Stable transformation of xeroderma pigmentosum ...

Carcinogenesls vol.17 no.9 pp.1909-1917, 19%

Stable transformation of xeroderma pigmentosum group A cells with an XPA minigene restores normal DNA repair and mutagenesis of UV-treated plasmids

Scott P.Myrand1, Robert S.Topping2 and J.Christopher States3 Center for Molecular Medicine and Genetics, Wayne Stale University, 2727 Second Avenue, Detroit, MI 48201, USA Present addresses: 'institute of Chemical Toxicology, Wayne State University, Detroit, MI 48201 and 2Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109, USA 'To whom correspondence should be addressed

Introduction Understanding human nucleotide excision repair (NER*) is important because NER plays a major role in the maintenance of genomic stability in response to environmental genotoxicants. The failure of the NER system to remove a wide variety of DNA damage causes genetic instability, which leads to the accumulation of mutations and, ultimately, tumor •Abbreviations: NER, nucleotide excision repair, XP, xeroderma pigmentosum; CPDs, cyclobutane pyrimidine dimers; aMEM, a-modified minimal essential medium; PBS, phosphate-buffered saline; CAT, chloramphenicol acetyltransferase. © Oxford University Press

Materials and methods Cell lines and stably transfected clones SV40-transformed normal (GMO637) and XP-A (XP12BE-SV aKa GM4429) human skin fibroblasts were obtained from the NIGMS Human Genetic Mutant Cell Repository (Camden, NJ). XP-A cells stably transformed with an XPA minigene (XAN1) and by a gene unrelated to XPA (2-O-A2; 12) were also used in this study. Both XAN1 and 2-O-A2 cells (11) were derived from XP12BE-SV cells. The 2-O-A2 cell line was the kind gift of Dr R.Stephen Lloyd (University of Texas Medical Branch, Galveston, TX). Cell lines l-O and 8-D are derived from GM4429 and GM0637 respectively by stable transformation with pRSV-NEO and a CYP1A1 expression vector (13). GM0637 and XP12BE-SV were grown in a-modified minimal essential medium (aMEM) (MediaTech, Washington, DC) containing 10% Fetal Clone II bovine serum (HyClone, Logan, UT), 10 mM HEPES, pH 7.0, 100 U/ml penicillin, 0.1 mg/ml streptomycin (Sigma Chemical Co., St Louis, MO) at 37°C in humidified 5% CO2. XAN1, 2-O-A2, l-O and 8-D were maintained in aMEM containing 25 or 300 ng/ml geneticin (Gibco BRL, Gaithersberg, MD). UV survival Survival of each of the four cell lines was assayed as colony forming ability after UV irradiation at the doses indicated. The cells were plated into 10 cm dishes 24 h before UV irradiation and allowed to attach. For the XAN1 and GM0637 cell lines, 200 cells were plated for 0 and 1.5 J/m2 and 500 cells for 3.0-6.0 J/m2. For the 2-O-A2 and XP12BE-SV cell lines, 200 cells were plated at 0 and 1.5 J/m2, 1000 cells at 3.0 J/m2 and 5000 cells at 4.5 and 6.0 J/m2. The cells were washed with phosphate-buffered saline (PBS) and

1909

Downloaded from carcin.oxfordjournals.org at Biblioteka Jagiellonska on July 14, 2011

The ability of an XPA minigene construct to complement the DNA repair defect in xeroderma pigmentosum group A (XP-A) cells was demonstrated. XP-A cells (XP12BESV) were stably transformed with an XPA minigene linked to a neomycin resistance (neo1^) expression cassette. The G418-resistant clone XAN1 was isolated and its DNA repair phenotype compared with XP12BE-SV cells transformed with a cosmid containing a human chromosome 8 gene and a neor cassette and selected for G418 resistance (2-0-A2), DNA repair-normal human fibroblasts and untransfected XP12BE-SV cells. Colony forming ability after UV irradiation, reactivation of a UV-irradiated chloramphenicol acetyltransferase (CAT) expression vector and UV-induced mutagenesis in a supF tRNA shuttle vector (pSP189) were all restored to normal levels in XAN1 cells. In addition, mutation spectra in the supF gene of pSP189 after replication in all four cell lines were compiled at low (100 J/m2) and high (1000 J/m2) UV doses. The majority of mutations were point mutations and these were predominately G:C—»A:T transitions regardless of dose for all cell lines. Dose-dependent differences were observed in the positions of mutation hot spots in pSP189 mutation spectra after replication in all four cell lines. Mutation spectra for XAN1 and GM0637 cells had only minor differences. An increase in the proportion of transversions was observed only in plasmids irradiated with a low UV dose and replicated in XAN1 cells. 2-0-A2 cells were reported to have partial restoration of DNA repair that was later suggested to be caused by a reversion. 2-0-A2 cells were nearly identical to XP12BE-SV cells in all aspects investigated, indicating that transformation to neor had no effect on DNA repair in these cells.

formation. The onset of tumor formation is greatly accelerated in patients with a defective DNA repair system, including those with xeroderma pigmentosum (XP). XP is an autosomal recessive disorder consisting of seven complementation groups (XP-A-XP-G) deficient in NER and one complementation group (XP-V) with normal NER but defective in post-replication repair (1). The NER defect in XP cells is in the recognition and incision of DNA damage. XP patients have a 2000-fold elevated incidence of epidermal neoplasia caused by exposure to sunlight. We have focused our studies on the XPA gene (2,3). The DNA repair defect in XP-A, in which cells are extremely sensitive to UV and chemical DNA damaging agents (4), is caused by mutations in XPA. The XPA protein is a central player in DNA lesion recognition (5). XPA protein has a nuclear localization signal and a zinc finger motif (6), which suggest involvement in binding damaged DNA. XPA protein binds (6-4)-pyrimidine-pyrimidone photoproducts in UV-irradiated DNA, but has little affinity for cyclobutane pyrimidine dimers (CPDs) (7). XPA protein affinity for CPDs can be enhanced by human single-strand binding protein (8). XP-A cells with DNA repair capabilities restored by gene transfection have been prepared using XPA cDNA constructs (6,9). A second human gene unrelated to XPA was reported to partially restore DNA repair capability to transfected XP-A cells (10). 2-0-A2 is a clone of these putatively complemented XP-A cells derived from XP12BE-SV (11). Human XPA was also recently cloned in our laboratory (3) and an XPA minigene construct was assembled and transfected into XP-A cells (XP12BE-SV). A clone of XP-A cells stably transformed with the XPA minigene and that has normal UV survival (XAN1) is the subject of this report.

S.P.Myrand, R.S.Topping and J.CStates overlaid with PBSG (PBS with with 1 mM glucose). The cells were irradiated and refed with fresh aMEM. The cells were allowed to proliferate for 7-11 days, allowing colony formation. The colonies were fixed with methanol, stained with 0.04% methylene blue and counted. Each cell line was assayed in triplicate for each dose. RNA analyses Poly(A) + RNA was prepared using FastTrack RNA preparation kits (InVitrogen, San Diego, CA). This method utilizes direct binding of poly(A)+ RNA to oligo(dT)-cellulose after lysis of cells in buffer containing SDS and digestion of proteins with proteinase K (14). RNA was quantified and purity checked by measuring A260 and A26o'A2go ratios (1.8-2.0). The presence of specific mRNAs was determined by RT-PCR (15). Briefly, first strand cDNA was synthesized using AMV reverse transcriptase (US Biochemicals, Cleveland, OH) in a 10 nl reaction containing 0.5 (ig mRNA. RNA was hydrolyzed by adding 1 ul 1 M NaOH and heating at 55°C for 5 min. The reaction was neutralized with 1 |il 1 M HC1 and 0.5 |il was used as template in each PCR reaction for XPA and ADA cDNAs. Oligonucleotide primers were specific for exons of the respective genes and designed using the NAR program (16). PCR products were resolved by agarose gel electrophoresis. Oligonucleotide primers used were CAGGTCACTGAACTAAA and GGCTAATGTAAAAGCA for XPA and ATTGAGCACCAGATTT and ACCCTATGTGTCCATT for ADA (17). Reactions were cycled 50 times at 94°C for 1 min, 45°C for 1 min and 72°C for 3 min, followed by a 10 min incubation at 72°C. PCR reaction buffer contained 10 mM Tris-HCl, pH 8.3, 50 mM KC1, 1.5 mM MgCl2, 0 1 |iM each oligonucleotide, 200 \iM dNTPS and 0.25 U Taq DNA polymerase (Amersham, Arlington Heights, IL) per 10 ul reaction. Chloramphenicol acetyltransferasc (CAT) assays

Sequencing of mutant plasmids The mutations induced by UV photoproduct formation and subsequent replication in human cells were determined by sequencing the target region of the pSP189 plasmid containing the mutant tRNA gene. Sequencing was performed using the Sequitherm cycle sequencing kits (Epicentre, Madison, WI), essentially according to the manufacturer's instructions. A sequencing primer specific for the non-coding strand (5'-TTTGTGATGCTCGTCAGGGG3') of pSP189 was designed using the NAR program (16). This oligonucleotide was 5'-end labeled using T4 polynucleotide kinase and (y-32P]ATP (3000 Ci/mmol; Dupont, Boston, MA). Sequencing reactions were performed in a Perkin Elmer DNA thermal cycler in two consecutive stages: the first consisted of 10 cycles of 95°C for 30 s, 55°C for 30 s and 70°C for 30 s; the second consisted of 10 cycles of 95°C for 30 s, 60°C for 30 s, 70°C for 30 s, 95°C for 30 s and 70°C for 1 min. The reaction products were resolved by electrophoresis in a 6% polyacrylamide-8 M urea 80 cm exponential wedge (0.2-0.6 mm) gel (CBS Scientific, Del Mar, CA) at 2500 V. The use of the wedge gel allowed the entire tRNA gene and signature sequence to be read using one film (35X43 cm) (26). Statistics Statistical comparisons were performed using Fisher's exact test for differences in proportions. The P values for a one-tailed test are presented.

1910

100

o o

.o a

o o

UV Dose to Plasmid (J/m2) Fig, 1. UV survival in a colony forming assay of four human cell lines. Survival (% control) of cells was determined after exposure to increasing doses of UV irradiation. The means ± SE of triplicate determinations are plotted. The error bars are obscured by the symbols when not visible. UV survival was measured in the following cell lines: XAN1 (A); 2-O-A2 (V); XP12BE-SV (O); GM0637 (D).

Results An XPA minigene complements XP-A cells An XPA minigene was constructed in a vector containing a neor expression cassette. The XPA minigene was constructed by joining three DNA fragments: a 1.8 kb SacII genomic DNA fragment containing 1.6 kb of XPA 5'-flanking sequence and 0.2 kb of XPA exon 1 (3); a 0.8 kb SacU-Ndel XPA cDNA fragment containing sequences from exon 1 to exon 6; a 0.9 kb Ndel-Xbal genomic DNA fragment containing the remaining 0.6 kb of exon 6 and 0.3 kb of XPA 3'-flanking sequence. The minigene vector was transfected into XP12BESV cells and geneticin-resistant cells were selected. The geneticin-resistant cells were then subjected to serial rounds of UV selection and individual clones of UV-resistant cells were isolated. The first of these clones, XAN1, was tested for colony forming ability after UV irradiation and found to be similar to repair-normal GM0637 cells (Figure 1). 2-0-A2 cells were as sensitive to UV as XP12BE-SV cells. The continued presence of both mutations in the XPA alleles of XP12BE-SV (27) was confirmed in XAN1 (data not shown). In addition, the level of XPA mRNA was examined by RTPCR (Figure 2). XPA mRNA is present at extremely low levels in wild-type human fibroblasts (6,28). The RT-PCR results show that XPA mRNA is at similar low levels in XAN1 and GM0637 cells, but undetectable in XP12BE-SV and 2-0-A2 cells under these amplification conditions. ADA mRNA was analyzed in parallel as a control for input cDNA template. ADA is expressed at low levels in human fibroblasts (29). The signal from ADA was greater than that from XPA. Slightly lower ADA cDNA in lane 2 indicates that the cDNA template input was slightly lower for XP12BE-SV than for the other three cell lines, which gave comparable ADA signals. Thus, stable transfection with the XPA minigene which expresses at

Downloaded from carcin.oxfordjournals.org at Biblioteka Jagiellonska on July 14, 2011

Plasmid pRSV-CAT (18) was UV irradiated and co-transfected with unirradiated pRSV-fiGAL (19) using LipofectAMINE (Life Technologies, Gaithersburg, MD) in serum-free medium. UV irradiation of pRSV-CAT was as described below for pSP189. CAT assays and normalization to figalactosidase activities were as described elsewhere (3,19). Transfection of cells with irradiated pSP 189 The plasmid pSP189 (20) was a gift from Dr Michael M.Seidman (Otsuka American Pharmaceutical Inc., Rockville, MD). Plasmid pSP189 is derived from pZ189 (21) by inclusion of a signature sequence to distinguish sibling and independent mutations in replicated plasmids. UV photoproducts were induced (22) in pSP189 DNA (30 ng/ml in TE 50:1; 50 mM Tris, 1 mM EDTA, pH 8.0) by 0, 100, 200, 500, 700 and 1000 J/m2 LTV using a UV photoproducts mineral lamp (peak wavelength 254 nm). Cells were plated into 6-well dishes (1.5X10* cells in 3 ml aMEMAvell) and allowed to attach and acclimate for 24 h. The cells were washed with serum-free medium and then transfected with 0 75 ng pSP189 DNA using 5 nl LipofectAMINE reagent (Gibco BRL, Gaithersberg, MD) for 5 h at 37°C in serum-free medium. Plasmid survival and mutation frequency data were compiled separately using 0.65 ng pSP189 and 0.15 ng unirradiated pZ189k DNA (a gift from Dr Steven Akman, City of Hope, Duarte, CA) to control for differences in retrieval of plasmid DNA from transfected cells. The cells were refed with 3 ml aMEM and after 48 h incubation plasmid DNAs were harvested using a modified Hirt extraction protocol (23). The retrieved plasmid DNAs were digested with Dpnl to remove non-replicated DNA (24) and introduced into the Escherichia coli indicator strain MBM7070 (21) by electroporation (25).

=•

An XPA minigene restores DNA repair and mutagenesis

A.

B.

M 1 2 3 4

m1

M

1

2

3

4 GM0637 c o

600-

10 200-

o XP12BE-SV

•Q*

Fig. 2. RT-PCR analyses of XPA and ADA mRNAs in four human fibroblast lines. RT-PCR was performed as detailed in Materials and methods. (A) XPA and (B) ADA assays were performed in parallel sets of assays. Lanes 1, GM0637; lanes 2, XP12BE-SV; lanes 3, 2-O-A2; lanes 4, XAN1; lanes M, 100 bp marker DNAs (Life Technologies, Gaithersburg, MD).

A:T A:T->G:C Transversions G:C-»T:A G:C-»C:G A:T->T:A A:T->C:G Total

2

100 J/m

2

20 (63) 45 (87) 20 (63) 42 (81) 0(0) 3 (6) 12 (38) 7 (13) 6(19) 2 (4) 4(13) 3 (6) 1(3) 2 (4) 1(3) 0 (0) 32 (100) 52 (100)

1000 J/m 53 (87) 52 (85) 1 (2) 8 (13) 2 (3) 2 (3) 2 (3) 2 (3) 61 (100)

2

0 J/m

2

XP12BE-SV 100 J/m

2

11 (50) 47 (73) 11(50) 47 (73) 0(0) 0(0) 17* (27) 11 (50) 8(36) l l b (17) 3(14) 3(5) 0(0) 2(3) 1(2) 0(0) 22 (100) 64(100)

1000 J/m 46(84) 45 (82) 1(2) 9(16) 3(5) 0(0)

5(9)

1(2) 55 (100)

2

0 J/m

2

100 J/m

2-O-A2 2

1000 J/m

2

17 (57) 63 (93) 48(86) 17 (57) 62 (91) 46(82) 1 (1) 2(4) 0(0) 5 (7) 8(14) 13 (43) 3 (4) 3(5) 7(23) 1 (1) 0(0) 3(10) 1 (1) 4(7) 0(0) 0 (0) 1(2) 3(10) 30 (100) 68 (100) 56 (100)

0 J/m2

100 J/m2 1000 J/m2

9(45) 74(95) 85 (83) 9(45) 74 (95) 83 (81) 0(0) 0(0) 2(2) 11 (55) 4(5) 17° (17) 4(20) 3(4) 9(8) 6(30) 0(0) 3(3) 1(5) 5(5) KD 0(0) 0(0) KD 20 (100) 78 (100) 102 (100)

"Significantly different from: XP12BE-SV 100 J/m2, P = 0.01; 2-0-A2 100 J/m2, P < 0.002. Significantly different from: XP12BE-SV 100 J/m2, P < 0.03; 2-O-A2 100 J/m2, P < 0.02; GMO637 100 J/m2, P < 0.04. Significantly different from 2-O-A2 100 J/m2, P = 0.025.

Discussion XP cells are hypersensitive to the cytotoxic and genotoxic effects of UV irradiation because they have defective NER. Previous studies by others have shown that transfection of XP-A cells (XP2OS-SV) with high level XPA cDNA expression vectors restores UV resistance of the cells (6) and the capability to repair UV-irradiated plasmids (34). We have expanded upon these observations by examining restoration of DNA repair in a different XP-A cell line (XP12BE-SV) by stable transfection with an XPA minigene (XAN1 cells). XPA mRNA levels in XAN1 cells are comparable with the low levels in wild-type GM0637 cells (Figure 2). We have also further investigated the repair properties of a clone (2-O-A2) of XP12BE-SV cells transfected with a gene unrelated to XPA that purportedly confers the ability to repair CPDs (10). In addition, we have investigated the influence of UV dose on the mutation spectra by comparing mutation spectra obtained from plasmids irradiated with low (100 J/m2) and high (1000 J/m2) UV doses. Complementation of XP-A cells by transfection with human gene constructs XAN1 cells behaved identically to the repair-normal GM0637 cells in assays for colony forming ability after UV irradiation (Figure 1), reactivation of a UV-irradiated CAT expression vector (Figure 3) and replication (Figure 4) and mutagenesis

(Figures 5 and 6) of a UV-irradiated shuttle vector. There were only minor differences in the shuttle vector mutation spectra from XAN1 and GMO637 (Figure 6). Thus, stable transfection of XP12BE-SV cells with the XPA minigene has completely restored DNA repair capability to normal as measured by these parameters. The DNA repair capacity of 2-0-A2 cells has been a topic of controversy (11). When first characterized, 2-0-A2 cells were reported to have improved UV resistance (12) and also ability to remove CPDs (10). The samples of 2-0-A2 cells we analyzed clearly have been transfected because they survive and grow in medium containing geneticin up to 300 (ig/ml, indicating they express the transfected neor expression cassette. However, our results indicate that the 2-0-A2 cells we analyzed exhibit no evidence of DNA repair greater than that exhibited by the true parental cells, XP12BE-SV (11). It is unlikely that 2-O-A2 cells ever had increased UV resistance. 2-0-A2 cells were originally isolated based on improved UV resistance compared with XP2OS-SV cells, the originally assumed parental XP-A line (12). Re-examination of the original published data on UV resistance of 2-O-A2 cells reveals that survival after UV was very similar to that of XP12BESV cells, consistent with our data (Figure 1). The reported observation of XPA protein in extracts of 2-0-A2 cells (11) is not the result of a reversion of a mutant XPA allele because both mutations present in XP12BE-SV cells are also present in 2-0-A2 cells (27). XP12BE-SV cells have modestly better UV resistance than XP2OS-SV cells (35), as do several other XP-A cell lines derived from patients with delayed onset of neurological disease (36). It is likely that the XPA protein detected in 2-O-A2 cells is related to low levels of altered XPA protein produced in the parental XP12BE-SV cells from low levels of mutant XPA mRNAs that have a small insertion (27). Unfortunately, the level of XPA protein in XP12BE-SV cells was not examined by Jones et al. (11). Relation to studies in complemented XP-A cells overproducing XPA protein Our results are similar in some respects and dissimilar in other respects to the findings previously reported by Levy et al., who used XP-A cells transfected with a high level XPA cDNA expression vector (34). The similarities include the predominance of G:C-»A:T transitions and the major mutation hot spots at positions 155, 156 and 163. Dissimilarities 1913

Downloaded from carcin.oxfordjournals.org at Biblioteka Jagiellonska on July 14, 2011

at positions 155, 156 and 163 were common to all cell lines when the data are compiled without partitioning by UV dose. Position 172 was a hot spot in XP12BE-SV, 2-0-A2 and GM0637. Positions 124 and 168 were hot spots in XP12BESV and 2-0-A2. Position 129 was a hot spot in XP12BE-SV and GM0637, but only at the low UV dose. Position 139 was a hot spot only in XAN1 and only at high UV dose. The hot spot at 156 was present in all cell lines at both low and high UV doses. In contrast, the major hot spots at positions 155 and 163 were variable depending on cell line and UV dose. Dose-dependent changes are present in the mutation spectra from all four cell lines. In XP12BE-SV, hot spots are present at positions 124 and 129 at low UV dose. These disappear at high UV dose. GM0637 cells have a hot spot at position 129 only at low UV dose. XAN1 cells have a hot spot at position 139 only at high UV dose. The frequency of mutations at position 172 was increased at the high UV dose in three cell lines: XP12BE-SV, XAN1 and GM0637.

S.RMyrand, R^.Topplng and J.CStates

include our not observing a significant decrease in G:C—>A:T transitions in DNA repair-proficient cell lines, as did Levy et al. The unchanging proportions of transitions and transversions in our experiments were most evident in plasmids irradiated with a high UV dose. In plasmids irradiated with the low UV dose (100 J/m2) there was a significant increase in transversions (predominantly G:C->T:A) in XAN1 cells but not GM0637 cells compared with the repair-deficient cell lines XP12BESV and 2-0-A2. Levy et al. concluded that mutation hot spots are not caused by DNA repair because the positions of the hot spots showed no changes with repair status of the cells. However, shuttle vector mutation spectra were altered in the XP-A cells, with 14-fold overexpression of XPA (XP2OS-pCAH19WS) as compared with either untransfected XP-A cells or repairnormal cells (34). Overexpression of XPA was associated with the loss of a major mutation hot spot (position 156) and the gain of a new mutation hot spot (position 124). It is possible that the change in the mutation spectrum is a consequence of overexpression of XPA in the XP2OS-pCAH19WS cells. High levels of a single component of the NER complex may cause an imbalance that alters the lesion recognition properties of the complex. Similar loss of a common hot spot in the pSP189 XP12BE-SV I

• J M t f W M IDH (M-tO)

I 19 0 I

| 110 I CT

T

AA A A

C

I

C

A

AT

2-0-A2 summon IOM (M-io>

I 1 1 •

I

I

I 1 » 0 I

I 1 3 0 I

OJfcBl A A A ~~A A

a

A

I

I

I

1 • 0

1 4 0

1 t 0 I

I

I 0

T T T T

A A 1 A A

T T

AA k A k k

A

A

T

T

A A

r

T T

1914

T T T T

T T T T

A A

T A

T

A

T C T

T

T

lOOJAnS

A

A C A

O T T

A

T

T A

A

C

1 • 0 I

1 • 0 1

1

T T

I t 0 0

A.

110

1000 Jtm2

B.

A A

110 1

1000 J/mJ

t

Downloaded from carcin.oxfordjournals.org at Biblioteka Jagiellonska on July 14, 2011

A.

mutation spectrum occurred in XP-D cells complemented with XPD cDNA in a high level expression vector (33). In this instance, there were two clones of complemented XP-D cells. The clone with highly overexpressed XPD (17.6-fold) had alterations in the mutation spectrum, including a loss of the major hot spot at position 155, compared with repair-normal cells, but the clone with modest overexpression (4.8-fold) did not. Our complemented XP-A cells (XAN1) are complemented with an XPA minigene that uses the native XPA promoter and have XPA mRNA levels very similar to the wild-type (Figure 2). Thus, it is unlikely that there is a gross imbalance in the components of the NER complex. When we compare mutation spectra with or without dose fractionation, we find no significant differences at either positions 124 or 156 between spectra from XAN1 and GMO637 cells. We did see a significant decrease in mutations at position 172 in XAN1 cells, but only when the low and high dose spectra are combined. Comparisons at only low or high dose show no significant differences between the mutation spectra from XAN1 and GMO637. Thus, transfection of XP12BE-SV cells with an XPA minigene has made these cells behave nearly identically to the repair-normal GM0637 cells.

An XPA minigene restores DNA repair and mutagenesis

C.

GM0637

1 100 1

1

1 1 » 0 I

12 0

I

AA

.£

I

I 1 • 0 I

A^Q

D. XAN1 •UPPMSKM MM #»1UjI

i

i 1 0 • 1

A.

1 1 1 0 1

d

1 1 2 0 I

1

1 1 3 0 1

1 1 • 0 1

1 t 1 0 1

1 4 0 1

1 1 T 0 1

1 1 1 • • 1

OJtml A A

A A T T

AA T T T

A

T

AT A A T A

a

A T T

T T

A

TAC A T

A A A A

0 J

A A T T T T

T T T T

T A T T

OT T T T T T

r

T

a

lOOJftal

A A

TT T T A T

AA

OT T

A A A A

J_A

T

T

T

A

T A

Fig. 6. Location of single and tandem base substitution mutations found in the supF gene of UV-irradiated pSP189 after replication in four human cell lines. (A) XP-A (XP12BE-SV), (B) 2-O-A2, (C) repair-normal (GM0637) and (D) XANl (XPA minigene-transfected XP-A) cell lines. The mutation spectra are separated by UV dose. Suppressor tRNA sequences start at bp 99 and end at bp 183. Each letter indicates a single base pair substitution mutation in an independent plasmid. Tandem base or closely spaced substitution mutations are underlined.

1915

Downloaded from carcin.oxfordjournals.org at Biblioteka Jagiellonska on July 14, 2011

Influence of UV dose on mutation spectra We have chosen to partition our mutation spectra by UV dose to examine the potential for a dose effect on the mutation spectrum. 6-4 Photoproducts and CPDs form preferentially in different dipyrimidine sequences and the UV dose influences the likelihood that either lesion will form at a particular dipyrimidine (reviewed in 1). For instance, 6-4 photoproducts form predominantly in TC and CC dinucleotides and CPDs form predominantly in TT and CT dinucleotides. At UV doses of 2000 J/m2 and less, CPDs form at TT three to four times more frequently than at all other dipyrimidines (38) and 6-4 photoproducts form predominantly at TC, with a minor portion formed at CC (39). We have preliminary data (not shown) on photoproduct mapping in pSP189 showing distributions of specific photoproducts consistent with these prior reports. Thus mutations occurring at CT (and TT) are likely caused by CPDs and mutations occurring at TC (and to some degree CC) are likely caused by 6-4 photoproducts. Furthermore, it has been shown that 6-4 photoproducts are normally present as a lower percentage of total dimers than CPDs in a given strand of DNA. However, the total fraction of dimers that are 6-4 photoproducts increases at higher UV doses. Therefore, by stratifying mutation spectra by UV dose, we hypothesized differences in the spectra would be found. Significant dose-

In contrast to the lack of difference in mutation spectra between repair-deficient and repair-normal cells observed by Levy et ai, in our experiments both DNA repair-deficient cell lines (XP12BE-SV and 2-0-A2) had mutation hot spots at positions 124 and 168 that were not evident in either of the repair-proficient cell lines (GM0637 and XANl). In addition, a new hot spot appeared in XANl cells at position 139. The hot spot at position 156 was prominent in all four cell lines in our system. Thus, our results, although not contradictory, are not entirely consistent with the observations by Levy et al. The differences in the experimental systems used should be noted. We have used XP12BE-SV as our starting XP-A cell line. This XP-A line has modestly better UV resistance than XP2OS-SV used by the other group (35) and likely expresses a very low level of altered XPA (27). This report is the first time mutation spectra have been compiled in GM0637 and XP12BE-SV cells transfected with pSP189 using LipofectAMINE. Different mutation hot spot patterns can result from different transfection agents (37). In addition, we have complemented the NER defect of the XP-A cells by transfection with an XPA minigene that uses the weak native XPA promoter to control transcription of the XPA cDNA, rather than a strong viral promoter. Thus, XPA is expressed at normal levels in XANl cells.

S.P.Myrand, ItS.Topping and J.CStates

specific hot spots were found in two cell lines: XP12BE-SV at position 124 (/> = 0.022); XAN1 at position 139 (P = 0.024). Dose-specific differences at position 164 in 2-0-A2 (P = 0.06) and position 172 in XP12BE-SV (P = 0.056) are suggestive, but not statistically significant. Thus, there are dose-specific differences in the mutation spectra within a cell line, but the differences are small. Others have previously compiled spectra with mutants from either a single UV dose or combined mutants from a number of different UV doses. It has been inferred that mutation spectra and specific lesions in a shuttle vector did not correlate by comparison of mutation spectra gathered at 100-500 J/m2 with lesions formed at 1000 J/m2 (40). However, others have shown that in SV40, 6-4 photoproducts and CPDs induce different mutation spectra that share some sites (41). A detailed analyses of lesion-specific mapping at both low (100 J/m2) and high (1000 J/m2) doses in the supF gene will determine whether there are dose-dependent differences for photoproduct formation that may correlate with the small dose-dependent differences we observed in the mutation spectra. Acknowledgements

References l.Friedberg.E.C, Walker.G.C. and Siede,W. (1995) DNA Repair and Mutagenesis. ASM Press, Washington, DC. 2.Satokata,I., Iwai.K., MatsudaX, Okada,Y. and Tanaka,K. (1993) Genomic characterization of the human DNA excision repair-controlling gene Xpac. Gene, 136, 345-348. 3.Topping,R.S., Myrand,S.P, Williams.B.L., AlberU.C. and StatesJ.C. (1995) Characterization of the human XPA promoter. Gene, 166, 341-342. 4. Kraemer,K.H. (1980) Xeroderma pigmentosum. In Demis,D.J., Dobson.R.L. and McGuireJ. (eds), Clinical Dermatology, Unit 17. Harper & Row, Philadelphia, PA, pp. 1-33. 5.Robins,P, Jones.CJ., Biggerstaff.M., Lindahl.T. and WoodJ*.D. (1991) Complementation of DNA repair in xeroderma pigmentosum group A cell extracts by a protein with affinity for damaged DNA. EMBO J., 10, 3913-3921. 6.Tanaka,K. et al. (1990) Analysis of a human DNA excision repair gene involved in group A xeroderma pigmentosum and containing a zinc-finger domain (see comments). Nature, 348, 73-76. 7.Jones,CJ. and Wood.R.D. (1993) Preferential binding of the xeroderma pigmentosum group A complementing protein to damaged DNA. Biochemistry, 32, 12096-12104. 8.Li,L., Lu,X.Y., Peterson.C.A. and Legerski.RJ. (1995) An interaction between the DNA repair factor XPA and replication protein a appears essential for nucleotide excision repair. Mol. Cell. Biol., 15, 5396-5402. 9. Miyamoto,I., Miura,N., Niwa,H., MiyazakiJ. and Tanaka.K. (1992) Mutational analysis of the structure and function of the xeroderma pigmentosum group A complementing protein. J. Biol. Chem., 267, 12182-12187. lO.Mori.T., Rinaldy/T.L., Athwal.R.S., Kaur.G.R, Nikaido.O., Lloyd.R.S. and Rinaldy,A. (1993) A xeroderma pigmentosum complementation group A related gene: confirmation using monoclonal antibodies against the cyclobutane dimer and (6-4) photoproduct. Mutat. Res., 293, 143-150. 11. Jones.CJ., Lloyd,R.S. and Wood.R.D. (1994) Analysis of cells harboring a putative DNA repair gene reveals a lack of evidence for a second independent xeroderma pigmentosum group A correcting gene. Mutat. Res., 324, 159-164. 12.Rinaldy,A., BeUew.T, Egli,E. and Lloyd,R.S. (1990) Increased UV resistance in xeroderma pigmentosum group A cells after transformation with a human genetic DNA clone. Proc. Natl Acad, Sci. USA, 87, 6818-6822. 13.StatesJ.C, Quan,T, Hines.R.N., NovalcR.F. and Runge-Morris,M. (1993) Expression of human cytochrome P45O 1A1 in DNA repair deficient

1916

Downloaded from carcin.oxfordjournals.org at Biblioteka Jagiellonska on July 14, 2011

The authors thank Dr Dan Levy for his assistance by supplying various protocols, computer software and suggestions and Dr Michael M.Seidman for his critical review of the manuscript. Technical assistance was provided by Miikelle S.Mitchell and Jack C.Albert, who were supported by NCRR grant RR0332 to JCS.

and proficient human fibroblasts stably transformed with an inducible expression vector. Carcinogenesis, 14, 1643—1649. 14.Schwab,M., Alitalo,K., Varmus.H.E., BishopJ.M. and GeorgeJ). (1983) A cellular oncogene (c-Ki-rar) is amplified, overexpressed, and located within karyotypic abnormalities in mouse adrenocortical tumour cells. Nature, 303, 497-501. 15.Kawasaki,E.S. and Wang,A.M. (1989) Detection of gene expression. In Erlich.H.A. (ed.), PCR Technology: Principles and Applications for DNA Amplification. Stockton Press, New York, NY, pp. 89-97. 16.Rychlik,W. and Rhoads.R.E. (1989) A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic Acids Res., 17, 8543-8551. 17.Freeman,B.C. and StatesJ.C. (1991) An STS in the human adenosine deaminase gene (located 2Oql2-ql3.11). Nucleic Acids Res., 19, 5084. 18.Gorman,C.M., Merlino.G.T, WiUingham.M.C, Pastan.I. and Howard.B.H. (1982) The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. Proc. Natl Acad. Sci. USA, 79, 6777-6781. 19. StatesJ.C. (1988) Complementation of xeroderma pigmentosum group A cells by DNA mediated gene transfer. In Friedberg.E.C and Hanawalt,P.C. (eds), Mechanisms and Consequences of DNA Damage Processing. Alan R.Liss Inc., New York, NY, pp. 307-311. 2O.Parris,C.N. and Seidman,M.M. (1992) A signature element distinguishes sibling and independent mutations in a shuttle vector plasmid. Gene, 117, 1-5. 21.Seidman,M.M., Dixon.K., Razzaque,A., Zagursky.RJ. and Berman.M.L. (1985) A shuttle vector plasmid for studying carcinogen-induced point mutations in mammalian cells. Gene, 38, 233-237. 22. Parris.C.N and Kraemer.K.H. (1992) Ultraviolet mutagenesis in human lymphocytes: the effect of cellular transformation. Exp. Cell Res., 201, 462^*69. 23.Hirt,B. (1967) Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol Biol, 26, 365-369. 24.Peden,K.W.C, PipasJ.M., Pearson-White.S. and Nathans.D. (1980) Isolation of mutants of an animal virus in bacteria. Science, 209,1392-1396. 25.Hanahan,D., JesseeJ. and Bloom.F.R. (1991) Plasmid transformation of E. coli and other bacteria. Methods EnzymoL, 204, 63-113. 26. StatesJ.C., Patel.L.R and Li,Q. (1991) A gel electrophoresis system for resolving over 500 nucleotides with a single sample loading. Biotechniques, 11, 46-^7. 27. StatesJ.C. and Myrand.S.P. (1996) Splice site mutations in a xeroderma pigmentosum group A patient with delayed onset of neurological disease. Mutat. Res., in press. 28.CleaverJ.E., Mitchell.D.L., Feeney.L. and Afzal.V. (1996) Chromatid exchanges may be induced by damage in sites of transcriptional activity. Mutagenesis, 11, 183-187. 29.Lattier,D.L., StatesJ.C, HuttonJJ. and Wiginton.D.A. (1989) Cell typespecific transcriptional regulation of the human adenosine deaminase gene. Nucleic Acids Res., 17, 1061-1076. 3O.Protic-Sabljic,M. and Kraemer.K.H. (1985) One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells. Proc. Natl Acad. Sci. USA, 82, 6622-6626. 31.Moriwaki,S.I., Tarone.R.E. and Kraemer.K.H. (1994) A potential laboratory test for dysplastic nevus syndrome: ultraviolet hypermutability of a shuttle vector plasmid. J. Invest. Dermatol., 103, 7-12. 32.Protic-Sabljic,M., Seetharam,S., SeidmanJvl.M. and Kraemer.K.H. (1986) An SV40-transformed xeroderma pigmentosum group D cell une: establishment, ultraviolet sensitivity, transfection efficiency and plasmid mutation induction. Mutat. Res., 166, 287-294. 33.Gozukara,E.M., Parris.C.N., Weber.C.A., Salazar.E.P, Seidman.M.M., WatkinsJ.F., Prakash.L. and Kraemer.K.H. (1994) The human DNA repair gene, ERCC2 (XPD), corrects ultraviolet hypersensitivity and ultraviolet hypermutability of a shuttle vector replicated in xeroderma pigmentosum group D cells. Cancer Res., 54, 3837-3844. 34.Levy,D.D., Saijo,M., Tanaka,K. and Kraemer.ICH. (1995) Expression of a transfected DNA repair gene (XPA) in xeroderma pigmentosum group A cells restores normal DNA repair and mutagenesis of UV-treated plasmids. Carcinogenesis, 16, 1557-1563. 35. Kraemer.K.H., Coon,H.G., Petinga,R.A., Barrett,S.F., Rahe,A.E. and RobbinsJ.H. (1975) Genetic heterogeneity in xeroderma pigmentosum: complementation groups and their relationship to DNA repair rates. Proc. Natl Acad. Sci. USA, 72, 59-63. 36. RobbinsJ.H. etal. (1991) Neurological disease in xeroderma pigmentosum. Brain, 114, 1335-1361. 37. Seetharam,S. and Seidman,M.M. (1991) Modulation of an ultraviolet mutational hotspot in a shuttle vector in xeroderma cells. Nucleic Acids Res., 19, 1601-1604.

An XPA mlnigene restores DNA repair and mutagenesis 38.Bourre,F., Renault.G., Seawell,P.C. and Sarasin.A. (1985) Distribution of ultraviolet-induced lesions in simian virus 40 DNA. Biochimie, 67, 293-299. 39.Bourre,E, Renault.G. and Sarasin,A. (1987) Sequence effect on alkalisensitive sites in UV-irradiated SV40 DNA. Nucleic Acids Res., 15, 8861-8875. 40. BrashX>.E., Seetharam,S., Kraemer.lCH., Seidman.M.M. and Bredberg^A. (1987) Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells. Proc. Nail Acad. Sci. USA, 84, 3782-3786. 41.Bourre,F., Benoit^A. and Sarasin.A. (1989) Respective roles of pyrimidine duner and pyrimidine (6-4) pyrimidone photoproducts in UV mutagenesis of simian virus 40 DNA in mammalian cells. J. Virol., 63, 4520-4524. Received on December 18, 1995; revised on May 8, 1996; accepted on May 29, 1996

Downloaded from carcin.oxfordjournals.org at Biblioteka Jagiellonska on July 14, 2011

1917

Downloaded from carcin.oxfordjournals.org at Biblioteka Jagiellonska on July 14, 2011