DNA repair in lymphoblastoid cell lines established from human genetic disorders

Chem.-Biol. Interactions, 33 (1980) 63--81 © Elsevier/North-Holland Scientific Publishers Ltd.

63

DNA REPAIR IN LYMPHOBLASTOID CELL LINES ESTABLISHED FROI~ HUMAN GENETIC DISORDERS

E.E. HENDERSON a and RENE RIBECKYb Department of Microbiology and Immunology and bFels Research Institute, Temple University School of Medicine, Temple University Life Sciences Center, Philadelphia, PA 19140 (U.S.A.) (Received March 5th, 1980) (Revision received August 3rd, 1980) (Accepted August 8th, 1980)

SUMMARY

Lymphoblastoid cell lines(LCLs) establishedfrom chromosomal breakage syndromes or relatedgenetic disordershave been used to study the effectsof mutagens on human lymphoid cells. The disorders studied include xeroderma pigmentosum, ataxia telangiectasia, Fanconi's anemia, Bloom's syndrome and Cockayne's syndrome. Three approaches were used to assess the cells'ability to cope with a particular mutagen: (I) assaying recovery of D N A snythetic capabilitiesas measured by [aH]thymidine (dT) incorporation; (2) measurements of classicalexcision D N A repair by isopyknic sedimentation of D N A density labeled with 5-bromo-2-deoxyuridine (BrdU); (3) determining cell survival by colony formation in microtiter plates. LCLs established from xeroderma pigmentosum showed increased sensitivitiesto ultraviolet (354 nm) lightand N-acetoxy-2-acetylaminofluorene (AAAF) as determined by D N A synthesisor colony formation and had diminished levels of excision-repair. Cockayne's syndrome LCLs, on the other hand, had increased sensitivitiesto ultraviolet(UV) light,A A A F and N.methyl.N'-nitroN-nitrosoguanidine ( M N N G ) while showing near normal levelsof DNA-repair after treatment with each agent. An L C L establishedfrom ataxiatelangiectasia had decreased D N A repair synthesisand defectivecolony-forming ability following treatment with M N N G . LCLs, in addition to ease of establishment, appear likely to provide useful material for the study of D N A repairreplication and itsrelationshipto carcinogenesis.

aSpecial Fellow of the Leukemia Society of America. Abbreviations: AAAF, N-acetoxy-2-acetoaminofluorene; BrdU, 5-bromo-2-deoxyuridine; dT, thymidine; EBV, Epstein-Barr virus; LCL, lymphoblastoid cell line; MNNG, N-methylN'-nitro-N-nitrosoguanidine; SSC, 0.15 M NaC1 plus 0.015 M sodium citrate.

64

INTRODUCTION A number of methods have been developed to determine a culture mammalian cell's ability to cope with an agent shown to be mutagenic or carcinogenic in another system. One is to measure the removal of identifiable lesions introduced by mutagens into DNA. The strategy has been used for lesions induced by X-rays [1], UV-irradiation [2] and a variety of nucleophilic reagents [3]. An extension of this approach has been to assay cellular extracts for enzymes capable of recognizing specific lesions introduced into superhelical bacteriophage DNA [4,5] or artificially constructed DNA templates containing modified bases [6]. Using intact cells, the effects of mutagens have been examined at the chromosomal levels through advanced cytogenic techniques. These include examining dividing cell populations for mutagen-induced chromosomal aberrations [7,8], sister chromatid exchanges [9] and micronuclei formation [10]. Another, possibly more direct approach to measuring cellular responses to mutagens has been attempts to quantitate DNA repair replication in viable cells following mutagen exposure. The methods used have included measuring unscheduled DNA synthesis [ 11], BrdU incorporation [ 12,13 ] and nucleotide incorporation by BND-cellulose chromotography [14]. The effect of unremoved lesions on DNA replication as measured by [3H]thymidine incorporation have also been used as an indicator for mutagen sensitivity [15--17]. Moore and Strauss [18] have refined this approach, sequencing particular bacteriophage DNA fragments, generated by endonuclease digestion, to determine sites of inhibition of DNA synthesis using cellular polymerase and mutagen-treated ~×-174 DNA. Biological approaches to the problem of measuring the 'fidelity' of DNA repair have also been sought. The first, simplest and most straightforward approach was to determine the effects of mutagens on cell survival as detected by colony formation. This basic m e t h o d was soon adapted to select for, and measure, morphologic transformation [19] as well as mutations at specific gene loci [20]. Host cell reactivation of mutagen-treated DNA viruses has been used in an analogous manner and has proven to be a useful tool for the study of cellular repair of biological functions [21]. Taken as a whole each of these methods has contributed both to the identification of potentially DNA-clamaging agents and to the understanding of the cellular mechanisms involved in the repair of this damage. In this preliminary report, we use several of these assays, namely, cellular survival, recovery of DNA synthesis and measurements of excision repair to analyze the effects of several mutagens on LCLs established from normal adults and chromosomal breakage syndromes. MATERIAI_~ AND METHODS Cell lines and agents The permanently proliferating lymphoblastoid cell lines used in these

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66 studies were established using the B95-8 transforming strains of Epstein-Barr virus (EBV) obtained from Dr. George Miller, Yale University, New Haven, CN. Peripheral blood leukocytes from individuals with various genetic defects, and from normal adults were transformed in microtiter plates using the limiting dilution method in collaboration with Dr. James German, New York Blood Center, New York (Table I) [22]. Transformation efficiencies were calculated using the Reed and Muench formula and expressed as the positive log of the virus dilution at which 50% of the microtiter wells would yield transformed colonies. The resulting LCLs were then maintained in Rosewell Park Memorial Institute medium (RPMI-1640) supplemented with 20% heat inactivated fetal bovine serum and penicillin and streptomycin. Under these culture conditions LCLs do not produce detectable transforming virus. The human fibroblast line WI-38, at passage 16, as obtained from Dr. Renato Baserga, Temple University. Viability was determined by Trypan blue dye exclusion (Trypan Blue Stain 0.4% in normal saline) during hemocytometer counts and exceeded 90% or the cultures were not used. For the UV source, total emission from a 15-W GE 15T8 low-pressure mercury germicidal lamp controlled by a rheostat was used. Incident (254 nm) UV dose rate for cellular inactivation was 0.50 J/m:/sec (5.0 ergs/mm2 / sec) as determined by a Laterjet dosimeter, calibrated and loaned by Alan Conger, Fels Research Institute. Aliquots (1-ml) of cells suspended in phosphate-buffer saline placed in 60-mm petri dishes were exposed with gentle agitation to ensure uniform irradiation. Crystalline MNNG (Aldrich Chemical Co., Inc., Milwaukee, WI) and AAAF (the kind and generous gift of Dr. Prabhaker Lotlikar, Fels Research Institute) were dissolved to RPMI-1640 immediately before use. Cell lines were tested for mycoplasma by autoradiography and found to be free of obvious contamination. DNA synthesis Effects of agents on cell line DNA synthetic capabilities were determined by slight modification of the method described by Agarwal et al. [15] for phytohemagglutinin-stimulated human lymphocytes. Briefly, the cells were exposed to various doses of a particular mutagenic agent, in the case of chemical carcinogens, for up to 1 h, then aliquoted at 5 X l 0 s cells/ml into 100 X 15-mm rubber-stoppered glass tubes. These aliquots were then exposed to [3H]dT, 6 pCi/ml, 19 Ci/mmol, for 1 h at various times after treatment with the agent. The reaction was terminated by the addition of 4 ml cold Hank's balanced saline and the cells processed for cold trichloroacetic acidprecipitable material on millipore filters and the incorporated radioactivity determined in a liquid scintillation counter. The effects of mutagens on DNA synthesis are expressed as a percentage of untreated cultures. Cesium chloride gradients Replicated and unreplicated DNA was separated using BrdU and isopyknic sedimentation in neutral cesium chloride (CsC1), a method initially developed by Pettijohn and Hanawalt [23] for bacteria and later modified for cell

67 culture (HeLa cells) by Painter and Cleaver [12] and for lymphoid cell suspension cultures by Fox and Fox [24]. Cells were preincubated in BrdU (CalBiochem) (10 ~g/ml) for 30 min, then exposed to the mutagen for up to 1 h and [3H]dT added at 25 ~Ci/ml, 49 Ci/mmol (New England Nuclear). This BrdU : [aH]dT ratio is well within the range necessary to distinguish between hybrid, replicated DNA, from non-replicated DNA [14,25]. The cultures were then incubated for an additional 2 h, harvested, washed twice in and resuspended in 0.15 M NaC1/0.015 M sodium citrate (SSC). Cells were treated with 0.2% sodium dodecyl sulfate, ribonuclease, and pronase K, and the DNA was phenol extracted and dialyzed against SSC. Neutral CsC1 gradients were prepared by addition of 25--50 ~g of DNA plus SSC to give a final volume of 4.5 ml. Solid CsC1 was then added to give a final density of 1.703 g/cm 3, followed by hydrodynamic shearing by five successive passages through a 22-gauge syringe needle. This procedure results in DNA of average size about 25 S as measured in neutral sucrose gradient [14]. The samples were then centrifuged for 60 h in a Beckman SWS0. I rotor at 30 000 rev./min and 25°C. Gradients were collected from the bottom and the individual fractions analyzed for absorbancy at 260 nm and for radioactivity (Fig. 2). Selected regions of the density gradients were pooled, dialyzed against SSC to remove CsC1 and rebanded [11,24], recovery being approx. 80% after this procedure. Cell survival Measurements of cell survival were accomplished by cloning in agarose or cell plating in microtiter plates, a method modified from a transformed centers assay originally described by Moss and Pope [26]. Briefly, lymphoblastoid cells at known cell densities either mutagen treated or control nontreated were serially diluted with an automatic pipette into individual wells of a 96-well microtiter plate previously inoculated with complete media. The plates were then incubated in a humidified 5% CO2 in air atmosphere. Colonies which developed within 3 weeks were counted with an inverted tissue culture microscope and the initial colony-forming units per seeded cell determined using previously described calculations [ 27].

RESULTS Colony formation is usually the first parameter examined in an attempt to demonstrate an increased sensitivity to chemical or physical carcinogens. In this regard, LCLs readily lend themselves to these types of assays [ 28]. This laboratory has recently established a number of LCLs representing the classical chromosomal breakage syndromes as well as other genetic diseases (E. Henderson and J. German, in prep.). As a result, in Table II we can now present a summary of the data obtained from survival measurements (expremed as an LD37) using these LCLs and several representative chemical and physical mutagens. The values obtained as a result of these studies are in general agreement with those published in literature reviews for fibroblast

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70 lines established from individuals with these syndromes [ 29,30]. It is important to note that the phenotypes of our transformed LCLs, whether established from a clinically normal individual or an individual with an apparent repair defect, appear to remain unchanged and stable following continuous culture. This stability is of course necessary if these lines are to be used in attempts to analyze the underlying metabolic defects. It should be pointed out, however, that differences in sensitivity to UV-light have been found to exist between isogenic cell types such as B- and T-lymphocytes [31], and therefore differences could exist between a u t o c h t h o n o u s cell lines from the same individual. Agarwal et al. [15] has previously shown that deficits in DNA repair could be detected by monitoring [3H]dT incorporation into UV-irradiated phytohemagglutinin stimulated, human peripheral blood lymphocytes. The rate of incorporation of thymidine in irradiated l y m p h o c y t e s during the second and subsequent rounds of DNA replication is taken to be indicative of the ability of the cells to repair damage to DNA, unrepaired lesions being a block to DNA synthesis. In attempts to determine the efficiency of these methods for established cell lines, we have attempted similar experiments using permanently proliferating LCLs established from normal and repairdefective individuals. In Table III we have examined DNA synthesis following UV-irradiation in LCLs established from xeroderma pigrnentosum complementation group A (B5-7-1), Cockayne's syndrome (AA8-8) and a normal adult (AA87-2-4). As can be seen from the percents column, the initial inhibitory effects of UV-irradiation on DNA synthesis were very similar in each of the 3 cell lines tested, indicating the rates of lesion formation were very likely similar. However, at subsequent times after irradiation, 24 h and 48 h, the normal cell cultures had recovered much of their DNA synthetic capabilities, whereas DNA synthesis in both the xeroderma and Cockayne's LCLs showed a reduced ability to recover, with xeroderma appearing to be more sensitive. In three other experiments the same p a t t e r n of recovery was seen with xeroderma heterozygotes appearing to behave as normal cell lines. Similar experiments were next carried o u t using the chemical carcinogen A A A F and an additional LCL established from dyskeratosis congenita (B43-1). It was observed (Table IV) that following AAAF exposure for 1 h and an immediate [3H]dT pulse there were similar depressions of DNA synthesis in each of the cell lines. However, at later times following A A A F treatment the DNA synthesis of xeroderma, Cockayne's and to a lesser degree the dyskeratosis LCLs failed to recover to the levels obtained by the normal LCL. A third series of experiments was carried o u t using MNNG as an inhibitor (Table V). In this experiment a cell line established from an ataxia telangiectasia patient (BB91-3-1), having an increased sensitivity to MNNG as determined by colony formation, was included (Table II). Unlike results with UV-light and AAAF in which a sensitivity in colony formation was accompanied by an easily detected sensitivity with respect to DNA synthesis, a reproducible reduction in the capability of ataxia LCLs to recover DNA synthetic ability following MNNG treatment was not seen, indicating that

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73 the lesion(s) responsible for increased killing in the ataxia LCL may not be an immediate block to DNA replication. Physical removal of lesions introduced into DNA is one mechaniam available to a cell which would enable it to survive potentially lethal damage. This process, if it involves nucleotide insertion, can be detected and quantirated using radioactively labeled DNA precursors [23]. Therefore, we have employed labeling with a BrdU : [3H]dT mixture followed by CsCI rebanding to analyze excision repair in LCLs treated with UV-light, AAAF or MNNG. Recognizing that DNA repair is both dose- and time-dependent, in these

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SAMPLE NUMBER Fig. 1. Plots of neutral cesium chloride rebanding of DNA isolated from control or UVtreated LCLs. Approximately 2.5 × 10 ~ exponentially growing cells were incubated with BrdU, 10 ~glml for 30 rain, then exposed to UV-irradiation (254 nm) in a petri dish at a close rate of 5 ergs/mm=/sec as determined by a Laterjet meter for a total fluence of 300 ergs/mmL Immediately after irradiation, [SH]dT 40 Ci/mM, 25/~Ci/ml was added and the cultures incubated for 2 h at 37°C. Following cell harvest, DNA was extracted and banded in neutral CsCI; the 5 fractions showing peak absorbaney at a normal density of 1.'/0 g/cm ~ were collected, combined, then dialyzed ngainst cold SSC overnight, and then rebanded in neutral CsCI, collected and analyzed as described in Materials and Methods. Symbols: o, prordes of optical density at 260 nm; *, prot'des of radioactivity 3H. Increasing density from right to left.

74 experiments attempts were made to use mutagen doses and incubation times which result in maximal or near maximal levels of DNA repair synthesis in lymphoblast cultures [14,32]. In Fig. 1 we show BrdU : [3H]dT labeling patterns obtained following CsC1 rebanding of DNA extracted from LCLs established from xeroderma (B5-7-1), Cockayne's syndrome (AA8-8) and a normal adult (AA87-2-4) treated with UV-irradiation (300 ergs/mm2). Incorporation of radioactivity into non-replicated 'light' DNA is observed to be approx. 10 times greater in UV-irradiated preparations when compared to non-irradiated controls. As expected, much less radioactivity was found incorporated into non-replicated xeroderma DNA extracted from UVirradiated cel]s. The levels and pattern of repair replication seen in LCLs using neutral CsC] rebanding are similar to that seen in semiconfluent human firbroblast cultures (WI-38, passage 16) (Fig. 2). The specific activities of unreplicated DNA from these gradients were calculated to be 52 3H cpm//~g of DNA for control non-treated cells and 697 ~H cpm//~g of DNA for the UVtreated cells without a second banding in CsC1. These values are higher than the specific activities of DNA obtained from UV-treated normal and xeroderma heterozygote LCLs, which were closer to 450 cpm/#g of DNA, and may be explained by slight differences in thymidine incorporation pathways. In Table VI are presented specific activities calculated from non-replicated DNA obtained in these and similar CsC] rebanding experiments. Two points are worth mentioning here. First, we can apparently detect elevated levels of excision repair synthesis (15% of normal) in UV-irradiated LCLs estab-

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Fig.2. Neutral cesium chloride banding o f D N A isolated f r o m c o n t r o l or UV-treated WI-38 (passage 16) normal h u m a n fibroblasts. F o u r 1 0 0 - m m culture dishes o f first-day c o n f l u e n t cells were exposed to B r d U 10 # g / m l for 30 min. T w o culture dishes were t h e n e x p o s e d to UV-irradiation (300 e r g s / m m ~ ) and the o t h e r t w o culture dishes k e p t in the dark. I m m e d i a t e l y after irradiation, [3H]dT 41 C i / m m , 25 # C i / m l was added and the cultures i n c u b a t e d for 2 h at 37°C. the cells were harvested by rubber "policemen and the D N A e x t r a c t e d as in Fig. 1 and b a n d e d o n c e in CsCI, collected and analyzed as described. S y m b o l s : o, profiles o f optical density at 260 n m ; e, profiles of radioactivity 3H. Increasing density f r o m right to left.

75 TABLE VI SPECIFIC ACTIVITIES OF DNA REPAIR IN LYMPHOBLASTOID CELL LINES (LCLs) ESTABLISHED FROM CHROMOSOMAL BREAKAGE SYNDROMES OR RELATED DISORDERS TREATED WITH UV-LIGHT OR AAAF a LCL treatment b

3H cpm

Absorbancy (260 nm)

cpm/~g DNA c

1382 20 349 16 146

1.65 2.24 1.85

42 454 435

2320 13 725 13 323

1.76 1.50 1.59

66 455 418

1391 2165 3581

1.75 1.07 2.01

40 101 89

2461 10 408

1.68 1.33

73 389

1910 17 029

2.02 1.94

47 438

2501 9943

4.12 3.34

30 149

Normal (AA8 7-2-4) None UV AAAF

Cockayne's syndrome (AA8-8) None UV AAAF

Xeroderma pigmentosum group A (B5-7-1) None UV AAAF

Dyskeratosis congenita (B43-1) None UV

Xeroderma heterozygote (B5-3-1) None UV

Normal unstimulated peripheral blood leukcytes None UV

a2 h of incubation with BrdU, [SH]dT postinsult. bUV-light: 300 ergs/mm 2 ; AAAF: 25 ug/ml for 1 h. CCalculated for 5 peak, normal density fractions of CsCI rebands (0.40 ml total volume, assuming that a solution with an absorbancy of 1.0 at 260 nm has 50 ug/ml DNA) (Painter and Young [ 32 ] ). Specific activity of replicated hybrid density DNA on first CsCI banding approx. 75 000 cpm/ug.

lished from complementation group A xeroderma (Table VI). Petinga et al. [33] were also able to show increased rates of unscheduled DNA synthesis in a complementation group A xeroderrna fibroblast line in which unscheduled DNA synthesis had previously been thought to be totally absent. Secondly, insertion of radiolabeled precursor into normal density DNA of unirradiated cultures was also seen although the specific activity was an order of magnitude less than in irradiated cultures (Table VI). The authors have also found that this radioactivity remains associated with the light peak following banding in alkaline (pH 11.5) CsC1. Such insertion of precursor in nonmutagenized cultures has previously been observed in density labeling experiments employing [3H]BrdU [34] as well as a [3H]dT : BrdU mixture

76

[25]. Whether this phenomenon represents contamination between the hydrid and normal density fractions or bona fide excision repair in the absence of exogenous mutagens remains to be determined. AAAF-induced DNA damage has been reported to be repaired by cellular mechanisms shearing a rate limiting step with the processes involved in UV-induced repair [35]. Therefore, CsC1 rebanding experiments similar to those with UV-irradiation were carried out on LCLs using the chemical carcinogen and alkylating agent, AAAF, and are summarized in Table VI. As can be seen from the specific activities calculated from unreplicated DNA in Table VI, both normal and Cockayne's syndrome but not xeroderma LCLs showed extensive levels of excision repair following AAAF treatment (25 /~g/ml). In addition, it can be determined by the radioactivity incorporated into non-replicated DNA that AAAF treatment elicited specific activities of repair synthesis very near those obtained for UV-irradiation. MNNG is an alkylating agent which, when investigated by BND-cellulose chromatography, induced levels of DNA repair intermediate between that seen for UV-light and methyl nitrosourea [14]. We therefore attempted to quantitate MNNG-induced excision repair in LCLs using density labeling procedures. As can be seen from the data summarized in Table VII, after

TABLE VII SPECIFIC ACTIVITIES OF DNA REPAIR IN LYMPHOBLASTOID CELL LINES (LCLs) ESTABLISHED FROM CHROMOSOMAL BREAKAGE SYNDROMES OR RELATED DISORDERS TREATED WITH MNNG a LCL treatment b

3H cpm

Absorbancy (260 nm)

cpm/ug DNA c

Normal (AA87-4-1) None MNNG

1933 6208

2.79 1.67

35 186

Cockayne 's syndrome (AA 8 ~ ) None MNNG

1334 4239

2.29 1.12

29 188

Xeroderma pigmentosum group A (A64-2-1) None MNNG

651 2687

1.20 1.03

27 129

Ataxia telangiectasia (BBg l-3-1) None MNNG

958 2360

1.36 0.94

35 125

a 2 h of incubation with BrdU, 10 ~g/ml, [SH] dT 25 ~Ci/ml, 56 Ci/mmol postinsult. bMNNG: 25,00/zg/ml. CCalculated for 5 peak, normal density, gradient fractions of CsCI rebands (0.40 ml total volume, assuming that a solution with an absorbancy of 1.0 at 260 nm has 50 ~g/ml DNA) (Painter and Young [32]. Specific activity of replicated, hybrid density, DNA on first CsCI banding approx. 85 000 cpm/~g.

77 MNNG treatment (25/~g/ml), each of the cell lines tested displayed excision repair. Slight differences were seen in the specific activities among the cell lines tested, which were 25--35% of the activity that was obtained with UV-light. Interestingly, both xeroderma and ataxia LCLs had only a slightly reduced level of repair following MNNG treatment. Scudiero has also reported a reduced level of DNA repair in 4 of 6 ataxia fibroblast lines [36]. This is in contrast to marked differences in cell survival among the cell lines following MNNG treatment (Table II). This discrepancy could be explained by differences between cell lines in repair mechanisms not detectable by labeling methods which employ DNA precursors, such as the type of repair observed by Karmn and Lindahl [37] in MNNG adapted E. coli. DISCUSSION A group of genetic diseases has been recognized which are characterized by chromosomal instability and increased instances of neoplasia [38]. These disorders have been collectively referred to as 'chromosomal breakage syndromes' and include xeroderma pigmentosum, Fanconi's anemia, ataxia telangiectasia and Bloom's syndrome. Each of these disorders has been intensely studied, and some of their phenotypes have been identified at the cellular level, yet the exact enzymatic defects associated with these diseases remain unknown although DNA repair mechanisms are thought to be defective. It should be pointed out, however, that Bloom's syndrome appears normal in all assays of DNA repair. In this study we have employed LCLs established from individuals with these chromosomal breakage disorders, as well as from clinically normal individuals, with the prospect of demonstrating the usefulness of LCLs for these types of investigations. LCLs may be the only cell type that permits growth of large numbers of cells from these genetic disorders for biochemical experiments.

LCL phenotype following transformation LCLs established following in vitro exposure of lymphocytes to EBV, harbor the viral genome in latent form [39] and express the Epstein-Barr directed nuclear antigen [40]. Although only 5% of the EBV genome appears to be transcribed into a form of RNA which can be translated [41], the possibility still exists that EBV-infection could alter the DNA repair capabilities of the cell. For example, partial inhibition of postreplication repair processes has been seen by Waters et al. [42] in rat fibroblast lines infected with Rauscher leukemia virus. The fact that large viruses like herpes simplex and EB carry genes for thymidine kinase and DNA polymerase makes it certain that there will be some points of difference in virally transformed cell lines affecting DNA metabolism (replication or repair), if the techniques used are sensitive enough. However, in our preliminary analysis of LCLs as regards their sensitivity to both chemical and physical carcinogens (Table II), we found that each LCL showed the same sensitivity which had previously been identified with the syndrome using

78 fibroblast lines. We conclude from these data that presently known DNArepair pathways remain basically unaltered in cells transformed in vitro by EBV.

Excision repair of DNA damage UV- and AAAF-induced DNA damage are repaired by mechanisms characterized as long patch (80--100 nucleotides inserted), as opposed to short patch (1--5 nucleotides inserted) used to repair some types of damage such as that induced by X-rays [43]. However, disagreement exists as to whether the long patch mechanisms which recognize the major lesions are the same for both UV- and AAAF-induced damage [35,44]. We have found, using xeroderma LCLs, as have others using xeroderma fibroblasts [45,46], that cultured xeroderma cells show increased cellular sensitivity and reduced excision repair for both UV-light and AAAF. On the other hand, Cockayne's LCLs which have reduced cell survival have normal levels of excision repair following either UV or AAAF treatment. This adds support to the hypothesis that a major lesion induced by UV-light and AAAF is recognized by mechanisms which share a similar rate-limiting step or which are under the same genetic control. We further found that the chemical carcinogen, MNNG, induced an intermediate level of excision repair measured by density labeling, agreeing with data obtained by BND-cellulose chromatography [ 14].

Cockayne's syndrome Cockayne's syndrome is a rare autosomal recessive disease characterized by dwarfism, acute sun sensitivity and progressive neurological abnormalities [47]. The possibility that Cockayne's syndrome patients have a DNA repair defect was first suggested by Schmickel et al. [48] when they reported increased sensitivity to UV-irradiation in post-UV colony-forming experiments and subnormal levels of UV-induced unscheduled DNA synthesis in Cockayne's cells. Cultural fibroblasts from Cockayne's patients have since been shown by Andrews et al. [49] to have decreased post-UV colonyforming ability but normal rates of unscheduled DNA synthesis, suggesting normal levels of repair. Our data agree with that of Andrews et al. [49], in that, using density labeling, Cockayne's LCLs dispalyed normal levels of UVand AAAF-induced excision repair. These results allow several hypotheses regarding the nature of the cellular defect in Cockayne's, e.g.: (1) the UVinduced excision process in Cockayne's is normal, the defect being unrelated to DNA repair; (2) the DNA repair process associated with long patch repair, while being quantitatively normal is qualitatively abnormal (error prone). Initial experiment using host cell reactivation of UV-irradiated herpes simplex virus type 1, and subsequent recovery of deoxypyrimidine kinase mutants, resistant to iododeoxycytidine, suggests that Cockayne's syndrome cells may have error-prone long patch repair (E. Henderson and W. Long, in prep.)

79 ACKNOWLEDGEMENT

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