Regular Article

Journal of Investigative Dermatology (2000) 114, 1022–1029; doi:10.1046/j.1523-1747.2000.00952.x

Reinvestigation of the Classification of Five Cell Strains of Xeroderma Pigmentosum Group E with Reclassification of Three of Them

Toshiki Itoh*,†, Stuart Linn†, Tomomichi Ono§ and Masaru Yamaizumi*

  1. *Department of Cell Genetics, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kumamoto, Japan
  2. §Department of Dermatology, Kumamoto University School of Medicine, Kumamoto, Japan
  3. †Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, U.S.A.

Correspondence: Dr Toshiki Itoh, Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, 401 Barker Hall, University of California, Berkeley, CA 94720-3202. Email: toshiki@uclink4.berkeley.edu

Received 1 July 1999; Revised 13 January 2000; Accepted 24 January 2000.

Top

Abstract

Xeroderma pigmentosum is a photosensitive syndrome caused by a defect in nucleotide excision repair or postreplication repair. Individuals of xeroderma pigmentosum group E (xeroderma pigmentosum E) have a mild clinical form of the disease and their cells exhibit a high level of nucleotide excision repair as measured by unscheduled DNA synthesis, as well as biochemical heterogeneity. Cell strains from one group of xeroderma pigmentosum E patients have normal damage-specific DNA binding activity (Ddb+), whereas others do not (Ddb–). Using a refinement of a previously reported cell fusion complementation assay, the previously assigned Ddb+ xeroderma pigmentosum E strains, XP89TO, XP43TO, and XP24KO, with various phenotypes in DNA repair markers, were reassigned to xeroderma pigmentosum group F, xeroderma pigmentosum variant, and ultraviolet-sensitive syndrome, respectively. The Ddb– xeroderma pigmentosum E strains, XP82TO, and GM02415B, which showed almost normal cellular phenotypes in DNA repair markers, however, remained assigned to xeroderma pigmentosum group E. With the exception of the Ddb+ strain XP89TO, which demonstrated defective nucleotide excision repair, both Ddb– and Ddb+ xeroderma pigmentosum E cells exhibited the same levels of variation in unscheduled DNA synthesis that were seen in normal control cells. By genome DNA sequencing, the two Ddb– xeroderma pigmentosum E strains were shown to have mutations in the DDB2 gene, confirming previous reports for XP82TO and GM02415B, and validating the classification of both cells. As only the Ddb– strains investigated remain classified in the xeroderma pigmentosum E complementation group, it is feasible that only Ddb– cells are xeroderma pigmentosum E and that mutations in the DDB2 gene are solely responsible for the xeroderma pigmentosum E group.

Keywords:

Cockayne syndrome, damage-specific DNA binding protein, nucleotide excision repair, postreplication repair, ultraviolet–sensitive syndrome

Abbreviations:

CS, Cockayne syndrome; DDB, damage-specific DNA binding; NER, nucleotide-excision-repair; PRR, postreplication repair; RDS, recovery of replicative DNA synthesis; RRS, recovery of RNA synthesis; TK, thymidine kinase; UVsS, ultraviolet-sensitive syndrome; UDS, unscheduled DNA synthesis; XP, xeroderma pigmentosum

Xeroderma pigmentosum (XP) is a rare autosomal recessive disease characterized by a clinical and cellular hypersensitivity to ultraviolet (UV) light (Cleaver & Kraemer 1995). Patients exhibit dermatologic abnormalities, including thickening and hyperpigmentation of sun-exposed skin, and develop sunlight-induced malignancies at an earlier age. By cell fusion analyses, XP has been classified into seven genetic complementation groups (A–G), which are defective in nucleotide excision repair (NER). A separate group, XP variant (XPV) has proficient NER but exaggerated delay in recovery of replicative DNA synthesis (RDS) after UV irradiation (Lehmann et al. 1975,1977;Rude & Friedberg 1977;Cleaver & Kraemer 1995). Recently, the XPV gene was cloned, and identified as DNA polymerase (Masutani et al. 1999;Johnson et al. 1999). In the seven NER defective groups, the genes which are defective in XP with severe clinical manifestations (XPA, B, and G) and with the classical type of XP (XPC, D, and F) have been cloned (Friedberg et al. 1995;de Sijbers et al. 1996). The XPE gene, however, has yet to be cloned because of several difficulties: (i) the clinical manifestations in XPE patients are very mild; (ii) as cells derived from XPE patients have high levels of unscheduled DNA synthesis (UDS), it is very difficult to distinguish between XPE, XPV, and normal cells; and (iii) the high level of UDS makes analysis of a complementation assay using cell fusion to determine the classification of XPE strains problematic.

Biochemical heterogeneity in binding to damaged DNA has been reported for XPE cell free extracts (Kataoka & Fujiwara 1991;Keeney et al. 1992;Friedberg et al. 1995). Cell strains from two of 13 unrelated XPE individuals lack a damage-specific DNA-binding (DDB) activity in nuclear extracts (Chu & Chang 1988;Keeney et al. 1992) and are termed Ddb– XPE. Recently, three additional Ddb– XPE patients were reported (Otrin et al. 1998). Mutations have been identified in the DDB2 gene in the three XPE Ddb– strains examined, but not in XPE Ddb+ strains (Nichols et al. 1996). The question has been proposed as to whether Ddb– and Ddb+ XPE strains actually comprise two separate XP complementation groups, with the DDB2 gene being responsible for the NER defect in Ddb– XPE patients. To clarify this question, we have extensively assessed DNA repair markers in five previously reported XPE strains using a refinement of a cell fusion assay (Itoh et al. 1994,1995a,1996b). We report here that three XPE Ddb+ strains have been misclassified, whereas two Ddb– strains are XPE. Hence it is feasible that DDB2 gene is the true XPE gene and XPE Ddb+ does not exist.

Top

Materials and Methods

Cells and culture conditions

The cell strains used in this study are shown in Table 1. All cells were cultured in Dulbecco's modified Eagle's minimum essential medium (ICN, Irvine, CA) supplemented with 10% (vol/vol) fetal bovine serum (ICN), penicillin G (100 units per ml), and streptomycin (100 mug per ml) in a humidified 5% CO2 incubator at 37°C.


UV survival assay

Colony forming ability was performed as previously described (Itoh et al. 1994,1995a). Cells were trypsinized and were plated on to 60 mm plates. After incubation for 10 h, they were washed with phosphate-buffered saline, irradiated with 3–10 J per m2 of UVC (254 nm) at a fluence rate of 0.7 J per m2 per s without medium present, and then fresh medium was applied. After 10–14 d, the plates were washed with phosphate-buffered saline, fixed with 80% (vol/vol) methanol, and stained with Giemsa, colonies were counted, and analyzed by survival curves. To determine the effect of caffeine on UV survival, cells were plated, washed, and irradiated as described above except that caffeine was added to a final concentration of 1 mM just after UV irradiation and the plates were incubated for 2–3 wk.

Post-replication repair (PRR) assay

Semiconservative DNA replication after UV irradiation was measured by a previously described method (Itoh et al. 1995a,1996b). Cells were irradiated with UV light (254 nm) at a dose of 5 J per m2, returned to fresh 10F-Dulbecco's modified Eagle's minimum essential medium, and incubated for 60 min before being labeled for 30 min with 0.37 MBq per ml (10 muCi per ml) of [3H]thymidine. Cells were harvested, suspended in phosphate-buffered saline, then X-irradiated at a dose of 20 Gy on ice. The suspension was layered on to 5 ml of a 5–20% (wt/vol) alkaline sucrose gradient containing 0.1 M NaOH, and centrifuged at 237,600 times g for 90 min at 4°C in a RPS55T rotor (Hitachi, Tokyo, Japan). After centrifugation, drop fractions were collected on to Whatman Grade 17 paper strips, and the acid-insoluble radioactivity was counted in a liquid scintillation counter.

Measurement of DNA synthesis after UV irradiation

The rate of DNA synthesis after UV irradiation was measured as described (Itoh et al. 1996b,1999). Briefly, cells were irradiated or mock irradiated with UVC (254 nm) at a dose of 5 J per m2 in fresh 10F-Dulbecco's modified Eagle's minimum essential media. After incubation for 2–3 d, they were pulse-labeled with [3H]thymidine (10 muCi per ml) for 30 min, and then lyzed with 0.2 M NaOH, harvested, and applied on to Whatman Grade 17 paper strips. After washing with 5% trichloroacetic acid, the acid-insoluble radioactivity was counted by liquid scintillation. Each point represents an average of four experiments.

Measurement of UDS, recovery of RNA synthesis (RRS) and RDS after UV irradiation

UDS, RRS, and RDS were measured as previously described (Itoh et al. 1994,1995a,b,1996a,b,1999). Twenty microliter aliquots of the test and control cell suspension were plated with a micropipette in two rows on a single coverslip (18 mm times 18 mm) for each experiment, as variations among coverslips has been observed to interfere with comparisons between cell strains. For UDS cells were irradiated with UVC (254 nm) at a dose of 10 J per m2 or 30 J per m2, immediately labeled with [3H]thymidine (40 muCi per ml) for 2.5 h, and fixed. Coverslips were mounted on glass slides, dipped in Kodak NTB-3 emulsion, and exposed for 24 h at 4°C. Grains above nuclei were counted under a microscope. For RRS, cells were irradiated with UVC (254 nm) at a dose of 15 J per m2. After UV irradiation, they were incubated for 23 h, and labeled with [3H]uridine (40 muCi per ml) for 1 h. Autoradiography was performed as above. For RDS cells were irradiated with UVC (254 nm) at a dose of 15 J per m2. After UV irradiation, they were incubated for 6 h and then labeled with [3H]thymidine (15 muCi per ml) for 1 h. Autoradiography was performed as above.

Complementation assay

Cell fusion was performed on a small scale as described previously (Itoh et al. 1994,1995a,b,1996a). Twenty microliter aliquots (1–2 times 104 cells) of cell suspension fused with Sendai virus were plated in the center of coverslips (18 mm times 18 mm) in 30 mm dishes and 20 mul (1–2 times 104 cells) of parental cells was plated on each side of the fused cells. Cells were incubated for 20 h and then irradiated with UV light at a dose of 30 J per m2 (UDS) or 15 J per m2 (RRS). Then, UDS or RRS was measured as above.

DNA sequencing

DNA sequencing was performed as described (Itoh et al. 1999). Genomic DNA was analyzed for mutations at nucleotide 730 (XP82TO) and 818 (GM02415B). Polymerase chain reaction was used to amplify the DNA using the following two primers: 632: 5'-CTA- GTAGCCGAATGGTGGTC and 920: 5'-TCGGATCTCGCTCTTCT- GGT. The polymerase chain reaction fragments were sequenced with primers 632, 920, and 1000: 5'-ATGGGTGTGAGGTGCTGGA using a Dye terminator cycle sequencing kit (PE Applied Biosystems, Foster City, CA) with a Model 373S DNA sequencer (PE Applied Biosystems).

Top

Results

Heterogeneity of UV sensitivity in cells previously classified as XPE

As patients with XPE show mild clinical manifestations and heterogeneity (Kataoka & Fujiwara 1991;Keeney et al. 1992;Cleaver & Kraemer 1995;Friedberg et al. 1995), we first examined the UV sensitivity of five cell strains classified as XPE by the colony-forming assay. We observed that the UV sensitivities of GM02415B, XP82TO, and XP43TO cells were indistinguishable from that of normal (Mori) cells, whereas XP89TO cells was slightly more sensitive than normal (Figure 1). XP24KO cells were significantly more sensitive to UV than the normal or the cells classified as XPE; they were as sensitive as the UVsS Kps3 cells, but less so than the CSA Mps1 cells (Figure 1d). These results indicated that UV sensitivity of previously classified XPE cells showed significant heterogeneity.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

UV survival. Appropriate numbers of cells were seeded on to 60 mm dishes. After UV irradiation at a fluence rate of 0.7 J per m2 per s, cells were incubated for 10–14 d, fixed with 80% methanol, and stained with Giemsa. Caffeine was added as indicated at a final concentration of 1 mM just after UV irradiation and the plates were incubated for 2–3 wk. Each point represents an average of three independent experiments; error bar, SEM. Cells utilized were: (a–d) Mori cells (square) a normal control. (a) GM02415B (triangle); XP82TO (filled circle). (b) XP43TO (filled circle); XP2SA (XPV) (circle); XP43TO 1 mM caffeine (filled triangle); XP2SA 1 mM caffeine (triangle). (c) XP89TO (filled circle); Nps8 (XPF) (circle). (d) XP24KO (filled circle); Kps3 (UVsS) (circle); Mps1 (CSA) (triangle).

Full figure and legend (18K)

Heterogeneity of UDS levels in cells previously classified as XPE

Although it has been reported that UDS levels of XPE cells were 40–60% those of normal cells (Bootsma et al. 1970;Kleijer et al. 1973;de Weerd-Kastelein et al. 1974;Kraemer et al. 1975b;Fujiwara et al. 1985;Okuno et al. 1994;Yamaizumi & Sugano 1994;Cleaver & Kraemer 1995), we have found that these relative levels vary among measurements (Itoh and Yamaizumi, unpublished data), and we attributed this variation mainly to differences among coverslips. To solve this problem, we have recently developed a simple method for measuring relative UDS, RRS, or RDS after UV irradiation by simultaneously plating two cell lines on the same coverslip (Itoh et al. 1994,1995a,1996b). The UDS levels are shown in Table 2. Among the cells previously classified as XPE, only XP89TO cells had significantly reduced UDS. AsFujiwara et al. (1985) measured UDS levels in XP24KO cells at the UV dose of 10 J per m2, we also repeated the experiment at that dose in experiment V of Table 2. Although the UDS levels in the cells previously classified XPE were statistically reduced compared with those in normal Mori cells in some cases (p <0.001) (Table 2), when we examined the normal range of UDS levels among several normal control cells (Mori, Turu, Sono, Goryo, Umi, and Mura (experiments III and IV of Table 2), similar variations in UDS levels were observed. Therefore, with the exception of XP89TO, the UDS levels of the strains previously classified as XPE strains were within the range of normal cells (Table 2).


Heterogeneity of RRS levels in cells previously classified as XPE

RRS levels are summarized in Table 3. XP43TO, XP82TO, and GM02415B cells showed normal RRS levels. RRS levels of XP89TO and XP24KO cells, however, were clearly reduced.


Reassignment of XP89TO cells as XPF

XP89TO cells showed slight UV sensitivity (Figure 1c) and a clear reduction in both UDS and RRS levels (Table 2 and Table 3), reductions more characteristic of XPD or XPF cells (Itoh and Yamaizumi, unpublished data). Therefore, we performed complementation assays (Itoh et al. 1994,1995a,b,1996c) between XP89TO cells and XPD (Kps5) or XPF (Kps6 and Nps8) cells. The results reclassified XP89TO cells as XPF (Figure 2a, b, Table 4).

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

RRS levels after UV irradiation in fused cells. The procedure for the complementation assay is described in Materials and Methods. To measure RRS levels, cells were irradiated with 15 J per m2 UV light, incubated for 23 h, and then labeled with [3H]uridine (40 muCi per ml) for 1 h. Arrows show fused cells (a) between XP89TO and Kps5 (XPD); (b) XP89TO and Kps6 (XPF); (c) XP24KO and GM10905 (CSB); (d) and XP24KO and Kps3 (UVsS). Scale bar: 5 mum.

Full figure and legend (100K)


Heterogeneity of the rate of DNA synthesis after UV irradiation in cells previously classified as XPE

To investigate the remaining four cell strains in more detail, we compared the rates of post-UV DNA synthesis (Figure 3). The results of these experiments were: (i) the post-UV DNA synthesis rates for XP24KO cells were similar to those for UVsS (Kps3) and CSB (GM10905) cells (Figure 3a); (ii) the pattern for XP43TO cells was similar to that of XPV (XP2SA) cells (Figure 3b); and (iii) the patterns for XP82TO and GM02415B cells were similar to that of normal (Mori) cells (Figure 3c).

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Rate of DNA synthesis after UV irradiation. Cells were irradiated with UV light at a dose of 5 J per m2. After 0, 2, 4, 6, or 8 h incubation, they were pulse-labeled with [3H]thymidine (10 muCi per ml) for 30 min. Cells utilized were: (a–c) Mori cells, normal control (square); (a) XP24KO (filled circle); Kps3 (UVsS) (circle); GM10905 (CSB) (triangle). (b) XP43TO (filled circle); XP2SA (XPV) (circle). (c) GM02415B (triangle); XP82TO (filled circle). Each value is the mean for three or four experiments; error bar, SEM.

Full figure and legend (20K)

Reassignment of XP24KO cells as UVsS

XP24KO cells showed normal UDS (Table 2), reduced RRS (Table 3), and a similar pattern in the rate of post-UV DNA synthesis compared with UVsS and CS cells (Figure 3a), suggesting that they belong to UVsS or CS (Itoh et al. 1996b). We performed complementation assays (Itoh et al. 1994,1995a,b) between XP24KO cells and CS or UVsS cells. These results reclassified XP24KO cells as UVsS (Figure 2c, d, Table 5).


Reassignment of XP43TO Cells as XPV

XP43TO cells showed almost normal UDS (Table 2), almost normal RRS (Table 3), and a pattern for the rate of post-UV DNA synthesis that was similar to that of XPV (Figure 3b), properties that are each characteristic XPV cells (Itoh et al. 1996b). Therefore, we measured RDS after UV irradiation with or without caffeine and conducted a PRR assay. The RDS level of XP43TO cells was reduced in the absence of caffeine, with an additional reduction in the presence of 1 mM caffeine (Table 6). This combination of three repair markers (normal UDS, normal RRS, and reduced RDS which is enhanced by caffeine) is characteristic specifically of XPV (Itoh et al. 1996b). Furthermore, by the PRR assay, the DNA synthesized after UV irradiation in XP43TO cells was found to be shorter than that in unirradiated cells (Figure 4a). In normal cells DNA synthesized after UV irradiation is the same size as that of unirradiated normal cells (Itoh et al. 1995a,1996b,1999). Moreover, a diminution by caffeine on UV survival was also found (Figure 1b). These results taken together indicate that XP43TO cells ought to be reassigned as XPV.

Figure 4.
Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Comparison of the sizes of DNA synthesized after UV irradiation in XP43TO, GM02415B, XP82TO, and XP2SA cells to that with no irradiation. Cells were irradiated with UV light at a dose of 5 J per m2, grown for 1 h, and then incubated for 30 min with [3H]thymidine (10 muCi per ml). Centrifugation of the DNA through a 5–20% (wt/vol) alkaline sucrose gradient was performed for 1.5 h at 237,600 times g. The top of the gradient is to the right. (a) XP43TO; (b) GM02415B; (c) XP82TO. square, unirradiated cells (control); filled square, UV irradiated cells.

Full figure and legend (25K)


Identification of mutations in the DDB2 Gene in XP82TO and GM02415B cells

As both XP82TO and GM02415B cells had similar phenotypes for DNA repair markers (Figure 1a and Figure 3c, Table 2 and Table 3), we examined whether the two strains belonged to the same complementation group. Although it seemed that both GM02415B and XP82TO cells complemented NER-defective XPA, XPC, XPD, XPF, and XPG cells (data not shown), the grain numbers in fused cells were almost the same as those of unfused GM02415B or XP82TO cells, because these cells showed almost normal ranges of UDS (Table 2) and RRS (Table 3). Indeed similar ranges of results were found in complementation assays between NER-defective XPA, XPC, XPD, XPF, and XPG cells and normal or XPV cells. Furthermore, UDS levels in fused cells were sometimes slightly higher than those of individual XPV or normal cells (Itoh and Yamaizumi, unpublished data). Therefore, by complementation we could not definitively classify XP82TO or GM02415B cells.

To rule out whether these cells belong to XPV, we examined RDS levels with or without caffeine (Table 6) and conducted a PRR assay (Figure 4b, c). Both XP82TO and GM02415B cells showed almost normal RDS levels, and no effect of caffeine was found (Table 6). Furthermore, the synthesized DNA after UV irradiation was the same size as that of unirradiated cells (Figure 4b, c). The results of these experiments indicate that XP82TO and GM02415B cells do not belong to XPV.

The absence (Ddb–) of a damage-specific DNA binding (DDB) activity has been identified in six of the 16 cell strains previously classified XPE (Kataoka & Fujiwara 1991;Keeney et al. 1992;Otrin et al. 1998;Itoh et al. 1999). DDB protein is a heterodimer of 127 kDa (DDB1) and 48 kDa (DDB2) subunits (Keeney et al. 1993;Dualan et al. 1995) and mutations in DDB2 have been reported in XP2/3RO (GM02415B) and XP82TO Ddb– XPE cells by sequencing analysis of both DDB2 cDNA and genomic DNA (Nichols et al. 1996). We have characterized the genomic organization of the DDB2 gene and found it to be comprised of 10 exons (Itoh et al. 1999). We have reconfirmed the previously report that XP82TO carried a homozygous Aright arrowG transversion at nucleotide position 730 which is now assigned to exon 6 (data not shown). In the cell strain GM02415B, we located a homozygous Gright arrowA transversion at nucleotide position 818, also in exon 6 (data not shown). Therefore, both of these cell strains have mutations in the DDB2 gene, in addition to being Ddb– and XPE.

Top

Discussion

XPE was designated as the fifth complementation group based on a cell fusion technique; however, XPE strains exhibit the highest UDS level of all the NER defective XP groups (Kraemer et al. 1975a,b). The patients, XP2RO (GM02415B) and XP3RO were first assigned to XPE and were clinically classified as ''classical XP, light to moderately severe'' (Bootsma et al. 1970;Kleijer et al. 1973;de Weerd-Kastelein et al. 1974). Next, XP24KO cells were assigned to XPE using GM02415B cells as the complementing cells in the cell fusion assay (Fujiwara et al. 1985). After that, XP80TO, XP81TO, and XP82TO cells were assigned to XPE using XP24KO cells (Kondo et al. 1988). The patient XP24KO was a 15 y old Japanese female who showed acute sun sensitivity reaction without blistering at age 1. Small pigmented freckles and telangiectasia on her face were observed at about age 11 or 12. She had no benign or malignant neoplasias (Fujiwara et al. 1985). Judging by these clinical manifestations, XP24KO was clinically similar to patients with UVsS (Itoh et al. 1995a,1996d), which has been confirmed by the reassignment of XP24KO cells to UVsS in this study.

XP80TO, XP81TO, and XP82TO cells were previously assigned to XPE using XP24KO cells as the complementing cells in the cell fusion assay. From the current study, it does appear that XP82TO is indeed XPE. As we determined that XP24KO cells are in fact UVsS and show almost normal UDS levels, however, the classification of XP80TO and XP81TO remains to be re-evaluated.

Currently, six XPE patients have been reported to have defects in DDB activity (Chu & Chang 1988;Keeney et al. 1992;Otrin et al. 1998). All of these patients exhibit a mild clinical form of XP and UDS levels of approximately 50% of those of normal cells have been reported. UDS is usually measured on single cell-seeded coverslips (Bootsma et al. 1970;Kleijer et al. 1973;de Weerd-Kastelein et al. 1974; 1975b;Kraemer et al. 1975a;Okuno et al. 1994;Yamaizumi & Sugano 1994). Variations among coverslips, however, are recognized as a limitation in measuring UDS accurately. Grain numbers on nuclei, which show UDS levels upon autoradiography, are affected by various factors, such as UV dose, labeling time, amount of label, kind of emulsions, thickness of emulsions, exposure time, and the temperature at time of development. Although most of these factors are kept uniform, it is difficult to control differences in the thickness of emulsions on different coverslips. Therefore, it is necessary to compare cells on the same coverslip and to confirm the results using other criteria. In particular, variations among preparations make the counting of grain numbers difficult especially in the case of high UDS. To resolve this problem, we adopted an alternative assay system that includes both XP and normal control cells on the same coverslip, and confirmed the results by an additional experiment (Itoh et al. 1994,1995a,1996a,b,c). Moreover, we used NTB-3 emulsion, as this is an emulsion with the highest sensitivity, allowing even slightly reduced DNA repair synthesis to be detected as a reduced number of grains.

Other factors, such as cell senescence, thymidine kinase (TK) levels, and intracellular nucleotide pools, also affect the incorporation of label in UDS experiments. Recently, it was reported that DDB protein binds to E2F1, thus modulating the activity of E2F1 as a transcription regulator (Hayes et al. 1998). As TK is one of the genes that are controlled by E2F1, TK activity may be lower in DDB-defective cells. Taken together, we feel that it is very difficult solely to use UDS complementation criteria for cells with near-normal NER activity, such as XPE cells to assign groups.

A summary of the results of this study are shown in Table 7. The three cell strains with defective DNA repair markers, each of which is of the Ddb+ phenotype, have been reassigned to XPF, XPV, or UVsS. The remaining two cell strains, which have a Ddb– phenotype, showed slightly reduced UDS levels, but almost normal phenotypes of other DNA repair markers. Together these results show that (i) at least three XPE Ddb+ strains were misclassified, and (ii) the clinical photosensitive syndrome, XP, has three subgroups based upon UDS: a low UDS subgroup (NER-defective XP), a near normal UDS subgroup [DDB2-defective XP (Ddb– XPE)], and a normal UDS subgroup [DNA polymerase eta-defective XP (XPV)]. Because of this heterogeneity, to diagnose photosensitive patients with/without skin cancer(s) and/or neurologic abnormalities, it is necessary to consider whether they suffer from XP, UVsS, or CS. Indeed, we previously reported that two CS patients (GM10903 and GM10905) have apparent XP phenotypes (Greenhaw et al. 1992;Itoh et al. 1996a). Of the five XPE strains tested in this study, only the Ddb– strains remain classified as XPE. This suggests that XPE is possibly a single group with DDB2 implicated as the defective gene. As DDB has not been demonstrated to be involved directly in NER, we are currently investigating possible alternative role(s) for it in response to DNA damage.


Top

References

  1. Bootsma, D, Mulder, MP, Pot, F, Cohen, JA: Different inherited levels of DNA repair replication in xeroderma pigmentosum cell strains after exposure to ultraviolet irradiation. Mutat Res, 1970 9, 507–516,  | PubMed | ISI | ChemPort |
  2. Chu, G & Chang, E: Xeroderma pigmentosum group E cells lack a nuclear factor that binds to damaged DNA. Science, 1988 1242, 564–567,
  3. Cleaver, JE & Kraemer, KH: Xeroderma pigmentosum. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds). The Metabolic and Molecular Basis of Inherited Disease, 1995 7th edn. New York: McGraw-Hill, 4393–4419,
  4. Dualan, R, Brody, T, Keeney, S, Nichols, AF, Admon, A, Linn, S: Chromosomal localization and cDNA cloning of the genes (DDB1 and DDB2) for the p127 and p48 subunits of a human damage-specific DNA binding protein. Genomics, 1995 29, 62–69,  | Article | PubMed | ISI | ChemPort |
  5. Friedberg, EC, Walker, GC, Siede, W: Nucleotide excision repair in mammalian cells In: DNA Repair and Mutagenesis. American Society for Microbiology, Washington, 1995 283–365,
  6. Fujiwara, Y, Uehara, Y, Ichihashi, M, Yamamoto, Y, Nishioka, K: Assignment of 2 patients with xeroderma pigmentosum to complementation group E. Mutat Res, 1985 145, 55–61,  | PubMed | ISI | ChemPort |
  7. Greenhaw, GA, Hebert, A, Duke-Woodside, ME, Bulter, IJ, Hecht, JT, Cleaver, JE, Thomas, GH, Horton, WA: Xeroderma pigmentosum and Cockayne syndrome: overlapping clinical and biochemical phenotypes. Am J Hum Genet, 1992 50, 677–689,  | PubMed | ISI | ChemPort |
  8. Hayes, S, Shiyanov, P, Chen, X, Raychaudhuri, P: DDB, a putative DNA repair protein, can function as a transcriptional partner of E2F1. Mol Cell Biol, 1998 18, 240–249,  | PubMed | ISI | ChemPort |
  9. Itoh, T, Cleaver, JE, Yamaizumi, M: Cockayne syndrome complementation group B associated with xeroderma pigmentosum phenotype. Hum Genet, 1996a 97, 176–179,  | ISI | ChemPort |
  10. Itoh, T, Fujiwara, Y, Ono, T, Yamaizumi, M: UVs syndrome, a new general category of photosensitive disorder with defective DNA repair, is distinct from xeroderma pigmentosum variant and rodent complementation group 1. Am J Hum Genet, 1995a 56, 1267–1276,  | PubMed | ISI | ChemPort |
  11. Itoh, T, Mori, T, Ohkubo, H, Yamaizumi, M: A newly identified patient with clinical xeroderma pigmentosum phenotype has a nonsense mutation in the DDB2 gene and incomplete repair in (6–4) photoproducts. J Invest Dermatol, 1999 113, 251–257,  | Article | PubMed | ISI | ChemPort |
  12. Itoh, T, Ono, T, Yamaizumi, M: A new UV–sensitive syndrome not belonging to any complementation groups of xeroderma pigmentosum or Cockayne syndrome: siblings showing biochemical characteristics of Cockayne syndrome without typical manifestations. Mutat Res, 1994 314, 233–248,  | PubMed | ISI | ChemPort |
  13. Itoh, T, Ono, T, Yamaizumi, M: A simple method for diagnosing xeroderma pigmentosum variant. J Invest Dermatol, 1996b 107, 349–353,  | Article | PubMed | ISI | ChemPort |
  14. Itoh, T, Shiomi, T, Shiomi, N, Harada, et al. Rodent complementation group 8 (ERCC8) corresponds to Cockayne syndrome complementation group A. Mutat Res, 1996c 362, 167–174,  | ISI | ChemPort |
  15. Itoh, T, Watanabe, H, Yamaizumi, M, Ono, T: A young woman with xeroderma pigmentosum complementation group F and a morphoeic basal cell carcinoma. Br J Dermatol, 1995b 32, 122–127,
  16. Itoh, T, Yamaizumi, M, Ichihashi, M, Hiro-oka, M, Matsui, T, Matsuno, M, Ono, T: Clinical characteristics of three patients with UVs syndrome, a photosensitive disorder with defective DNA repair. Br J Dermatol, 1996d 134, 1147–1150,  | Article | PubMed | ISI | ChemPort |
  17. Johnson, RE, Kondratick, CM, Prakash, S, Prakash, L: hRAD30 mutations in the variant form of xeroderma pigmentosum. Science, 1999 285, 263–265,  | Article | PubMed | ISI | ChemPort |
  18. Kataoka, H & Fujiwara, Y: UV damage-specific DNA-binding protein in xeroderma pigmentosum complementation group E. Biochem Biophys Res Commun, 1991 175, 1139–1143,  | Article | PubMed | ISI | ChemPort |
  19. Keeney, S, Chang, GJ, Linn, S: Characterization of a human DNA damage binding protein implicated in xeroderma pigmentosum E. J Biol Chem, 1993 268, 21293–21300,  | PubMed | ISI | ChemPort |
  20. Keeney, S, Wein, H, Linn, S: Biochemical heterogeneity in xeroderma pigmentosum complementation group E. Mutat Res, 1992 273, 49–56,  | PubMed | ISI | ChemPort |
  21. Kleijer, WJ, de Weerd-Kastelein, EA, Sluyter, ML, Keijzer, W, de Wit, J, Bootsma, D: UV-induced DNA repair synthesis in cells of patients with different forms of xeroderma pigmentosum and of heterozygotes. Mutat Res, 1973 20, 417–428,  | PubMed | ISI | ChemPort |
  22. Kondo, S, Fukuro, S, Mamada, A, Kawada, A, Satoh, Y, Fujiwara, Y: Assignment of three patients with xeroderma pigmentosum to complementation group E and their characteristics. J Invest Dermatol, 1988 90, 152–157,  | Article | PubMed | ISI | ChemPort |
  23. Kondo, S, Mamada, A, Miyamoto, C, Keong, CH, Satoh, Y, Fujiwara, Y: Late onset of skin cancers in 2 xeroderma pigmentosum group F siblings and a review of 30 Japanese xeroderma pigmentosum patients in group D, E and F. Photodermatology, 1989 6, 89–95,  | PubMed | ISI | ChemPort |
  24. Kraemer, KH, Coon, HG, Petinga, RA, Barrett, SF, Rahe, AE, Robbins, JH: Genetic heterogeneity in xeroderma pigmentosum: complementation groups and their relationship to DNA repair rates. Proc Natl Acad Sci USA, 1975a 72, 59–63,  | Article | PubMed | ChemPort |
  25. Kraemer, KH, de Weerd-Kastelein, EA, Robbins, JH, Keijzer, W, Barrett, SF, Petinga, RA, Bootsma, D: Five complementation groups in xeroderma pigmentosum. Mutat Res, 1975b 33, 327–340,  | PubMed | ISI | ChemPort |
  26. Lehmann, AR, Kirk-Bell, S, Arlett, CF, Harcourt, SA, de Weerd-Kastelein, EA, Keijzer, W, Hall-Smith, P: Repair of ultraviolet light damage in a variety of human fibroblast cell strains. Cancer Res, 1977 37, 904–910,  | PubMed | ISI | ChemPort |
  27. Lehmann, AR, Kirk-Bell, S, Arlett, CF, Paterson, MC, Lohman, PH, de Weerd-Kastelein, EA, Bootsma, D: Xeroderma pigmentosum cells with normal levels of excision repair have a defect in DNA synthesis after UV-irradiation. Proc Natl Acad Sci USA, 1975 72, 219–223,  | Article | PubMed | ChemPort |
  28. Masutani, C, Kusumoto, R, Yamada, A: et al. The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta. Nature, 1999 399, 700–704,  | Article | PubMed | ISI | ChemPort |
  29. Nichols, AF, Ong, P, Linn, S: Mutations specific to the xeroderma pigmentosum group E ddb– phenotype. J Biol Chem, 1996 271, 24317–24320,  | Article | PubMed | ISI | ChemPort |
  30. Okuno, Y, Tateishi, S, Yamaizumi, M: Complementation of xeroderma pigmentosum cells by microinjection of mRNA fractionated under denaturing conditions: an estimation of sizes of XP-E and XP-G mRNA. Mutat Res, 1994 314, 11–19,  | PubMed | ISI | ChemPort |
  31. Otrin, VR, Kuraoka, I, Nardo, T: et al. Relationship of xeroderma pigmentosum group E DNA repair defect to the chromatin and DNA binding proteins UV-DDB and replication protein A. Mol Cell Biol, 1998 18, 3182–3190,  | PubMed | ISI | ChemPort |
  32. Rude, J & Friedberg, EC: Semiconservative deoxyribonucleic acid synthesis in unirradiated and ultraviolet-irradiated xeroderma pigmentosum and normal human skin fibroblasts. Mutat Res, 1977 42, 433–442,  | PubMed | ISI | ChemPort |
  33. de Sijbers, AM, Laat, WL, Ariza, RR: et al. Xeroderma pigmentosum group F caused a defect in a structure-specific DNA repair endonuclease. Cell, 1996 86, 811–822,  | PubMed |
  34. de Weerd-Kastelein, EA, Keijzer, W, Bootsma, D: A third complementation group in xeroderma pigmentosum. Mutat Res, 1974 22, 87–91,  | PubMed | ChemPort |
  35. Yamaizumi, M & Sugano, T: UV-induced nuclear accumulation of p53 is evoked through DNA damage of actively transcribed genes independent of the cell cycle. Oncogene, 1994 9, 2775–2784,  | PubMed | ISI | ChemPort |
Top

Acknowledgments

We are grateful to Dr. Yoshisada Fujiwara and Dr. Seiji Kondo for providing XP24KO, XP43TO, XP82TO, and XP89TO cells. We also thank Dr. Hironori Niki and Mrs. Yuka Itoh for preparation of the manuscript. This work was supported in part by Grands-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan (to T.I. and M.Y.), by the Naito Foundation (to T.I.), by the Kao Foundation for Arts and Sciences (to T.I.), and by the USPHS (P30ES08196; to S.L.).

Extra navigation

.
ADVERTISEMENT