mannheim, HAT medium. Surviving hybrid clones (putative partial hybrids) were isolated with cloning cylinders and their UV-sensitivity was checked by the cell suspension spotting method (Shiomi et al., 1982). One UV-sensitive partial hybrid clone named 6LlO30 was isolated and assigned to the same complementation group as US31 cells by the method reported elsewhere (Shiomi et al.. 1988).

2. I. Cells and culture conditions The cell lines used in this study were mouse L5 178Y, its UV-sensitive mutant derivatives US 17, US3 1 and US46 which belong to rodent complementation group 5. 8 and 6 (Shiomi et al., 1982; Thomp-

2.3. Measurement of thymine photoproducts by ELBA

dimers

and

(6-4)

Direct binding of monoclonal antibodies to thymine dimers or (6-4) photoproducts was mea-

T. ltoh et al. /Mutation

sured by ELISA as described by Mizuno et al. ( 1991). Briefly, DNA was extracted from UV-irradiated (20 J/m’) or unirradiated cells at various time intervals after irradiation. The extracted DNA was sonicated (TOMY, Tokyo), denatured and applied to 1% protamine sulfate-pretreated polyvinyl chloride microtiter plates. TDM2 or 64M2 was used as the first antibody and biotinylated F(ab)’ fragment of goat anti-mouse IgG (H + L) (Zymed Laboratories, San Francisco, CA) was used as the second antibody. After incubation with streptavidin-peroxidase conjugate (Zymed Laboratories). samples were incubated in a substrate solution containing 0.04% n-phenylene diamine and 0.007% HT02. After 30 min incubation at 37°C. 2 M H:SO, was added to stop the reaction, and absorbance at 490 nm was measured with an Immuno Reader NJ-2000 (Japan Spectroscopic Co., Tokyo, Japan). The mean values of three wells were calculated and values obtained from unirradiated samples were subtracted as background. 2.4. Cell fusion complementation

test

Cell fusion was performed on a small scale as described elsewhere (Itoh et al., 1994). Briefly, l-2 X 10” of 6L1030 cells, l-2 X lo4 reference XP or CS cells, 400 HAU of UV-irradiated Sendai virus and 45 ~1 of BSS-Ca (137 mM NaC1/5.4 mM KC1/0.44 mM KH,PO,/O.34 mM Na,HPO,/l mM CaCl?/ 13 mM Tris-HCl, pH 7.6) in a total volume of 53 ~1, were mixed in 2.0 ml microcentrifuge tubes for 5 min at 4°C and then incubated for IO- 15 min at 37°C with vigorous shaking. After adding 70 ,ul of IOF-DMEM. the cells were centrifuged, and the resultant cell pellet was resuspended in 70 ~1 of IOF-DMEM. To facilitate judging the results of the complementation test, 20 ~1 aliquots of the fused cell suspensions were plated with micropipettes in the center of coverslips (18 X 18 mm) in 30 mm dishes and 20 ~1 of parental cells were plated on each side of the fused cells. The dishes were incubated for 2-4 h in a CO, incubator, then 2 ml of IOF-DMEM was added gently, followed by incubation for 20 h. Recovery of RNA synthesis (RRS) after UV-irradiation was measured as follows. The coverslips on which fused cells were plated were washed with phosphate-buffered saline (PBS) and irradiated with UV (254 nm) at a dose of 15 J/m’.

Research 362 (19961 167-I 74

169

At this time, half of the coverslip was covered with a small metal plate to protect the cells from UV-irradiation. After irradiation, the cells were incubated for 23 h in the culture medium and then labeled for 1 h in a medium containing [ 3H]uridine ( 100 @/ml). Coverslips were mounted on glass slides, dipped in Kodak NTB-3 emulsion, and exposed for 24 to 48 h at 4°C. Grains in UV-irradiated cells stained with Giemsa were counted under a microscope. 2.5. DNA transfection

arld selection of transformants

One day before DNA transfection 5 X lo5 of 6L1030 cells were seeded into 60-mm dishes. Transfection was carried out using lipofectamine (Gibco BRL. USA), essentially as described by the manufacturer. Plasmid ~98.2 (Peterson and Legerski, 1991) was the generous gift of B. Sugden and are described in Yates et al. (1985). Plasmid pCSA5 was an Epstein-Barr virus-based expression vector (pEBS7; Peterson and Legerski, 1991) containing CSA cDNA fragment (Henning et al.. 1995). 6 pg of ~98.2 and 24 pg of lipofectamine were separately added to Opti-MEM I (Gibco BRL, USA); thereafter, the two were mixed and left at room temperature for 30 min. Cells were washed with phosphate-buffered saline (PBS) before addition of the Opti-MEM-DNA-lipofectamine mixture. Cells were incubated for 5 h; thereafter equal volume of IOF-DMEM was added. Medium was refreshed with lOF-DMEM after 24 h. Cells were trypsinized (and, depending on cell density, divided over more dishes) and placed on selection medium containing G418 (concentration 600 pg/ml) 72 h after transfection. Colonies, designated 6L1030EB, were isolated and grown into mass cultures. Subsequently, pCSA5 was transfected into 6L1030EB cells as described above and selected by the medium containing 150 pg/ml of hygromycin B. Colonies were isolated and grown mass cultures. 2.6. UV sun:kil

assay

Colony-forming assay was measured by the method described elsewhere (Itoh et al., 1994). Briefly, appropriate numbers of cells were inoculated onto 60-mm dishes and left to attach for 10 h. Subsequently, cell were rinsed with PBS and exposed to UV-light (254 nm) at a fluence rate of 0.7

170

T. ltoh rt d/Mutation

J/m’/s. 10 d after UV-irradiation, colonies were fixed with 80% methanol and stained with Giemsa.

Resrarch 362 (lYY6i

167-174

normal. To overcome this difficulty, we adopted recovery of RNA synthesis (RRS) after UV-irradiation. which is severely depressed in US3 1 cells (data

3. Results 3.1. RemoL,af kinetics for two major UV-induced lesions in mouse W-sensitive US31 cells To compare the repair phenotype of mouse UVsensitive US3 1 cells (rodent complementation group 8) with other UV-sensitive cells, we measured kinetics of removal of cyclobutane pyrimidine dimers and (6-4) photoproducts in three mouse UV-sensitive mutant cells, US 17 (group 51, US46 (group 6) and US31 (group 81, using ELISA coupled with specific monoclonal antibodies against these two DNA lesions. As shown in Fig. la, L5178Y (normal cell line). US46 and US31 cells showed removal of almost all (6-4) photoproducts within 6 h after UVirradiation, while in US17 cells about 80% of (6-4) photoproducts remained even after 12 h. Thus, removal of (6-4) photoproducts is defective in US 17 cells but not in US46 or US31 cells. Repair of cyclobutane pyrimidine dimers is less efficient than removal of (6-4) photoproducts even in parental L5178Y cells. More than 30% of cyclobutane pyrimidine dimers remained in L5178Y cells 24 h after UV-irradiation (Fig. lb). However, in US17, US31 and US46 cells. more than 70% of cyclobutane pyrimidine dimers remained unrepaired after 24 h of incubation. Thus, all three mutants were defective in repair of cyclobutane pyrimidine dimers. Since the repair kinetics of US3 1 cells were similar to those of US46 cells (group6/CSBl (Troelstra et al.. 1992) but not to those of US17 (groupS/XPG) (O’Donovan and Wood, 1993; Shiomi et al., 19941, it was suggested that US31 cells were mouse counterparts of CS cells.

0

12

18

24

Repair time (h) 120 y

80

60

01 0

3.2. Complementation transfectiorz

6

I

6

12

18

24

tests by cell fusion and

We performed complementation tests by cell fusion between US31 and some XP or CS cells. Cell fusion complementation tests measuring unscheduled DNA synthesis (UDS) as a cellular marker are difficult with CS cells since UDS levels of them are

Repair time (h) Fig. 1. Removal kinetics of (6-4) photoproducts (a) and pyrimidine dimers (b) in L5 178Y ( v ).US 17 (A ),US3 I (0 1,and US46 (0) after UV-irradiation. After cells were irradiated with UV at a dose of 20 J/m’. DNA was extracted after various periods of incubation and the photoproducts were assayed by ELBA using specific antibodies. Points represent mean values of three independent preparations: bars indicate SEM.

T. Itoh et 01. /Mutatim Table I Complementation

between 6L1030 and mouse UV-sensitive

Fused

Experiment Standard

no.

line

lJS17TORh

No.

cell

of surviving

after

group)

clones

No.

of

hybrid

(6)/6L1030

SC)

I32

24

I06

US3lTOR

(8)/6L1030

0

04

US17TOR

(5)/6L1030

31

NDc

US46TOR

(6,/6L

US31TOR

(8)/6LlO30

‘I 0

hz> hlj

1030

not shown), as a marker for complementation tests (Itoh et al., 1994). However, US31 cells were unsuitable for complementation tests for two reasons: (1) they grow in suspension, and (2) fusion efficiency by Sendai virus is very low. Thus, we constructed a

were plated on 0.5% soft agar plates

cell

Cell type

RRS

Complementation

(grains/nucleus+SEiLI)”

CSZSE(CSA)/6L1030

Mps I (CSA)/6L1(130

CSlb10(CSB)/6L1030

Yppsl(XPF)/6LIO30

CS2SEICS

I MO

Mps I /cs I MO

heterokaryons CSZSE 6LlO30 heteroknryons Mps I 6L1030 heterokaryons

21.3k1.8 65.Ok3.4

CSI>lO

9.w

6LlO30 heterokaryons

16.9+1 .9 J7.0&1.7

1Npsl

18.7kl.3 8.6kO.c) 1s.s+1.4 23.911.7

8. Ii-O.6

10.2+0.8

I ,o

6L1030

15.1+1

her-rokaryons

25.2?2.0

CS?SE

ll.lil.7 3.650.5 24.7fl. 7.620.7

CSlMO heterokaryons Mps

I

CSlMO

6.7kO.6

.6

I 1 1 1 1

P>O. I Ob

P>o. 10”

P
and reference

cells were compared

Ih

P
+ T t

P
I

i 1

P
’ Data are means + SEM of 50 determinations. a Each pair of heterokaryons

( 100mm).

UV-sensitive partial hybrid termed 6L 1030 by fusion of US31 cells with X-irradiated normal mouse fibroblast line LTA; 6L1030 cells had a fibroblastic shape and were as sensitive to UV as the parental US31 cells. and were assigned to complementation

test using 61~1030 and reference cells.

Fused

clones

(HAT’oua’)

line

’ UV selection: Five cycles of UV radiation (5 J/m’ ) were carried out. ’ TOR, TG and ouabain-bouble resistant. ’ ND, not determined. Note: 6L1030 (10’ cells) and IJV-sensitive standard lines (IOh cells) treated with PEG1500

Table 2 Complementation

171

UV-selection”

(5)/6LlO30

US46TOR II

f 1996)167-174

standard cell lines

(complementation /Test

I

Research M2

by two sample t-tests.

172

T. Itoh et al./Mutatiorr

group 8, since they did not complement the defect of US31 cells as shown in Table I. Furthermore, they fused easily with other cells mediated by Sendai virus. Among the nine excision repair-defective complementation groups in humans, only three genes involved in XPE, XPF and CSA, had not been identified. Since the characteristics of XPE cells are quite different from those of 6L 1030 cells, cell fusion was performed with XPF and CS cells. These two cell types also have impaired RRS after UVirradiation. Since the fused cells were placed between 6L1030 cells and reference XPF or CS cells on the same coverslip in the complementation test (Itoh et al.. 1994), minor changes in grain numbers of the fused cells were recognized easily by comparison with the grain numbers of the two parental cell

Research 362 (1996)

167-174

IO

5

10

UV (J/ms) Fig. 3. UV survival of 6L1030EB and 6Ll030EB-CSA cells. The procedure for UV survival assay is described in Materials and Methods. Each point represents an average of three experiments. WI38VA13 cells (~1 were used as a normal control. 6L1030EB cells (0 ): 6Ll030EB-CSA cells (0 ).

Fig. 2. Recovery of RNA synthesis (RRS) after UV-irradiation in 6LlO30 cells fused with CS cells. (a) 6L1030 cells fused with CSIMO (CSB) cells; fb) 6L1030 cells fused with Mpsl (CSA) cells. Arrowheads indicate heterokaryons. Note that nuclei of 6LlO30 cells indicated by arrows are easily discriminated from those of primary CS cells because the former have more nucleoli and showed deeper staining with Giemsa.

types plated on each side of the fused cells. Increased RRS was observed in heterokaryons of 6L1030 cells with XPF (Npsll or CSB (CSIMO) (Fig. 2a). but not with CSA (Mpsl) (Fig. 2b). To confirm the fidelity of inspection, we counted the grains over the fused cells and the results are summarized in Table 2. Although the grain number in UV-irradiated mononuclear 6L 1030 cells varied in different experiments, possibly due to the differences in culture conditions, the grain number in heterokaryons was higher than in the parental cells in all combinations, except 6L1030 cells with CSA (CS2SE. Mpsl) cells (grain numbers in heterokaryons were approximate 70-100% of unirradiated cells). These results indicate that 6LlO30 cells share a defect with CSA. More recently (during preparation of this manuscript), CSA gene has been just cloned (Henning et al., 1995). To confirm our results of cell fusion complementation tests described above, we transfected pCSA.5 (containing CSA cDNA fragment) into 6L 1030EB cells (designated 6L 1030EBCSA) and examined UV-survival assay. UV-sensitiv-

T. Itoh et al. /Mutation

ity of 6L1030EB-CSA cells were recovered to that of a normal cells (WI38VA13) (Fig. 3). From this result, it was confirmed that ERCC8 corresponded to CSA.

4. Discussion US3 1 cells were originally isolated from a mouse T cell line, L5178Y, as a UV-sensitive mutant (Shiomi et al., 1982). T cell lines grow in suspension and generally have a low efficiency of incorporation of exogenous DNA by CaPO,-DNA coprecipitation. In addition, L5 178Y and mutant derivatives were unable to form viable hybrids after fusion with any kind of human cells (one exception has been reported to date: Hori et al., 1983) for unknown reasons. All of these characteristics of US31 cells were not suitable for either cell fusion for complementation tests or transfection for gene cloning. To overcome these problems. we have developed a method in which repair-deficient mutant cells are fused with transfection-proficient cells such as LTA which have been irradiated with appropriate doses of X-rays (1 O-25 Gy). followed by screening of hybrid clones to identify those with a mutant phenotype, in which chromosomes containing the normal repair genes of LTA are lost (Shiomi et al., 1988). 6L1030, a hybrid clone derived from US31 and X-irradiated LTA cells grew on plastic dishes with a fibroblastic shape, was also hypersensitive to UV, and belonged to the same complementation group as US31 cells (Table 1). In a preliminary experiment to determine whether human repair genes can compensate for the defect in group 8 mutants, 6L1030, instead of US31 cells were fused with human fibroblast WI38VA13 cells. Many UV-resistant hybrid clones remained even after several weeks of culture to eliminate human chromosomes after the first UV-selection. These results indicate that our method is potentially useful for reconstruction of new cells retaining an original phenotype of interest and that a human repair gene. ERCCS, can complement the defect in mouse group 8 mutants. The lack of complementation in crosses between 6L 1030 and CSA cells indicates that 6L1030 cells belong to the same complementation group as CSA.

Research 362 (19961 167- I74

173

Thus, ERCCS. which complements the defect of group 8 rodent cell mutants, should be involved in CSA. Removal kinetics of both cyclobutane pyrimidine dimers and (6-4) photoproducts in US31 cells are quite similar to those in US46 cells. Cells from CS patients have also been shown to be defective in the repair of cyclobutane pyrimidine dimers. but normal in that of nondimer photoproducts such as (6-4) photoproducts (Parris and Kraemer, 1993). ERCC6, which complements the defect of group 6 rodent mutants such as US46 cells, has been demonstrated to be involved in CSB (Troelstra et al.. 1992). Recent studies using cell-free systems for complementation tests (Biggerstaff et al.. 1993; van Vuuren et al., 1993; Park and Sancar, 1994) have suggested that the gene defective in XPF was ERCC4. Furthermore, CSA gene complemented the defect of 6L1030 cells (Fig. 3). All of these findings support our complementation test results.

Acknowledgements We are grateful to Mrs. Yuka Itoh for preparation of the manuscript. This work was supported by grants from the Ministry of Education, Science and Culture of Japan and the Okukubo Memorial Fund for Medical Research in Kumamoto University School of Medicine.

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