EXPERIMENTAL

CELL

RESEARCH

191,

256-262 (1990)

Transformation and Immortalization of Diploid Xeroderma Pigmentosum Fibroblasts B. KLEIN,

A. PASTINK,*

Laboratory for Molecular Carcinogenesis 2300 RA Leiden, The Netherlands;

H. ODIJK,~

and *Department and tDepartment

A. WESTERVELD,?’

Diploid xeroderma pigmentosum (XP) skin fibroblast strains from various XP-complementation groups (B, C, G, and H) were transformed with an origin-defective SV40 early region or with the pSV3gpt plasmid. In the latter case, transfected cells were selected for their ability to express the dominant xgpt gene. Immortalized cell lines were obtained from XP-complementation groups C (WA, 3MA, and 20MA; XP3MA and XPSOMA were formerly considered to belong to complementation group I), G (2BI and 3BR), and H (2CS). No immortalized cells could be isolated from complementation group B (11BE). The immortalization frequency of wild-type diploid fibroblasts and diploid cultures from XP patients was not significantly increased by cotransfection with the SV40 early region plus several selected viral and cellular oncogenes. In fact, cotransfection with some of the oncogenes caused a marked decrease of the transformation frequency. The observed immortalization occurred at a frequency of approximately 5 X lo-'. (21 lsso Academic PWS, I~C.

INTRODUCTION

Xeroderma pigmentosum (XP) is an autosomal recessive disease characterized by abnormal sensitivity to sunlight and by multiple cutaneous neoplasms [ 11. Cells derived from XP patients are hypersensitive to killing by uv-irradiation and show a diminished capacity to repair uv-damaged DNA. Genetic heterogeneity in XP could be demonstrated by somatic-cell fusion. The data obtained so far have resulted in the assignment of eight complementation groups for XP, designated A through H. The extreme differences in uv survival between normal human and XP cells provide an excellent tool by which repair-proficient transfectants, and thus XP-correcting genes, can be isolated. However, these studies are hampered by two ’ Present address: Academical Medical Center, Department of Anthropogenetics, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. ’ To whom requests for reprints should be addressed. 0014.4827190 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form

major problems. First, human cell strains have a limited in vitro life span ranging from about 10 to 45 generations; and second, they show low transfection efficiencies. These problems can be avoided by the use of immortalized XP cells which exhibit higher transfection efficiencies than the corresponding nonimmortalized cells (this study and unpublished results). Recently, Tanaka and colleagues succeeded in cloning the repair gene correcting XP-A by using SV40-immortalized XP-A cells [2]. Obviously, isolation of immortalized XP cell lines would also be of great help for the cloning of other genes that are defective in XP. Human diploid fibroblasts have a limited in vitro lifespan and do not immortalize spontaneously. Induction of an unlimited lifespan has been achieved at a very low frequency, by treatment with 6oCo y rays [3] or, more commonly, by transformation with SV40 or SV40 DNA [4]. SV40-transformed cells exhibit a characteristic morphology and criss-cross growth pattern, and express the SV40 T-antigen. However, although the cultures show an extended lifespan of up to 20-50 population doublings [4] the cells are not immortalized and invariably enter the crisis stage in spite of the fact that they are transformed by SV40. An extremely small fraction of the cells escapes senescence and gives rise to immortal cell lines. Not only human fibroblasts but also human skin keratinocytes have been successfully transformed and immortalized by SV40 DNA [4]. Other viruses that have been used to immortalize human cells are Epstein Barr virus (for B-lymphocytes only; [5, 6]), human papillomavirus types 16 and 18 (for skin keratinocytes; [7] H. zur Hausen, personal communication), and human adenovirus types 5 and 12 (for human embryonic retinoblast cells [S-lo]). It should be noted that, whereas adenovirus-5-transformed embryonic kidney cells were found to enter a crisis stage, the adenovirustransformed human retinoblast cells did not show signs of a crisis phase. Several groups have isolated immortalized XP fibroblast cell lines [ll-161. However, immortalized fibroblasts of complementation groups B and H were still lacking. In the present report we describe immortalization studies of XP complementation groups B, C, G, and H. The experiments resulted in the 256

Inc. reserved.

AND A. J. VAN DER EBB

of Radiation Genetics and Chemical Mutagenesis, Syloius Laboratory, P.O. Box 9503, of Cell Biology and Genetics, Erasmus Uniuersity, Rotterdam, The Netherlands

TRANSFORMATION

AND

isolation of immortalized cell lines from XP-C, -G, and -H. The immortalization frequencies of SV40-transformed human fibroblasts are very low and virtually nothing is known about the mechanisms by which cells escape from senescence. To obtain information about the steps involved in this process and to improve the efficiency of immortalization we have cotransfected diploid cells with SV40 DNA and several other viral or cellular oncogenes. The data show that none of the combinations lead to an increase of the frequency of immortalization of the XP cells, nor of wild-type fibroblasts. MATERIALS

AND

METHODS

Cell strains. The normal diploid fibroblast strains VHlO and VH12 and the diploid XP fibroblast strains were grown in Ham’s F10 medium supplemented with 10% fetal calf serum. During and after transfection with SV40 early region DNA, the cells were grown in Dulbecco’s minimal essential medium/Ham’s F10 (1:l) supplemented with 10% fetal calf serum. Cultures transfected with the pSV3gpt plasmid were maintained in medium containing 25 pg/ml mycophenolic acid (mpa), 10 pg/ml xanthine, 15 pglml hypoxanthine, 0.2 pgl ml aminopterin, 5 @g/ml thymidine, and 2.3 fig/ml deoxycytidine. The XP cell strains used were XP25RO of complementation group A; XPllBE of group B; XP8CA, XP3MA, and XP20MA of group C; XPBBI and XP3BR of group G; XPSCS of group H. The XP3MA and XPBOMA cells originally were assigned to a distinct complementation group I. After this work was completed, it became apparent that the two strains actually belong to complementation group C [17]. Plasmid DNAs. The SV40 origin-defective (orii) early-region clone [ 181 used in these experiments has been described by Dinsart et al. [ 191. The following clones containing viral and cellular oncogenes were used for the cotransfection experiment: EJras, a plasmid containing the activated c-H-rus’* gene regulated by its own promoter [20]; c-myc and v-myc, plasmids containing the cellular and viral myc genes regulated by the SV40 promoter [21]; Ad5ElA and Ad5E1, plasmids containing the adenovirus type 5 ElA or El regions regu lated by their own promoters [22, 231; and PyLT, a plasmid containing the polyoma virus large T-antigen coding region regulated by the polyoma early promoter [24]. pSV2neo is a plasmid that harbors the neomycin’ gene (conferring resistance against the antibiotic G418) under the control of the SV40 early promoter [25]. pSV3gpt is a plasmid harboring the SV40 early region with a functional origin of replication and the bacterial gene for xanthine-guanine phosphoribosyl transferase (zgpt) [26]. pSVlacZ is a plasmid containing the bacterial 1acZ gene [27]. Clone 33.15 contains a moderately repeated human DNA sequence recognizing hypervariability in the arrangement of repeat units in genomic DNA [28]. DNA transfections and propagation of transformed cells. DNA transfections were carried out essentially as described by Graham and van der Eb [29]. The cells were seeded at a concentration of 5 X lo5 cells per 5-cm dish or 3 X lo6 cells per 650.cm3 tissue culture flasks, 48 h before transfection. Cultures were transfected with 5 pg/ dish of SV40 ori- early-region plasmid or 2 kg/dish of the corresponding purified viral DNA insert. Other viral or cellular oncogenes were transfected at 2.5 pg plasmid/dish. The 650~cm3 flasks were transfected with 30 bg per flasks of the SV40 DNA or 60 pg pSV3gpt. Six weeks after transfection with SV40 early-region DNA either single foci were isolated or polyclonal cultures were established by trypsinizing whole cultures containing multiple transformed foci. Cultures transfected with pSV3gpt were trypsinized 2 days after transfection and reseeded in two 650-cm3 flasks, after which they were maintained in medium containing 25 pg/ml mycophenolic acid (mpa) to select for expression of the rgpt gene. All other transformed cultures were

IMMORTALIZATION

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257

maintained either in lo-cm dishes or in 650.cm3 flasks. Each of the isolated transformed clones, the polyclonal cultures, and the pSV3gpt-transfected cultures were individually passaged until they reached crisis and ceased growing. The transformed cultures were passaged 1:4, when they had just reached confluency. Of each transformed clone, one culture was generally maintained. Of the polyclonal cell lines and pSV3gpt-transformed cells, two cultures were maintained unless otherwise stated. Untransformed diploid cultures were passaged 1:2 once a week. After a 1:2 split, the cell doubling number was increased by 1, after a 1:4 split (transformed cells only) the cell doubling number was increased by 2. Unscheduled DNA synthesis, immunofluorescence, and Southern blotting. Unscheduled DNA synthesis was measured according to Westerveld et al. [30]. Indirect immunofluorescence was carried out as described by Zantema et al. [31]. Southern blotting was carried out as described by Southern 1321.

RESULTS

Diploid XP fibroblast strains of complementation groups B (llBE), C (%A, 3MA and 20MA), G (2BI and 3BR), and H (2CS), and normal diploid fibroblast strain VHlO and VH12 were transformed by an origin-defective (ori-) SV40 early region plasmid [18]. Foci of morphologically transformed cells were isolated individually, or polyclonal cell lines were obtained by trypsinizing whole cultures in 650-cm3 flasks containing 100-300 transformed colonies. XP fibroblasts of complementation groups B, C (3MA and 20MA), and H were also transformed with plasmid pSV3gpt, which harbors the SV40 early region with a functional origin of replication (ori+) and the bacterial xgpt gene which confers resistance to mycophenolic acid. Transfectants expressing the pSV3gpt plasmid were selected for their ability to proliferate in the presence of mycophenolic acid. Transformation with pSV3gpt was included in this study as an alternative method to immortalize with SV40. Mayne et al. [33] have reported that diploid human fibroblasts can be immortalized efficiently by transfection with pSV3gpt and selection in medium containing mycophenolic acid. The isolated transformed cell clones and the polyclonal cell cultures were individually passaged 1:4 until the cultures reached the crisis phase. This became apparent by a decrease of the growth rate, followed by a total growth arrest and a degeneration of the cultures. The clones transformed by the orii SV40 early region exhibited an extended lifespan: crisis appeared after an estimated total number of cell doublings varying between 55 and 90. The transformed XP-G cultures, however, stopped dividing already after 30-40 cell doublings. (These values include the number of doublings prior to transformation but not those required to form a primary transformed focus.) In our study, untransformed diploid skin fibroblasts practically stop dividing between cell doubling 25 and 35, depending on the cell strain. Immortalized clones became visible as rapidly growing colonies, often appearing several weeks after

258

KLEIN

ET AL.

TABLE Transformation

and Immortalization by the SV40 ori-

Early

1

of Wild-Type Human Region (A) or with

and Xeroderma Pigmentosum the pSV3gpt Plasmid (B)

Fibroblasts

(4

Cell strain

Cell doubling nr at transfection

Approximate number of transformantsl~g SV40 orii early region DNA/lo’ cells

VHlO XPllBE (group B) XP8CA (group C) XP3MA (group C) XP20MA (group C) XPPBI (group G) XP3BR (group G) XPZCS (group H)

16 23 16 10 9 22 18 18

50 10 25 10 10 10 10 10

Transformed clones or polyclonal cultures isolated 24 2 1 2 2 24 24 2

Immortalized clones

(polyclonal)” (polyclonal)” (polyclonal)” (polyclonal)”

(polyclonal)”

(B) Cell strain

Cell doubling number at transfection

XPllBE (group B) XP3MA (group C) XPZOMA (group C) XPPCS (group H)

23 10 9 18

mpa-Resistant polyclonal culture’ 2 2 2 2

Immortalized clones 0 Id Id 0

a Each polyclonal culture consisted of 100-300 transformed colonies. * Multiple immortalized colonies appeared after a short crisis period. It is unknown whether these colonies arose from multiple independent immortalization events or from a single event. ’ The number of mycophenolic acid (mpa)-resistant colonies per culture could not be estimated because the cells were subcultured 2 days after transfection. d See footnote b.

the onset of the crisis when most of the cells in the cultures had died. The results are summarized in Table 1. In each set of 24 individually propagated transformed clones from normal VHlO cells, XPBBI cells, and XP3BR cells (the latter 2 both from group G), one single immortalized clone appeared. Thus, 23 out of each set of 24 transformed cultures senesced without giving rise to a detectable immortalization event. A single immortalized clone also appeared in the polyclonal culture from XP8CA (group C) and in one of the two polyclonal cultures from XP2CS (group H). However, multiple immortalized colonies arose in 1 of 2 polyclonal cultures from XP3MA and in 1 out of 2 polyclonal cultures from XP20MA (both group C), after a short crisis period. It is not known whether these colonies arose as a result of single or multiple immortalization events. No immortalized clones were observed in the 2 polyclonal cultures from XPllBE (group B). Transformation with pSV3gpt (Table 1B) resulted in the appearance of multiple immortalized colonies in 1 out of 2 polyclonal cultures from XP3MA and in 1 out of 2 polyclonal cultures from XPBOMA. No immortalized clones were observed in pSV3gpt-transformed XPllBE (group B) and XPBCS (group H).

To ensure that the immortalized clones originated from the diploid cells from which they had been isolated the relationship between DNA of the immortalized clones and their diploid counterparts was tested with probe 33.15, the so-called “Jeffreys probe.” The results (Fig. 1) showed that DNA of the immortalized lines have basically the same hybridization patterns as those of the parental diploid cell strains. DNA repair capacity as measured by unscheduled DNA synthesis (UDS) of the various cell strains and immortalized lines is presented in Table 2. The UDS levels of the immortalized cell lines equals that of the parental diploid cultures. Influence of Other Cellular or Viral Oncogenes on SV40-Induced Immortalization Cellular oncogenes or transforming genes other than SV40 are not efficient as immortalizing agents for human fibroblasts [34]. It is conceivable, however, that a combination of the SV40 early region with other oncogenes might lead to an increase of the rate of immortalization. To test this possibility, VH12 normal diploid fibroblasts and XP25RO diploid fibroblasts (XP group

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IMMORTALIZATION

259

OF XP

express the SV40 T-antigen, but no integrated v-myc sequences could be detected (data not shown). To determine the efficiency of cotransfection, diploid VH12 cultures were transfected with the SV40 early region plus the plasmid pSVlacZ, carrying the bacterial 1acZ gene. Six weeks after transfection, lo-20% of the SV40-transformed colonies contained blue-stained cells, indicative of 1acZ gene expression [35]. This implies that in the cotransfection experiment with other oncogenes, between 10 and 20% of the clones might contain more than one oncogene. The actual cotransfection efficiency may have been somewhat higher in view of the fact that 20-30% of the colonies in cultures that had received the ras oncogene in addition to the SV40 early region, exhibited the ras-like morphology. Efficiency of DNA Transfection XP3BR-G Cell Line

12

3

4

5

6

FIG. 1. Detection of polymorphic DNA fragments in genomic DNAs from XP cells before and after immortalization. A Southern blot of HinfI digested cellular DNA from 3 untransformed XP cell lines (lane 1, 3,5) and their SV40-transformed and immortalized derivatives (lane 2, 4, 6) was hybridized with the radioactively labeled human probe 33.15 [28]. The DNA pairs were derived from XP8CA cells (lanes 1 and 2). XPPBI cells (lanes 3 and 4), and XPZCS cells (lanes 5 and 6).

A) were transformed with the SV40 early region, alone or in combination with viral or cellular oncogenes (Table 3). Transformed colonies appeared in most transfected cultures. The morphology and growth pattern of the cells in the colonies usually resembled that of the cells transformed by the SV40 early region alone. Several of the transformed clones were tested for the presence of the SV40 T-antigen and all were found to be positive (data not shown). Table 3 shows that transformation efficiencies were higher in the VH12 cultures than in the XP25RO cultures, possibly due to a difference in the intrinsic transfection efficiency between the cells. Furthermore, a drastic decrease of transformation efficiency was noted when other oncogenes were present in addition to the SV40 early region. Transformed clones were isolated from as many different transfection combinations as possible, and each clone was individually propagated until cell proliferation ceased. Although an extended lifespan was noted for most, if not all, transformed lines, none of them escaped senescence, with one exception. This unique immortalized clone appeared in a VH12 culture transfected with the SV40 early region plus the v-myc gene. It was found to

of Immortalized

The immortalized SV40-transformed XP3BR-G cell line was tested for its ability to incorporate exogenous DNA after Ca-phosphate-mediated DNA transfer. This cell line was chosen because of its high sensitivity to uv light, which makes it particularly suitable for the isolation of the correcting DNA repair gene. Table 4 shows that the efficiency of transfection of pSVBneo, containing the neomycin-resistance gene as selectable marker, was very low. Since the low transfectability was found to be correlated with a low cloning efficiency, attempts were made to isolate clones with increased transfectability by first selecting for improved cloning efficiency. Cell colonies that had appeared in the original cloning efficiency experiment were isolated and tested again for their cloning ethciency. One of these subclones reached a 25% efficiency of cloning and turned out to have also a greatly improved transfectability (Table 4). Attempts to

TABLE

2

Unscheduled DNA-Synthesis Levels in UV-Irradiated Diploid XP Fihroblast Strains and in the Corresponding SV40Transformed and Immortalized Cells UDS level (%)

Cell line Wild-type XP8CA (group C) XPBMA (group Cl XPZOMA (group C) XP2BI (group G) XP3BR (group G) XPZCS (group H)

Untransformed cells 100 16 13 18 6 9 34

SV40-Transformed and immortalized cells 100 13 17 19 10 9 29

260

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ET AL.

TABLE The

Cell strain VH12

XP25RO (group A)

Influence

of Viral

DNA used for transfection (SV40 ori- 5 pg/dish; other oncogenes 2.5 pg/dish) sv40 SV40 sv40 sv40 SV40 SV40 SV40 SV40 sv40 SV40 sv40 sv40 SV40 SV40 SV40 SV40

+ + + + + + +

EJ ros ” - myc c ~ m,yc EJ ras + EJ ras + EJ ras + EJ ras +

c - myc Ad5ElA Ad5El Py LT

+ + + + + + +

EJ ” cEJ EJ EJ EJ

c - myc Ad5ElA AdSEl Py LT

ras my myc MS + ras + ras + ras +

and Cellular

Oncogenes

Transformants per wg SV40 DNA per lo5 cells

XP3BR-G, original immortalized line XP3BR-G, subclone 4

Immortalized clones 0 0 1 0 0 0 0 0 0 0

2 0.5

5 2

22-37 12/H

0 0

0.5 0.5

1 2

26 30137

0 0

4

1 30

Approximate number of cell doublings before crisis 5520 7712 10-15 11-13 10-20 10-15 5-20 7-13 25-35 15-45

In the present study we describe the isolation by SV40 transformation of immortalized cell lines from XP fibroblasts of complementation group C, G, and H, as well as from normal VHlO and VH12 fibroblasts. Several attempts to establish immortalized lines from XP complementation group B have been unsuccessful, even

G418resistant clones/fig pSV2neo

Transformed clones isolated

Immortalization

5 10 10 10 10 I 10 10 5 10

DISCUSSION

TABLE

on SV40-Induced

85 19 3 10 1.5 2 5.5 5.5 11 6

further improve transfectability have not been successful. To determine the amount of DNA incorporated per cell, G418-resistant colonies obtained by transfection with pRSVneo were subjected to Southern blotting analysis. Only small amounts of pRSVneo had been incorporated (approx. 4 copies/cell; results not shown). Thus, we conclude that the uptake of exogenous DNA by the immortalized XP3BR-G line was low, although its efficiency of transfection expressed as numbers of G418-resistant colonies was reasonably high.

Relationship between DNA Transfectability Efficiency of Immortalized XP3BR-G

3

and Cloning Cell Line Cloning efficiency

0.1% 25%

Note. Cultures were transfected with pSV2neo (2 yg/9 cm dish), containing the neomycin gene conferring resistance against the antibiotic G418. Cells that had taken up the pSV2neo construct were selected in medium containing 150 fig G418/ml.

after treatment of the SV40-transformed cultures with the mutagen ENU (1, 2, and 3 mMlm1; result not shown). The XP3MA and XPSOMA cells showed an unusual feature in that immortalization of these cells was accompanied by the appearance of multiple transformed foci. A possible explanation is that the immortalization events had occurred a number of passages before the polyclonal cultures as a whole entered crisis. The immortalized cells then could have proliferated along with the nonimmortalized cells, and have multiplied to more than 100 progeny cells at the time the culture as a whole entered crisis. The data in Table 1 also show that transformation by pSV3gpt does not result in more efficient immortalization than the classical focus assay with SV40 ori- DNA. In our study, immortalized clones were obtained with both methods. The results presented in Table 3 show that the efficiency of immortalization could not be increased by cotransfecting the cells with the SV40 early region and other viral or cellular oncogenes. In fact, transformation efficiencies dropped considerably when other oncogenes were present in addition to the SV40 transforming genes. This may be due to the fact that the other oncogenes functionally interfere with the transformation pathway used by SV40. It is unlikely that it is caused by oversaturation of the cells with transforming DNA sequences since concentrations of SV40 DNA higher than 5 pg/5-cm dish still gave rise to an increase of the transformation frequency (unpublished results). It can be argued that the failure to demonstrate increased immortalization frequencies is due to the fact that none of the transformed clones contained an additional oncogene other than the SV40 early region. In-

TRANSFORMATION

AND

deed, we have not attempted to demonstrate the presence of functional oncogenes in the transformed clones, mainly because it is difficult to prove unambiguously that the protein of a particular cellular oncogenes is produced. On the basis of evidence presented under Results it can be estimated that the cotransfection efficiency may be 1530%. If this is correct, then at least 1 or 2 clones of each category of transformed VH12 cells (Table 3) should contain a second oncogene. If this had resulted in a drastic increase of immortalization it would have been noticed. Our results confirm earlier observations that immortalization is a rare event in human fibroblasts. We have made a rough estimation of the immortalization frequency from the data shown in Table 1. Considering the number of cells seeded per dish or flask at the last passage before crisis, and assuming that each appearance of multiple transformed colonies in the XP3MA and XPSOMA cultures is the result of a single immortalization event, then it can be concluded that seven immortalization events have occurred in about 140 X lo6 cells. This corresponds to an immortalization frequency of 5 X lo-‘, a value that does not differ greatly from the frequency of 3 X 1O-7 calculated by Shay and Wright [36] for an “immortalization-competent” clone. Furthermore, cell-fusion experiments indicate that senescence is a dominant trait [3i’, 381. Together, these data suggest a model in which the rare event responsible for immortalization of SV40transformed human fibroblasts involves the mutational inactivation of one allele of a putative senescence gene. The second allele of this gene may still be intact, but its activity is suppressed by the viral T-antigens present in the cells. Evidence that SV40 T-antigens can suppress the activity of the senescence gene is provided by the observation that SV40-transformed human fibroblasts have a considerably extended in vitro lifespan, as compared to untransformed fibroblasts. Mutational inactivation of both alleles of the senescence gene in the same cell is highly unlikely, since that would predict immortalization frequencies far lower than the observed 5 X IO-‘, and would also predict immortalization to be independent of SV40 transformation. Thus, according to this model, immortalization requires the combined effects of (1) partial inactivation by viral T-antigens of the senescence gene product, or partial suppression of transcription of the senescence gene; (2) mutational inactivation of one allele of the same gene. The observation that SV4O-t,ransformed keratinocytes apparently can be immortalized more easily than fibroblasts [4] suggests that regulation of senescence is cell-type- or cell-lineage-dependent. This is also indicated by the fact that human embryonic retinoblast cells can be immortalized by adenovirus transformation without even entering a crisis phase [lo]. Human cells differ from rodent cells in that the latter readily acquire an unlimited

IMMORTALIZATION

OF XP

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life span after transformation by certain viral or cellular oncogenes. Possible explanations for this phenomenon are (1) rodent cells are genetically more unstable than human cells and frequently show hemizygosity of genes [39], and (2) SV40 transforming genes and other oncogenes are more effective in suppressing the activity of senescence genes in rodent cells than in human fibroblasts. Our limited data on transfectability of the immortalized XP3BR-G cell line show that acquisition of an unlimited life span does not automatically result in a more efficient stable uptake of DNA. In the established XPSBR-G cell line high transfectability correlated with high cloning efficiency. In spite of a relatively high transfectability of our XP3BR-G subclone the amount of DNA incorporated per cell is low, equaling the amount of DNA taken up by Hela cells [40, 411. The results of this and other similar studies show that further work is required to understand the mechanism( s) of immortalization. The authors thank W. Keijzer (Rotterdam) for providing some of the XP cells; L. Mayne and her colleagues (Brighton) for their hospitality shown toward B.K. during her stay at their department and also for kindly providing her with details of their transformation method; R. van Ham and G. Weeda for allowing them to use their unpublished data on DNA uptake of the immortalized XP-G cells; and E. Bakker (Leiden) for advice on testing the DNA samples with the 33.15 probe, kindly provided to them by Dr. A. Jeffreys. This work was supported by the Institute of Radiopathology and Radiation protection (IRS).

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