Elevated hprt mutant frequency in circulating T-lymphocytes of xeroderma pigmentosum patients

Mutation Research, DNA Repair, 273 (1992) 171-178 © 1992 Elsevier Science Publishers B.V. All rights reserved 0921-8777/92/$05.00

171

MUTDNA 00188

Elevated hprt mutant frequency in circulating T-lymphocytes of xeroderma pigmentosum patients J a n e C o l e , Colin F. A r l e t t , P a u l G. N o r r i s a, G a b r i e l l a S t e p h e n s , Alastair P.W. W a u g h , D a v i d M. B e a r e a n d M i c h a e l H.L. G r e e n MCR Cell Mutation Unit, University of Sussex, Falmer, Brighton, East Sussex I~NI 9RR (Great Britain) and a Photobiology Unit, Institute of Dermatology, St. Thomas' Hospital, London (Great Britain) (Accepted 20 May 1991)

Keywords: Xeroderma pigmentosum; Hprt mutant frequency; T lymphocytes;UV sensitivity; Skin cancer

Summary The mutant frequency to 6-thioguanine resistance in circulating T-lymphocytes from 10 xeroderma pigmentosum patients (including complementation groups D and G and XP variants) has been determined. A highly significantly elevated frequency was observed, compared to age-matched, non-smoking control donors ( × 2.1-fold higher than the mutant frequency in normal control donors, adjusted for age and cloning efficiency, p < 0.001). The mutant frequency of 5 XP heterozygotes was in the normal range, when age, smoking habit and log cloning efficiency were taken into account. A number of possible factors which may account for the elevated mutant frequency seen in the XP donors (including an elevated spontaneous mutation rate, UV mutagenesis of the T-cells as they pass through the skin, an effect of environmental mutagens such as tobacco smoke,, or as a consequence of immune defficiency) are discussed.

Xeroderma pigmentosum (XP) is a rare recessire disorder characterised by exaggerated sunburning, increased susceptibility to cutaneous malignancics and in some cases progressive neuro-

Correspondence'. Dr. Jane Cole, MRC Cell Mutation U n i t , University of Sussex, Falmer, Brighton, East Sussex BNI 9RR

(Great Britain).

Abbret,iations: BCC, basal cell carcinoma; CE, cloning efficiencyl GUM, generalised linear interactive modelling;

HPRT, hypoxanthineguaninephosphoribosyltransferase;MF, mutant frequencyl NK, natural killer cell; SCC, squamous cell carcinoma; 6TG, 6-thioguanine; UDS, unscheduled DNA syn-

thesis; XP, xerodermapigmentosum.

logical disorders (Kraemer et al., 1987). Cellular studies have identified two groups of XP patients. In the first, excision repair of ultraviolet-induced DNA lesions such as pyrimidine dimers is defective and the cells from such patients show reduced levels of unscheduled DNA synthesis (UDS) following UV-irradiation. Cell-fusion studies have identified 7 ¢omplementation groups amongst these XP patients (Hoeijmakers ~,d Bootsma, 1990). In the other group of patients known as XP variants, excision repair is normal but the cells are defective in post-replication repair following UV-irradiation (Lehmann et al., 1975). It has been demonstrated that cultured skin fibroblasts (Maher and McCormick, 1976; Arlett and Harcourt, 1983), EBV transformed

172

lymphoblastoid cells (Tatsumi et al., 1987) and T-lymphocytes (Arlctt and Cole, 1989; Arlett et al., 1992) from both excision-defective and XP variant patients are hypersensitive to the lethal effects of UV-irradiation (254 nm) although the degree of sensitivity varies considerably between patients, even within one complementation group (Hoeijmake:'s and Bootsma, 1990). It has also been demonstrated using cultured cells that while the spontaneo~:s frequency of mutants at the hypoxanthine guanine phosphoribosyl transferase (hprt) locus is within the range seen in cells derived from normal individuals, cultured cells from both excision defective and excision competent XP donors are hypermutable following 254nm UV-irradiation (Arlett and Harcourt, 1 9 8 3 ; Maher et al., 1976, 1977). We have reported preliminary results showing an elevated in rive hprt mutant frequency in circulating T-lymphocytes in a limited number of XP patients (Cole et al., 1989, 1990, 1991; Norris et al., 1990; Anstey et ai., 1991; Tares et al., 1989). In this paper we report the accumulated results of our studies over a number of years on 11) XP patients, including 5 patients from templementation group D, one of whom, XP8BR (M. Stcffanini, personal communic.'ttion) has symptoms of XP and Cockayne's syndrome, 2 in complementation group G (Norris et al., 1987), 2 XP variants, and one patient (XP6BR), complemenration group undetermined, with a histo~ of sell-healing melanoma (Anstey et al., 1991).

Materials and methods

consisting of T and B lymphocytes, were counted, diluted as appropriate and plated at 200 #l/well in 96-well microtiter ~ trays (Nunc) in RPMI 1640 Hepes-buffered medium (Dutch Modification, Gibco) supplemented with 10% human AB serum, 100 units per ml penicillin, 100/zg per ml streptomycin, 2 mM glutamine, 100 /xg per ml sodium pyruvate, 0.5% of the mitogen phytohaemagglutinin (HA15, Welcome diagnostics), recombinant interleukin-2 (kindly supplied by Cetus Corporation, U.S.A.) and 104 irradiated lymphoblastoid feeder cells per well (GMI899A, an HPRT- line supplied by the Human Genetic Mutant Cell Repository, Camden, N J, U.S.A.). To determine the cloning efficiency (CE) under non-selective conditions the lymphocytes were diluted to 15 cells per ml (3 cells per well, one plate). To determine the hprt mutant frequency (MF), the lymphocytes were plated at 1 x 105/ml (2 × 104 per well, 2-8 plates) in the presence of 5 × 10-* M 6TG. The plates were incubated for 17-20 days in a humidified 5% CO.,:air incubator before being scored microscopically for negative wells. The CE in the presence or absence of 6TG was calculated from the zero term of the Poisson distribution and the MF per clonable cell calculated as in Henderson e~ al. (1986). In several cases, the mutant frequency was determined from more than one blood sample, and in many cases the sample was split and it was determined in two or more separate experiments. In each experiment, the MF of one or more normal donors was deter. mined to provide control data.

Experimental protocol

Statistical analysis

Heparinised peripheral blood samples we,e obtained from the 10 XP patients and 5 XP heterozygotes listed in Tables 1 and 3. The mononuclear cell fraction was separated by standard procedures and stored in liquid nitrogen in foetal calf serum + 10% DMSO as described in Cole et al. (1988). The mutant frequency to 6thioguanine resistance (6TG R~') was determined essentially as in Cole et al. (1988). The MNCs were thawed, and cultured overnight in the absence of mitogen, when the m o n o c y t e / maerophage cells largely attached to the flask, The lbllowing day, cells in suspension, mainly

The data were analysed using the statistical package GLIM (Generalised Linear Interactive Modelling, Royal Statistical Society, London) (Henderson et al,, 1986; Cole et al,, 1988). The log MF in XP donors was compared to our control data base for children and non-smoking adults, We have found that MF increases with age. and is inversely related to log cloning efficieney (CE)(Cole et al., 1988). Analysis took both these factors into account, Since we have demonstrated that the MF in normal donors is elevated in smokers, the XP heterozygotes were compared against our normal data base for both smoking

173 TABLE 1 XERODERMA PIGMENTOSUM SUBJECTS Donor number

Age when studied (years)

Sex a

Designation (complementadon group)

UV-sensitivity and UDS h

Comments

82

i 4-16

M

XP124LO(G)

14% UDS

No skin cancer or neurologic abnormalities. Reduced NK activity

83

12- i 3

F

XP 125LO(G)

12% UDS

No skin cancer or neurologic abnormalities. Reduced NK activity

93

16

M

XP I 11LO(D)

Fibroblasts UV sensitive

Neurologic abnormalities 2 SCC. Reduced NK activity

95

7- 8

M

XP135LO(D)

Fibroblasts UV sensitive. 9% UDS

No skin cancer or neurologic abnormalities. Reduced NK activity

98

16-17

M

XP 107LO(D)

Lymphocytes UV sensitive. 18% UDS

Neurologic abnormalities. No skin cancers. Reduced NK activity

103

36

F

XP 1DU(D)

Lymphocytes and flbroblasts UV sensitive. 2t)~ UDS

11)4

48

F

XP3DU (variant)

Fibroblasts slightly

UV sensitive. Defective daughter strand repair 205

62

M

XPIIBR (variant)

Defective daughter strand repair

SCCs, BCCs, melanoma

treated with therapy for several years,

253

67

M

XP6BR

Lymphocytes and fibroblasts UV sensitive. 15q~ UDS

Multiple self-healing melanomas. Increased q~ NK cells ( = normal level of activity),

316

1

M

XPgBR(D)

Lymphocytes and fibroblasts VERY sensitive. 5()~, UDS, Very sensitive postUV RNA synthesis.

Symptoms of XP and Cockayne's syndrome. The patient died age 2 years,

M, male: F, female. b UDS, unscheduled DNA synthesis, c NK, natural killer cells; SCC, squamous cell carcinoma; BCC, basal cell carcinoma. The smoking habit of donors 104 and 205 is not known, donor 82 started smoking during the study and the remaining donors are non-smokers.

174

TABLE 2 HPRT MUTANTFREQUENCIES IN XP PATIENTS Donor

Expt.

No.a 82 (XPI24LO)

83 tXPI2:)LO) 93(XPIIILO)

1

2a b

1

C.E. (%) 72.0

38.4 38.4

95.3

Mutant frequency ×10 -~

7.74 7.66

6.07

3.63

2

44.8

3.52

1

32.7 13.0

5.72 9.08

2a b

13.0

24.67

la

59.7

4,08

b

57.7 35.6

4.07 6.09

I 2

20.4 21.1

40.49 21.82

1I)3(XPIDU)

1

45.5

24.22

104 (XP3DU)

2 ia b c

44.2 46,2 55.8 69,3

32.82 40.29 28,30 22,78

1 In b 2 I

72.2 29.0 37.7 s:.3 2.2

19.92 49.19 29.~!

95 (XPI35LO)

2

98(XP107LO)

3

2()5(XPI IBR) 253(XPhBR)

316 (XPSBR) "

9. !

20.98

2~,o9

10.~9

Numbers indicate r~pe.I blood samples, letters indicalu replicate experiments from one blood sample.

and non-smoking adults, taking age, log CE and smoking habit into account. Results The XP patients we have studied here, with their designations and complementation groups when known, UV sensitivity and clinical syruptoms are listed in Table 1. The results of the mutant frequency determinations with these padents and some of their heterozygous parents are listed in detail in Tables 2 and 3, and summarised in Figs. 1 and 2. In Fig. I, the log mean MF for each XP is shown, together with the log mean

MF of all the normal, non-smoking donors in our data base. The latter has been obtained over a period of years, and includes data from the experiments performed at the same time as the XP results, as well as other experiments on normal donors undertaken during this period. The majority of the data points from the normal donors are the mean of repeat and split blood samples (Cole et al., 1988, 1989) which enable us to estimate the variability within and between donors, as well as estimating the effect of donor age, donor category, smoking habit and cloning efficiency. An analysis of this substantial data base using GLIM reveals a highly significant increase in hprt murant frequency in the XP patients (×2.1 higher than normal donors, adjusted for age and CE, p < 0.001, t = 5.18). As a group, the XP heterozygotes do not show an elevated MF, although it should be noted that the non-smoking heterozygote with the highest MF (from 2 separate blood samples) is the mother of the XP child with the highest mutant frequency,

Discussion It has boon well-documented that XP patients have a considerable excess (over 1000-fold) of BCC and SCC in sunlight exposed a)eas of the skin, for example head, eyes, tongue and neck, and in addition an excess of malignant melanoma in non-exposed parts of the body (Cleaver, 1990; Hooijmakers and Bootsma, 1990; Kraemer et al., 1987; Lehmann and Norris, 1989). An excess of tumours, although considerably lower (10-20fold), has also been suggested for some internal areas of the body not exposed to sunlight (Kraemer vt al., 1987), However, the numbers are smaller and the evidence for non.UV induced cancer in XPs is not secure (Cairns, 1981; Krae. mer et al., 1984; Takebe et al., 1987). What is clear is that UV-induced mutation is implicated as one of the causes of skin cancer in XP patients. Several possibilities may be proposed for the elevated frequency of hprt mutants in circulating T-lymphocytes that we have observed in XP patients, Firstly it is possible that the spontaneous mutation rate might be higher in the XP patients than in normal donors. This does not seem to be

175 100

100

,

,

~-~

,

, .

10

~

lO

~

~ ~'

l

~"

&

~

.1

o

20

40 60 Age (yrs)

80

100

Fig. 1, Mean mutant frequency in XP donors compared to normal, non-smokers, n , XP (non-smoker); II, XP (started smoking during the study); l]l, XP (smoking habit unknown); o, normal non-smoking donors,

a likely explanation since there is no evidence from in vitro studies that XP cells have spontaneously elevated frequencies of mutants at the hprt locus (Arlett and Harcourt, 1983) or at the diphtheria toxin locus (Glover et al., 1979), or of chromosome aberratiotts, SCEs (de Weerd Kas~elein et al., 1977; Wolff et al., 1977) or cell transformants (McCormick et al., 1986). Cultured

cPo~r ~_o o q) ~" o'12]

Oo

o

o

o

1

.1

0

W~o

'

0

'

'

20

'

'

'

40 60 Age (yrs)

80

Fig. 2. Mean mutant frequency in XP heterozygotes compared to normal adult smokers and non-smokers. D, XP heterozygores, non-smoker; II, XP heterozygotes, smokers; ra XP heterozygote, smoking habit unknown; o, normal non-smoker; o, normal smoker.

XP cells are, however, hypersensitive to the induction of lethal and mutagenic effects of UVCirradiation and certain chemical carcinogens that form bulky adducts on DNA e.g. polycyclic aromarie hydrocarbons like benzo[a]pyrene, 4nitroquinoline-N-oxide or dietary mutagens such as tryptophan pyrolysis products (Arlett and Har-

TABLE 3 MUTANT FREQUENCY IN XP HETEROZYGOTES Donor No.

ABe

Sex

Smokin8 habit "

XP relative

CE

81

37

F

S

Mother of 82 and 83

70.6

9.48

94

37

F

S

Mother of 93

52.3 47.6 47.6

10.01 6.10 8,62 4.60

66.6

Mutant frequency ×10 ~

97

34

M

NS

Father of 95

80.8 78.6 63.8 98,5

3.65 4.70 4.11 1.11

99

41

F

NS

Mother of 98

74.3 31.8

36.57 24.16

141

30

F

UK

Mother of 95

18.0

12,45

a S, smoker; NS, non-smoker; UK, unknown.

100

176

court, 1983; Edwards et al., 1987; Heflich et al., 1980; Kraemer et al., 1984; Maher et al., 1 9 7 7 ; Maher and McCormick, 1976; McCormick and Maher, 1978; Patten et al., 1984; Simon et al., 1981; Wolff et al., 1977). This suggests a second possibility for the elevated in vivo frequency of hprt mutants, namely elevated induced mutation in lyraphocytes as a consequence of mutagen exposme and defective repair of the DNA damage, Thig DNA damage could occur as a result of solar irradiation (Patton et el., 1984) of lymphocytes as they passed through the skin, or as a result of exposure to chemical carcinogens, for example from cigarette smoke. We have shown that the mutant frequency in normal donors is elevated in smokers (Cole et al., 1988; Bridges et el., 1990). While most of our XP patients are documented as non-smokers, it is possible that environmental smoke ("passive" smoking) might be considerably more mutagenic for XP patients, and we note that some of the children in our study have smoking parents (Table 3). it is also conceivable that mutagens in the diet (ProticSabljie et al., 1985), or other environmental mutagens that induce bulky DNA adducts, are responsible for part of the elevated frequency seen in XPs. it may be possible to distinguish some of these alternatives by analysing the mutational spectrum seen in T-lymphocytes from normal and XP donors. The UV-induced mutational spectrum has been investigated in vitro in fibroblasts (Dorado et el., 1990) and we are at present undertaking a similar study of the in rive mutants in XP donors, A third possibility which might help to explain the elevated mutant frequency in XP patients is that it is a consequence of immune deficiency, either as the result of an intrinsic defect of immune surveillance or as a result of defective immune response to solar damage compared to normal donors (Kripke, 1984; Hersey et al., 1983). It was proposed some years ago by Bridges (1981) that such effects might be in part responsible for the high incidence of skin cancer seen in XPs. The immune status of a relatively small propertion of all XP patients has been studied, and some have been reported to have immune defects, including impaired cell-mediated immunity, combined immunodeficiency and NK cell abner-

malities (Kraemer et al., 1987; Oeaver, 1990; Norris et al., 1988, 1990; Lehlnann and Norris, 1989; Wysenbeck et ai., 1986; Morrison et al., 1985). Immune dysfunction has not been observed in other XPs, although in some cases not all immune functions have been investigated. It is possible that perturbations in the immune response may result in increased cell division rate in lymphocytes, which could result in elevated mutant frequencies. Rapidly dividing cells are more likely to mutate for two reasons. Firstly replication errors in cell division may lead to mutation and secondly, less time is available for the repair of DNA damage that is normally repaired in "resting" lymphocytes. Evidence that in rive mutations may indeed occur preferentially in dividing cells has been suggested by Aibertini et al. (1990). A final explanation for the elevated mutant frequency seen in circulating T-cells, that it could be the result of clonal expansion of hprt- mutants, is not supported by preliminary studies on mutational spectra, combined with T-ceU receptor analysis, of mutants from normal and XP patients (0. Stephens and H. Steingrimsdottir, unpublished observations). In conclusion, some mention may be made of the influence on mutant frequency of treatment of XP patients with retinoic acid derivatives for the prevention of skin cancers (Kraemer et el., 1988). One of our donors (XP variant donor No. 205, Berth-Jones et al., 1990) had been treated for a number of years with etretinate before his mutant frequency was tested, and we do not have an observation on his mutant frequency before treatment commenced. Four of the younger patients in our study were treated with etretinate for a number of months after the observations we report here were made, The mutant frequency in T-cells was mouitored during and after the therapy. Mutant frequencies were consistently lower following treatment, but the difference just failed to achieve significance (t = 2.00, n -- 27). We are now (one year after the last etretinate treatment) repeating these observations, but to date it does appear that etretinate therapy may indeed lead to a reduction in the frequency of hprt mutants in circulating lymphocytes. A larger sample size would clearly help to resolve this question.

177

Acknowledgements This work was supported in part by grant number EC4V0037.UK(H) from the European Community. W e would like to thank Professor B.A. Bridges and Dr. A.R. Lehmann for many helpful discussions, Drs. F. Gianneili and P. Botcherby (Guys), C. Moss and P. Friedmann (Newcastle), R. Moore (Belfast), M. Judge (Great Ormond St.), B. Johnson (Dundee) and J. Berth-Jones (Leicester) for supplying some of the blood samples, and all our

donors for their help and co-operation, Note added in proof X P 6 B R (donor 253) is now recognised as cornplementation group C (A. Joose and W. Wermeulen, personal communication),

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