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British Journal of Radiology (2006) 79, 510-517
© 2006 British Institute of Radiology
doi: 10.1259/bjr/83726649
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Full paper

Clinical and cellular ionizing radiation sensitivity in a patient with xeroderma pigmentosum

C F Arlett, PhD 1 P N Plowman, MD, FRCP, FRCR 2 P B Rogers, MB BS, MRCP, FRCR 2 C N Parris, PhD 3 F Abbaszadeh, MSc 3 M H L Green, PhD 4 T J McMillan, PhD 5,6 C Bush, BSc 6 N Foray, PhD 7 and A R Lehmann, PhD 1

1 Genome Damage & Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, , 2 Radiotherapy/Clinical Oncology, St Bartholomew's Hospital, 25 Bartholomew Close, West Smithfield, London EC1A 7BE, , 3 Division of Biosciences, School of Health Sciences and Social Care, Brunel University, Kingston Lane, Uxbridge, Middlesex UP8 3PH, , 4 School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Lewes Road, Brighton BN2 4GJ, , 5 Institute of Environmental and Natural Sciences, Faraday Building, Lancaster University, Lancaster LA1 4YA, , 6 The Institute of Cancer Research, Royal Cancer Hospital, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK, , 7 INSERM U647, ID17, European Synchrotron Research Facility, Rue Jules Horowitz, BP220 – 38043 Grenoble, France,


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
XP14BR is a cell line derived from a xeroderma pigmentosum (XP) patient from complementation group C. The patient was unusual in presenting with an angiosarcoma of the scalp, treated by surgical excision and radiotherapy. Following 38 Gy in 19 fractions with 6 MEV electrons, a severe desquamation and necrosis of the underlying bone ensued, and death followed 4 years later. The cell line was correspondingly hypersensitive to the lethal effects of gamma irradiation. We had previously shown that this sensitivity could be discriminated from that seen in ataxia-telangiectasia (A-T). The cellular response to ultraviolet radiation below 280 nm (UVC) was characteristic of XP cells, indicating the second instance, in our experience, of dual cellular UVC and ionizing radiation hypersensitivity in XP. We then set out to evaluate any defects in repair of ionizing radiation damage and to verify any direct contribution of the XPC gene. The cells were defective in repair of a fraction of double strand breaks, with a pattern reminiscent of A-T. The cell line was immortalized with the vector pSV3neo and the XPC cDNA transfected in to correct the defect. The progeny derived from this transfection showed the presence of the XPC gene product, as measured by immunoblotting. A considerable restoration of normal UVC, but not ionizing radiation, sensitivity was observed amongst the clones. This differential correction of cellular sensitivity is strong evidence for the presence of a defective radiosensitivity gene, distinct from XPC, which is responsible for the clinical hypersensitivity to ionizing radiation. It is important to resolve how widespread ionizing radiation sensitivity is amongst XP patients.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Xeroderma pigmentosum (XP) is a rare autosomal recessive disease [1] characterized by clinical and cellular sensitivity to ultraviolet (UV) radiation. The patients show hypersensitivity to sunlight and extreme susceptibility to sunlight-induced skin cancer [2, 3]. The majority of cases are defective in one of seven genetically distinct nucleotide excision repair genes (complementation groups A–G [3]) which confer increased cellular susceptibility to UV-induced killing and mutation [4–7]. In addition, cells are also hypersensitive to agents which generate bulky lesions in DNA [8]. Approximately 20% of cases are competent in excision repair, but defective in daughter strand repair [9] by virtue of a defect in a DNA polymerase ({eta}) [10]. Cells from such patients are minimally sensitive to the lethal effects of UV [11], but are hypermutable [12, 13].

Since ionizing radiation mainly produces single and double DNA strand breaks [14, 15] and types of base damage repaired by the alternative pathway of base excision repair [16, 17], hypersensitivity to ionizing radiation would not be anticipated as a feature in the XP syndrome. However, we have reported cellular hypersensitivity to the lethal effects of gamma radiation in fibroblasts from XP complementation group G, XP3BR [18] derived from a patient who never came to radiotherapy. There are two reports of patients from complementation group C who have been exposed to radiotherapy. DiGiovanna et al [19] recorded the uneventful radiotherapy of such a patient with an inoperable spinal cord astrocytoma, no assessment of cellular sensitivity to either UVC or ionizing radiation was provided (fibroblast cell line = XP23BE). In the second example [20], the patient died 3 months after a course of combined radiotherapy and chemotherapy for a thalmic glioma. Again, no assessment of cellular sensitivity was reported (fibroblast cell line = XP233VA).

We detail here radiosensitivity at both the clinical and cellular level in an XP patient assigned to genetic complementation group C. The homozygous mutation in the XPC gene generates a stop codon at codon 718, resulting in a truncated protein missing the C-terminal 233 of the 940 amino acids. To date, this patient is unique in carrying this mutation (see XP mutation database: URL http://xpmutations.org). The cell lines XP23BE and XP233VA acted as reference controls. Earlier published observations reported the clinical hypersensitivity in this patient as one of a series of four sensitive individuals encountered amongst 2000 paediatric radiotherapy patients during a 20 year period at St Bartholomew's Hospital [21]. Discrimination from ataxia-telangiectasia (A-T) was achieved on the basis of clinical criteria and by demonstrating a normal level of radiation resistant DNA synthesis.

We report here details of the pathology consequent to radiotherapy, further characterization of the cell line as XP, DNA repair studies following ionizing radiation and evidence for the existence of a separate radiosensitivity genetic defect.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
The patient
A clinical description of the patient, a female of Pakistani origin, is provided in Salob et al [22] together with details of the significant reduction to 9% in nucleotide excision repair in fibroblasts (designated 86/0029) and assignment to complementation group C. She was considered unusual in presenting with both XP and hypoplastic anaemia. At the age of 14 years, she developed an angiosarcoma of the scalp overlying the right parieto-occipital bone, a feature thought to be unique in XP [23]. Previous skin (epithelial) tumours had been treated with excision or with 5-fluorouracil cream.

Cells
Untransformed fibroblast cell lines from reference normal control donors, 1BR.3, 142BR, 149BR, 250BR and 251BR, were established in Brighton using standard procedures [24]. The normal cell line used at Sutton was GF11. Fibroblasts of the patient as used in the original complementation assignment [22] designated 86/0029 ( = XP14BR.1), were obtained from Dr P Botcherby (Guy's Hospital, London, UK). A second biopsy taken from an uninvolved region of skin during radiotherapy at St Bartholomew's Hospital generated a second cell line, XP14BR.2, in Brighton. Reference XP complementation group C fibroblast cultures from the studies noted above were obtained from Dr K Kraemer, NIH, Bethesda, MD (XP23BE) [19] and Dr A Sarasin, Villejuif, France (XP233VI) [20]. XP15BR is a reference XP cell line, generated in Brighton, from complementation group A (N Jaspers, Rotterdam, personal communication) cell line. Two A-T cell lines AT1BR and AT2BR [25] were used for scaling purposes. All these cells were maintained in culture using standard materials and procedures [11, 24] and were verified to be free of mycoplasma infection. All biopsies were obtained under the approved ethical procedures in place in the relevant institution at the time.

Fibroblast cell lines were transformed using the immortalizing vectors pSV3gpt and pSV3neo in Brighton in a programme using standard procedures [26]. Both pre-crisis (not immortal) and post-crisis (immortal) cultures of single cell origin were available for investigation. Reference normal material was generated using this vector from the cell lines 1BR.3 ( = 1BR.3-G, from pSV3gpt and 1BR.3neo from pSV3neo), and 142BR ( = 142BRneo, from pSV3neo) in addition to XP14BR ( = XP14BRneo17, from pSV3neo). Immortalized control cell lines MRC5V1 (from Dr P Debenham, former MRC Radiobiology Unit, Harwell, UK) and GM0637 (from NIGMS, Camden, NJ, USA) were also employed. The untransformed fibroblast cell line MRC5 was obtained from C Babbs, Huntingdon Research.

Table 1Go summarizes the designations of the strains used.


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  		Table 1. Designations of the cell lines compared in this investigation

 
Cell killing
Details of sources and dosimetry for UVC and a Cobalt 60 gamma ray facility, together with the design of cell killing experiments, have been described in detail elsewhere [24, 27]. The response to the cross-linking agent nitrogen mustard (Sigma Chemical Co., Poole, UK) was verified using standard protocols [28].

DNA repair
We have used two methods for the evaluation of repair following ionizing radiation. In the first, double strand break (DSB) induction and repair were evaluated at the Institute of Cancer Research, Sutton, using pulsed field gel electrophoresis (PFGE) [29–31], with a repair time of 4 h. A separate series of experiments evaluating DSB induction and repair was performed at Institut Gustave-Roussy, Villejuif, France. The experimental procedures are described in Foray et al [32]. A Caesium 137 radiation source was used for the Villejuif experiments. Here, the maximum repair time was 24 h. The control cell line was 149BR.

A second method, based upon a process of reduction in the frequency of excess chromosome fragments [33], was also undertaken at Villejuif. The premature chromosome condensation (PCC) technique permits the measurement of chromosome breaks in cells in G0/G1. Cells were irradiated in plateau phase of growth (6 Gy, {gamma}-ray 137Cs, 4°C) and were then returned to 37°C for repair. Chromatin condensation was generated by fusion of fibroblasts in plateau phase of growth with synchronized mitotic cells by using PEG 6000, as described previously [33], but with the notable exception that CHO cells were replaced by HeLa cells. After hypotonic treatment, cells were fixed in methanol:acetic acid and stained with Giemsa. At least 30 fusions were analysed by means of light microscopy. PCC data were expressed in chromosome fragments in excess (ECF) (total number PCC fragments minus 46). The reference normal cell line was 149BR.

Transfection with the XPC gene
The SV40-transformed XP14BRneo17 cell line was transfected with plasmid containing either the full-length XPC cDNA, generously supplied by P van der Spek (Rotterdam), or a clone truncated at the 5' end of the open reading frame, which nevertheless can complement the UV sensitivity of an XPC cell line [34]. Each plasmid was cotransfected with pSVgpt using calcium phosphate precipitation and selection was applied for the co-transfecting gpt marker using medium containing mycophenolic acid, aminopterin and xanthine. When colonies became visible to the naked eye, they were picked individually using cloning rings and expanded maintaining the selection for the gpt marker.

XPC protein measurements
Pellets of different cells were dissolved directly in Laemmli buffer and 15 µg protein run on 7.5% SDS-PAGE gels, followed by immunoblotting. The blots were probed with a polyclonal antibody raised against a peptide containing the C-terminal 18 amino acids of the XPC protein [35] and developed with the ECL kit (Amersham).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Radiation pathology
The angiosarcoma of the scalp was excised and the patient was referred for radiotherapy because of the closeness of the marginal excision. 6 MEV electrons were employed, which have a depth of penetration of approximately 2 cm in soft tissue and less in bone. Due to the characteristics of the electron beam, any further depth would be fast diminishing.

After 38 Gy radiation applied in 19 fractions, the child developed severe moist desquamation in the region in the scalp (Figure 1Go). Ulcerated and underlying necrotic bone at the depth of the ulcer was revealed and this never healed. This reaction is very far outside the normal expected range of response to this clinical radiotherapy dose prescription.


Figure 1
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  		Figure 1. The irradiated scalp portal. The irradiated skin remained depilated and became frankly ulcerated and necrotic in the operation wound site.

 
2 years following radiotherapy, the patient suffered a Grand Mal convulsion, and a CT scan of the brain showed extensive cerebral oedema arising from an oedematous focus directly beneath the radiation portal (Figure 2Go). However, the area of the oedema continued to spread through most of the hemisphere over the next few months. Steroids were employed and taken for a period of 22 months, but the child died from raised intercranial pressure and extending left hemisphere oedema 4 years after the radiotherapy. A diagnosis of "spreading cerebral necrosis, consequent upon the radiotherapy," was made.


Figure 2
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  		Figure 2. CT brain scan demonstrating widespread hemispheric oedema beneath the irradiated portal. Noteworthy is the widespread extent of the oedema throughout the hemisphere, not withstanding the restricted portal size, the moderate prescription dose(38 Gy) and the low energy electron portal (6 MEV) – that had to penetrate bone first. It was this unrelenting cerebral oedema, emanating from radio necrosis of brain under the radiation portal, that caused death.

 
Cell killing
The responses of XP14BR and a set of control and reference fibroblast cell lines to the lethal effects of UVC are illustrated in Figure 3Go. As anticipated XP14BR.2 fibroblasts and the earlier derived replicate culture, XP14BR.1 (86/0029) proved to be hypersensitive. Hypersensitvity was, as expected, also demonstrated with the reference cell lines XP23BE and XP15BR used for scaling purposes. With gamma irradiation, both versions of XP14BR were indistinguishable in their response and proved to be almost as sensitive as the two reference A-T cell lines (AT1BR and AT2BR). Two other cell lines derived from representatives of complementation group C (XP23BE and XP233VI), where no clinical sensitivity had been reported, were normal in their responses (Figure 4Go) as was XP15BR. With mitomycin C, no distinction in response between XP14BR.2 and the normal cell line 1BR.3 was observed (data not shown). For nitrogen mustard, a comparison between the immortalized XP14BRneo17 and the control lines MRC5V1, GM0637 and 1BR.3-G (Figure 5Go) suggested no increase in sensitivity in XP14BRneo17.


Figure 3
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  		Figure 3. Ultraviolet radiation below 280 nm (UVC) clonal survival of XP14BR fibroblasts. Key: Figure 3XP14BR.1 (biopsy 1); Figure 3XP14BR.2 (biopsy 2, mean ± standard error of 4 experiments). Normal fibroblast lines: Figure 31BR.3 (mean ± standard error of 3 experiments), Figure 3142BR, Figure 3250BR, Figure 3251BR. Additional XP cell lines: Figure 3XP15BR, Figure 3XP23BE. Single experiments except where indicated.

 

Figure 4
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  		Figure 4. Ionizing radiation clonal survival of XP14BR fibroblasts. Key as forFigure 3Go: Additional XP line; Figure 4XP233VA; Ataxia telangiectasia fibroblast lines; Figure 4AT1BR; Figure 4AT2BR. Means ± standard error based on numbers of experiments as follows: XP14BR.1, 1; XP14BR.2, 6; 1BR.3, 58; 142BR, 8; 250BR, 4; 251BR, 13; XP23BE, 2; XP233VA, 2; AT1BR, 5; AT2BR, 3.

 

Figure 5
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  		Figure 5. Nitrogen mustard sensitivity of SV40 transformed XP14BR fibroblasts. Key: Figure 5XP14BRneo17; Normal transformed fibroblast lines; Figure 5MRC5V1; Figure 5GM0637; Figure 51BR.3-G. Means ± standard error of 2–4 experiments.

 
DNA Repair
DSB were measured using PFGE. Immediately after irradiation there was a slight, but not significant, reduction in the level of induced damage in XP14BR in comparison with the control, GF11, over the dose range of 0–40 Gy. There was no evidence for a difference between the two cell lines after a 4 h period for repair (data not shown).

When, however, a time course of repair was generated (Figure 6Go) DSB repair in XP14BR.2 was more rapid at earlier times than in control cells. A similar pattern has been reported for A-T cells. At later times, however, the residual levels of DSB were greater than in normal cells, but less than in A-T cells. As a second measure of repair, the time course of repair of chromosomal damage, as reflected in the reduction of excess chromosome fragments, revealed a small, but consistent, deficiency in XP14BR.2 (Figure 7Go).


Figure 6
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  		Figure 6. DNA double strand break(DSB) repair rate after 30 Gy delivered at 4°C. Repair data are expressed as the fraction of radiation-induced DNA fragments (FAR) migrating in the gel that remained at the indicated repair times. Each point represents the mean ± standard error of at least three replicate experiments. Key: Figure 6XP14BR.2; Figure 6149BR, Normal non-transformed fibroblast line.

 

Figure 7
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  		Figure 7. Rate of repair of chromosome breaks after 6 Gy delivered at 4°C. Repair data are expressed as percentage of premature chromosome condensation fragments in excess (ECF) at the repair times indicated. Each point represents the mean ± standard error of at least three replicate experiments. Key: Figure 7XP14BR.2; Figure 7149BR, Normal non-transformed fibroblast line.

 
Transfection with the XPC gene
A series of single cell derived clones were established from the transfection of XP14BRneo17 with XPC cDNA. XPC protein was analysed by immunoblotting with an antibody to the C-terminus of the XPC protein. The XP14BR cell line has a mutation which truncates this protein [36], so that it lacks the C-terminus against which the antibody was raised. As expected, no XPC protein was detected in the XP14BRneo17 recipient cell line (Figure 8Go, lane 3). However, the expected 125 kDa band of XPC protein was restored to the level present in MRC5V1 (lane 4) in clones T921A (lane 1) and T943A (lane 2), but not in T941B or T948B (lanes 5 and 6). No protein band was visible in the T941B or T948B transfectants, using a low exposure of the X-ray film. Overexposure of the film revealed a very low level of C-terminal XPC protein in transfectant T941B, but not in T948B (Figure 8Go, lower panel, lane 5).


Figure 8
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  		Figure 8. Immunoblotting for the C-terminal end of the XPC protein. Lanes 1 and 2, T921A, T943A (transfectants); lane 3, XP14BRneo17 (recipient); lane 4, MRC5V1 (control); lanes 5 and 6, T941B and T948B (transfectants). Upper panel, 20 s exposure, lower panel, 5 min exposure.

 
Cell killing in transformed and transfected cells
In response to UVC, there was a significant enhancement of survival of T921A and T943A (the clones expressing substantial amounts of XPC protein) above XP14BRneo17. However, a similar level of restoration of UV resistance was also seen in clone T941B, which only expressed low levels of XPC protein (Figure 8Go). The UVC sensitivity recorded for these three clones was intermediate between XP14BRneo17 and GM0637 and MRC5V1 (Figure 9Go), the concurrent control normal cell lines.


Figure 9
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  		Figure 9. Ultraviolet radiation below 280 nm (UVC) sensitivity of XP14BRneo17 and its transfectants. Means ± standard error of the number of experiments indicated. Key: Figure 9XP14BRneo17 (non-transfected, 1 expt); Figure 9T921A (2 expts); Figure 9T943A (4 expts); Figure 9T941B (2 expts) (all transfectants); Figure 9MRC5V1 (1 expt); Figure 9GM0637 (2 expts).

 
The response of XP14BR.2, XP14BRneo17, three normal cell lines, 1BR.3, 142BR and MRC5 and their immortalized derivatives; 1BR.3neo, 142BRneo and MRC5V1 to graded doses of gamma irradiation is illustrated in Figure 10aGo. The enhancement of survival after ionizing radiation as a consequence of transformation was first described by Arlett et al [37]. This is particularly evident in XP14BR, with the consequence that the level of discrimination between it and the reference normals is less than in the corresponding untransformed cell lines. However, unlike the case with UVC, after exposure to gamma radiation no enhancement of survival above the parental cell line was observed. Indeed, all four clones appeared slightly more sensitive (Figure 10bGo).


Figure 10
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  		Figure 10. Ionizing radiation sensitivity of XP14BR, XP14BRneo17, and transfectants. Means± standard error of the number of experiments indicated. A. Effect of SV40 transformation on radiosensitivity. Key: Figure 10XP14BR (6 expts); Figure 10XP14BRneo17 (1 expt); Figure 101BR.3 (58 expts); Figure 101BR.3neo (4 expts); Figure 10142BR (8 expts); Figure 10142BRneo (3 expts); Figure 10MRC5 (1 expt); Figure 10MRC5V1 (2 expts). B. Comparison of transfectants with XP14BRneo17. Key: Figure 10XP14BRneo17 (1 expt); Figure 10T921A (2 expts); Figure 10T943A (3 expts); Figure 10T941B (2 expts); Figure 10T948B (1 expt).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Our patient was unusual amongst XPs in presenting with hypoplastic anaemia, and was originally thought to be a case of Fanconi's anaemia [22]. Her angiosarcoma was also considered to be unique [23] in association with XP. The clinical radiosensitivity and consequent pathology which followed treatment of this scapular lesion is peculiar to this individual [21].

At the cellular level, the response to UVC is unexceptional. However, the hypersensitivity to gamma radiation has, in our hands, only been demonstrated in one previous instance using fibroblasts (XP3BR) from an individual from complementation group G [18]. The lack of clinical sensitivity in the two other representatives of complementation group C, XP23BE and XP233VI, which act as reference controls, was reflected in their normal responses in our cellular assays (Figure 4Go). The cellular sensitivity of XP14BR to ionizing radiation may be contrasted to the lack of sensitivity to cross-linking agents and implies a cellular defect in repair specific to gamma radiation.

The study of repair of DSB in XP14BR generates results reminiscent of those obtained with A-T cells; an early, more rapid repair than in normals culminating at 24 h with less repair [32]. When combined with the outcome of the study of repair of chromosome fragments, these results are consistent with a defect in the repair of a small fraction of DSB as has been found for A-T cells [32, 38].

We have already acknowledged that the cellular radiosensitivity in XP14BR may be discriminated from that manifest in A-T cells by the normal level of radiation resistant DNA synthesis [21]. This alone is suggestive of the possibility of the presence of another non-A-T radiosensitivity gene, but first it was necessary to verify that the XPC mutation [36] itself was not responsible for the hypersensitivity.

The correction, by transfection with the XPC gene, of the XP genetic defect was, as expected, successful in restoring cellular resistance to UVC irradiation. In one transfectant, T941B, substantial resistance to UVC irradiation was restored, even though only very low levels of XPC protein were detected in the transfectant. This suggests that XPC protein is not rate-limiting for neuclotide excision repair and that this low level is sufficient to deal with significant levels of UV damage.

The psv3neo immortalization of XP14BR itself produced a large enhancement of ionizing radiation resistance. This effect of transformation, reported earlier [37], is specific to ionizing radiation and not UV, and may be generated by the abrogation of functional p53. However, the failure to enhance ionizing radiation resistance in clones, which, in addition to showing an enhancement of their UV response are also positive for the UVC gene product, supports the contention that XP14BR is a double mutant combining defects in both XPC and an unknown radiosensitivity gene. The resolution of this proposed genetic defect and the identification of the gene(s) becomes a priority.

The other unusual clinical features exhibited by the patient remain unexplained. There was no evidence for more specific defects in immunity.

The present demonstration of both clinical and cellular radiosensitivity in XP14BR raises the question of the frequency of such radiosensitivity in this syndrome. While XP14BR may be unique in being a double mutant, we have reported a previous instance of cellular sensitivity in XP3BR [18]. There was no relevant radiotherapy in this case. In this report we provide data showing normal cellular responses for XP15BR, from complementation group A and XP23BE and XP233VI from complementation group C. The record of the outcome of radiotherapy in the patient who generated the cell line XP23BE is unambiguous in excluding any clinical sensitivity [19]. The clinical information of the outcome of combined chemotherapy and radiotherapy for the second XPC patient is not so clear cut since the child died 3 months after treatment [20]. In the absence of any cellular radiosensitivity there remains the possibility of sensitivity to components of the chemotherapy regimen. As far as we are aware, the patient from whom cell line XP15BR was generated has never come to radiotherapy. In the light of the outcome of radiotherapy in our present case and the observation of cellular radiosensitivity in XP3BR, it becomes important to resolve the extent of radiosensitivity in XP. This is reinforced by the possibility that these patients, because of their high frequency of tumours, are more likely to experience radiation.

Current address for Dr P B Rogers: Royal Berkshire Hospital, London Road, Reading, Berkshire RG1 5AN, UK.


   Acknowledgments
 
We are grateful to Randy Legerski and Peter van der Spek for the XPC cDNA plasmids, and to Peter van der Spek for the anti-XPC antibody. NF was supported by an INSERM Interface grant, Electricité de France, Ligue Nationale Contre le Cancer and ARC fundations. CFA, ARL and MHLG were supported in part by Euratom grant B16-E1042-UK.

Received for publication October 28, 2005. Revision received December 23, 2005. Accepted for publication January 9, 2006.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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