Genetic Heterogeneity in Xeroderma Pigmentosum: Complementation Groups and Their Relationship to DNA Repair Rates - PNAS
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Proc. Nat. Acad. Sci. USA
Vol. 72, No. 1, pp. 59-63, January 1975
Genetic Heterogeneity in Xeroderma Pigmentosum: Complementation Groups
and Their Relationship to DNA Repair Rates
(ultraviolet radiation/mutations/unscheduled DNA synthesis/somatic cell genetics)
KENNETH H. KRAEMER*, HAYDEN G. COONt, ROBERT A. PETINGA, SUSANNA F. BARRETT,
ANN E. RAHEt, AND JAY H. ROBBINS
Dermatology Branch and t Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014
Communicated by Aaron B. Lerner, October 4, 1974
ABSTRACT Fibroblast strains from 12 patients with (11) and XPiLO (12) are identified by the nomenclature for
xeroderma pigmentosum had lower than normal rates of XP strains (13). Fibroblasts were grown without anti-
DNA repair, as determined by autoradiographic studies of biotics in modified Ham's F12 medium (14) supplemented
ultraviolet-induced unscheduled nuclear DNA synthesis.
The nuclei in binuclear cells, obtained by fusing fibro- with 5% fetal calf serum at 370 in a 5% C02-95% air incuba-
blasts from certain pairs of these strains, had a greater tor with more than 95% humidity. They were used after two
rate of DNA repair than the nuclei of either strain's un- to seven additional passages.
fused mononuclear cells. These results indicate that com-
plementary corrections of the strains' repair defects had Cell Fusion. One- to 3-day-old coverslip cultures in 60-mm
occurred in the fused cells. Four complementation groups petri dishes were used for irradiation and fusion, the latter
were found, indicating that at least four mutations caused
decreased DNA repair among these 12 strains. The unfused performed by a modification of the method of Yamanaka and
mononuclear cells of each group had a characteristic rate Okada (15). Cultures, cooled on ice, were washed with cold
of repair that differed from the rates of the other groups. Hanks' balanced salts solution and covered with 0.1 ml of
cold, serum-free medium containing about 100 hemagglutinat-
In xeroderma pigmentosum (XP), a rare disease of autosomal,
recessive inheritance, sun-exposed skin develops severe solar ikg units [standardized with 0.03% (v/v) chick erythrocytes]
of fl-propiolactone-inactivated Sendai virus (14). Ten minutes
damage, pigmentation changes, and neoplasms (1). XP cells later, about 0.1 ml of cold, serum-free medium containing
repair DNA damaged by ultraviolet (UV) light more slowly 5 X 104 cells (to be fused with those already on the cover-
than normal cells (1-3). Defective repair in cells from most slips) was dropped on each coverslip. After 20 mm in the cold,
XP patients can be detected by measuring the rate at which the dishes were placed in the 370 incubator. Three hours later
the cells incorporate [3H]thymidine into DNA segments the coverslips were covered with serum-containing medium
synthesized to replace regions containing UV-induced pyrimi- (3 ml per dish), and the incubation was continued.
dine dimers (1, 2, 4). This [3H]thymidine incorporation re-
flects DNA repair replication and is manifested autoradio- UV Irradiation. About 16 hr after addition of inactivated
graphically as unscheduled DNA synthesis (1, 2, 4-6). virus, the cultures were washed twice with phosphate-buffered
XP is one of the few human diseases (7, 8) in which somatic saline (pH 7.4) at room temperature and covered with
cell genetic studies have successfully shown different muta- phosphate-buffered saline (1 ml per dish). Immediately after
tions to be responsible for an observed phenotype. When irradiation with 300 erg/mm2 of 254 nm UV light (16), the
fibroblast strains from certain pairs of XP patients are fused phosphate-buffered saline was removed and 2 ml of Hanks'
in culture to form binuclear heterokaryons, both of the nuclei base medium 199 (Difco Laboratories or NIH Media Unit)
in the fused cells perform more unscheduled DNA synthesis containing bicarbonate, 20%0 fresh, heparinized, filtered
than the nuclei of the unfused mononuclear cells of either humah plasma, and 20 IACi of [methyl-3H]thymidine (specific
strain (1, 9). De Weerd-Kastelein et al. (9) first demonstrated activity, 17-25 Ci/mmol; Amersham-Searle) was added. The
this genetic heterogeneity in XP and have found three com- dishes were placed in the incubator for 3 hr. The cultures
plementation groups (10). We have found four complementa- were then washed and fixed. Unirradiated coverslip cultures
tion groups among the 12 XP strains we have studied, and were processed in parallel with the irradiated cultures.
we have shown that all the strains within a complementation
group have a similar rate of unscheduled DNA synthesis Autoradiograms and Their Analysis. Autoradiograms,
that is characteristic for each group. prepared with NTB-3 emulsion (Eastman Kodak), were e'x'-
posed at 40 for 7 days (8 day s in the case of Exp. 1 of Table
MATERIALS AND METHODS 1), then developed and stained. Grain production and count-
Fibroblast Strains. Twelve XP and three normal control ing efficiencies were constant for the range of grain counts
donor strains were from the American Type Culture Collec- in these experiments.
tion, Rockville, Mld. Ten of the XP strains were from patients On coverslip cultures not treated with virus, grains over
of the NIH series (1). These XP strains and strains XPKMISF "lightly labeled" (1, 2, 5, 6) nuclei from 100 consecutively
observed mononuclear cells were counted. Virus-treated
Abbreviations: XP, xeroderma pigmentosum; UV, ultraviolet. coverslips were scamied under low power for areas containing
*
Present address: Department of Dermatology, University of multinucleate cells that had the most light labeling. In such an
Miami School of Medicine, Box 520875, Miami, Fla. 33152. area, grains were counted over the 100 nuclei of 50 consecu-
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5960 Cell Biology: Kraemer et al. Proc. Nat. Acad. Sci. USA 72 (1976)
TABLE 1. Relationship of the XP complementation groups to the amount of UV-induced unscheduled DNA synthesis performed by
XP fibroblast strains during the first 3 hr after irradiation
Complen % of Normal unscheduled DNA synthesis* (mean grain count)t
mentation Fibroblast
group strain Exp. 11 Exp. 2t Exp. 3t Exp. 4t Exp. 5T
Control donor k 100 (114.8) 100 (117.2)
Control donor L 100 (114.9) - 100 (125)
Control donor P - 100 (130.2) 100 (99.9)
A XP1LOProc. Nat. Acad. Sci. USA 72 (1975) XP Complementation Groups and DNA Repair Rates 61
56
56
Control Donor R a
M Control Donor
R ..
30 L Mononuclear Cells
Mononuclear Cells
25 No UV UV
20
10 1 . 48
.~~ ~~~~~~~b_ d
69 72
...qH I= -
e-
,
1.
*
Group A 42 Group A ,rR;:' '
C
30: XPILO n XPI LO *>.
20
25- No UV
r-
Mononuclear Cell ks Mononuclear Cells
UV
_Il FIG. 2. Autoradiograms of a Sendai virus-treated culture of
two complementing XP fibroblast strains, XP1BE (group C)
and XP12BE (group A). The cells were irradiated and incubated
with [3H]thymidine as described in Materials and Methods. Cells
5.'J a-d are unfused mononuclear cells; e and f, binuclear cells. Cells
12.6
fl68 Group B 1. Group B a and b show unscheduled DNA synthesis typical of strain
XPIIBE XPI IBE XP1BE (Table 1 and Fig. lh); c and e have low grain counts
- 30 tf
25
Mononuclear Cells Mononuclear Cells typical of XP12BE (Table 1); d is "heavily labeled" in S-phase.
L) L
No UV UV synthesis; f is a complementing heterokaryon. X400.
Do [
= /
ing cells was not noticeably decreased by 10 mM hydroxy-
Io f urea, a concentration that inhibited S-phase DNA synthesis
L e
* in the cultures by more than 95%.
4.2
B76 Group C Group C 47
XPIBE
30o Mononuclear Cells v Mononuclear Cells XPILO x XPILO XPILO x XPILO
25 No UV UV Mononuclear Cells Binucleor Cells
20 rF Ki NJ''AUV
2
5
20'1Suvt-.T
-
UV
5.
X.I h
- C. a b
43
Group D Group D L. 7.77.7
30ri XP5BE XP5BE
Mononuclear Cells Mononuclear Cells LI 3
Group AxA 4 Group A x A
25
No UV UV
> !0; XPILO x XPKMSF XPILO x XPKMSF
Mononuclear Cells Binucleor Cells
50- aft
'0
O1 20 30So
4 0 5C 60 7W0 U .v3
4, r . C K- W c2 3C Av NC iW61 7X
GRAINS PER NUCLEUS
FIG. 1. Histograms showing UV-induced unscheduled DNA 5 ~~~~~~C
d
synthesis during the first 3 hr after UV irradiation with 300 erg/
mm2 in a fibroblast strain from a control donor and in a fibroblast o 10 20 30 40 50 60 70 80 90 l00 0 10 20 30 40 50 60 70 80 90 100
strain from each of the four complementation groups. Data are GRAINS PER NUCLEUS
from the autoradiograms of Exp. 1, Table 1. Histograms in left FIG. 3. Histograms for Sendai virus-treated, irradiated fibro-
column (a, c, e, g, and i) show nuclear background for each blasts showing lack of complementation when XP fibroblasts
strain's unirradiated cells. Histograms in the right column (b, d, are fused with cells from the same strain or with cells from an-
f, h, and j) show nuclear background plus any UV-induced un- other strain in the same complementation group. a, Unfused
scheduled DNA synthesis for each strain's irradiated cells. The mononuclear cells on a coverslip containing cells only from strain
grain class of 0 through 5 grains per nucleus includes nuclei with XP1LO. b, Binuclear cells (homokaryons) on the same coverslip.
no grains as well as those with as many as 5 grains; grain classes c, Unfused mononuclear cells on a coverslip containing cells from
then run successively as 6 through 10, 11 through 15, etc. When both strains XPlLO and XPKMISF. d, Binuclear cells (homo-
more than 35 nuclei are in a single grain class, the bar representing karyons of XP1LO and of XPKMSF; and heterokaryons with a
them is truncated, and the number of nuclei in that class is printed nucleus from each strain) on the latter coverslip. Each arrow
near the top of the bar. Each arrow indicates the mean grain indicates the mean grain count for the nuclei with 30 or fewer
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count for the 100 consecutively evaluated nuclei of each histo- grains per nucleus among the 100 consecutively observed nuclei
gram (50 in the case of b) except that occasional cells with an in each histogram. Histograms are presented as in Fig. 1 and
unrepresentatively high grain count were excluded. Materials and Methods.62
25
2-C
05
5n
1S
66
3WOXPILO x XPIIBE
of12
UA
8
o V
!~~~~~~~~~~~~3
=3
~~i
Cell ]Biology: Kraemer et al.
iru
Group A x B
Mononuclear Cells
UV
Group AxC
XPILDO x XPIBE
Grou B x
Mononuclear Cells
_._E u__.iS
2
,
Group A x B
XPILO
UV
4-1,-. ..A'
GroupAxC
XPILO XPIBE
UV
x
x
Binucleor Cells
Binuclear Cells
XPIIBE
,
0oo 6
A RmU
b
-30
=:
2 5-Proc. Nat. Acad. Sci. USA 72 (1975) XP Complementation Groups and DNA Repair Rates 63
TABLE 2. Assignment of the 12 XP fibroblast strains to their tion groups (8). On the other hand, for mutations in the
unscheduled DNA synthesis complementation groups* enzyme hypoxanthine-guanine phosphoribosyltransferase
(HGPRT) in cultured Chinese hamster cells, strains within
m
one complementation group had, as for XP, a similar level of
v c m mp
N
mpCX mp0 pp mtsm
LO co -
enzyme activity different from the level in the other com-
1- rq ra ret
rt
fr
P4 P-
Pt PL P4 P4 A
plementation group (22).
XPILO 0 Existence of five complementation groups in XP indicates
XPKMSF 0 n that at least five mutations result in decreased rates of UV-
XP12BE 0 00 induced unscheduled DNA synthesis. Attempts are being
XP1IBE + + 0 made to correlate each of these repair defects with abnormal
functioning of XP cells. Cutaneous manifestations of XP, in-
XP1BE + + + 0 cluding neoplasms, are present in patients of all groups, but
XP2BE + + + + 0 0
XP3BE + n + + 0 0 0 neurological abnormalities appear to be present only in pa-
XP8BE n n + + 0 00 n tients of groups A, B, and D (1). The capacity of XP fibro-
XP1OBE + n + + 0 00 0 0 blast strains to reactivate UV-damaged adenovirus is re-
XP5BE
lated to their complementation group assignment (23).
+ + n + + + + n + 0 Colony-forming ability after UV irradiation can differ among
XP6BE n n + + + n n + + 0 n
XP7BE + + + + + + + n + 000 XP fibroblast strains (K. H. Kraemer, S. F. Barrett, and J. H.
Robbins, unpublished data), and preliminary studies indicate
*
+, Complementing; 0, noncomplementing; n, not studied. that strains within a group have the same sensitivity to UV
Fibroblast strains listed in the vertical columns were fused to radiation. Thus, the identity of the complementation groups
those listed in the horizontal columns. Complementation was derived from studies of unscheduled DNA synthesis in fused
determined autoradiographically by visual inspection (e.g., see cells appears to be preserved in relation to other UV-associated
Fig. 2) or by grain count analysis (as in Figs. 3-5). processes.
DISCUSSION 1. Robbins, J. H., Kraemer, K. H., Lutzner, M. L., Festoff,
The light labeling seen after UV-induced unscheduled DNA B. W. & Coon, H. G. (1974) Ann. Int. Med. 80, 221-248.
synthesis in unfused, mammalian mononuclear cells (5, 6), 2. Cleaver, J. E. (1968) Nature 218, 652-656.
including XP cells (1-3, 11, 16, 18), reflects the insertion of 3. Epstein, J. H., Fukuyama, K., Reed, W. B. & Epstein, W.
L. (1970) Science 168, 1477-1478.
radioactive nucleosides during hydroxyurea-resistant (17) 4. Cleaver, J. E. (1973) in Advances in Radiation Biology, eds.
repair replication into gaps created by excision of DNA Lett, J. T., Zelle, M. R. & Adler, H. (Academic Press, Inc.,
segments containing UV-induced pyrimidine dimers. That New York), pp. 1-75.
the UV-induced unscheduled DNA synthesis we observed in 5. Rasmussen, R. E. & Painter, R. B. (1966) J. Cell Biol. 29,
11-19.
complementing binuclear XP fibroblasts represents DNA 6. Painter, R. B. & Cleaver, J. E. (1969) Radiat. Res. 37, 451-
repair replication is supported by resistance of the unscheduled 466.
DNA synthesis to hydroxyurea and by experiments that 7. Nadler, H. L., Chacko, C. M. & Rachmeler, M. (1970) Proc.
indicate that such complementing, fused XP cells have normal Nat. Acad. Sci. USA 67, 976-982.
repair replication (19), thymine dimer excision (20), and host- 8. Lyons, L. B., Cox, R. P. & Dancis, J. (1973) Nature 243,
533-535.
cell reactivation of UV-damaged adenovirus 2 (R. S. Day, 9. De Weerd-Kastelein, E. A., Keijzer, W. & Bootsma, D.
III, K. H. Kraemer, and J. H. Robbins, unpublished data). (1972) Nature New Biol. 238, 80-83.
Each of our four complementation groups has a characteris- 10. De Weerd-Kastelein, E. A., Keijzer, W. & Bootsma D.
tic rate of UV-induced unscheduled DNA synthesis given by (1974) Mutat. Res. 22, 87-91.
11. Cleaver, J. E. (1970) Int. J. Radiat. Biol. 18, 557-565.
the unfused mononuclear cells of its strains (Table 1). A 12. Parrington, J. M., Delhanty, J. D. A. & Baden, H. P. (1971)
similar relationship has been found (13, 21) for the three Ann. Hum. Genet. 35, 149-160.
groups of De Weerd-Kastelein et al. (9, 10). In experiments to 13. Kleijer, W. J., De Weerd-Kastelein, E. A., Sluyter, M. L.,
relate their three complementation groups with our groups, Keijzer, W., De Wit, J. & Bootsma, D. (1973) Mutat. Res.
fusion studies in their laboratory (21) and in ours have re- 20, 417-428.
14. Coon, H. G. & Weiss, M. C. (1969) Proc. Nat. Acad. Sci.
vealed that only one of their three groups, the one containing USA 62, 852-859.
their strain XP2RO, is different from all of our four groups. 15. Yamanaka, T. & Okada, Y. (1966) Biken J. 9, 159-175.
Thus, there are five known complementation groups in 16. Burk, P. G., Lutzner, M. A., Clarke, D. D. & Robbins, J. H.
XP (21). Our method for evaluating unscheduled DNA (1971) J. Lab. Clin. Med. 77, 759-767.
17. Cleaver, J. E. (1969) Radiat. Res. 37, 334-348.
synthesis shows that their strain XP2RO has a greater rate 18. Robbins, J. H. & Kraemer, K. H. (1972) Biochim. Biophys.
of unscheduled DNA synthesis than the strains in our group Acta 277, 7-14.
D, and the complementation group containing strain XP2RO 19. De Weerd-Kastelein, E. A., Kleijer, W. J., Sluyter, M. L.
has, therefore, been named group E. & Keijzer, W. (1973) Mutat. Res. 19, 237-243.
The only other human disease in which cell fusion studies 20. Paterson, M. C., Lohman, P. H. M., Westerveld, A. &
have revealed complementation groups relating to a defective Sluyter, M. L. (1974) Nature 248, 50-52.
21. Bootsma, D., De Weerd-Kastelein, E. A., Kleijer, W. J. &
biochemical process that can be quantitated in the unfused Keijzer, W. (1975) in Molecular Mechanisms for the Repair
cells is maple syrup urine disease (8). In this disease, however, of DNA, eds. Hanawalt, P. C. & Setlow, R. B. (Plenum
unlike XP, the strains within a complementation group do not Press, New York), in press.
have similar degrees of reduced enzyme activity, and strains 22. Sekiguchi, T. & Sekiguchi, F. (1973) Exp. Cell Res. 77, 391-
403.
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with apparently similar degrees are in different complementa- 23. Day, R. S.,- III (1974) Cancer Res. 34, 1965-1970.You can also read
