Colony-forming ability of ultraviolet-irradiated xeroderma pigmentosum fibroblasts from different DNA repair complementation groups

147

Bioehirniea et Biophysiea Acta, 442 (1976) 147--153 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98651

COLONY-FORMING ABILITY OF ULTRAVIOLET-IRRADIATED XERODERMA PIGMENTOSUM FIBROBLASTS FROM DIFFERENT DNA REPAIR COMPLEMENTATION GROUPS

KENNETH H. KRAEMER, ALAN D. ANDREWS, SUSANNA F. BARRETT and JAY H. ROBBINS Dermatology Branch, National Cancer Institute, National Institutes o f Health, Bethesda, Md. 20014 (U.S.A.) (Received January 13th, 1976)

Summary Patients with x e r o d e n n a pigrnentosum develop severe sunlight-induced damage, including malignant neoplasms, on sun-exposed skin. Some pa~ents also have neurological abnormalities. Xeroderma pigrnentosum cells are known to have impaired ability to repair ultraviolet light- or chemical mutagen-induced damage to their D N A , and cell-fusion studies have shown five complementation groups among the D N A excision repair-deficient strains. All xeroderma pigmentosum fibrob!ast strains we tested had lower colony-forming abilities after ultraviolet irradiation than normal strains. Furthermore, we have found that strains from different complementation groups can have different postultrvviolet colony-forming abilitiesand that strains from patients with neurological abnormalities are the most sensitive to ultraviolet light. These results suggest that extremely ineffective repair of damaged D N A in central nervous system neurons may be the cause of the neurological abnormalities. Introduction

Xeroderma pigmentosum (XP) is a rare, autosomal recessive disease in which patients develop severe sunlight.induced cutaneous damage including malignant cutaneous neoplasms. Some X P patients also have associated neurological abnormalities caused by the premature death of neurons in the central nervous system. Numerous studies,utilizingseveral different physico-chemical assays of D N A repair, including ultraviolet-induced thymidine incorporation (unscheduled D N A synthesis), have shown that fibroblastsfrom most X P patients have iraAbbrevlnUon:XP, z~od~'ma Pi~#ntoi~m.

148 p ~ired ability to perform excision repair of ultraviolet-induced damage to their DNA

[i].

Unscheduled DNA synthesis has been analyzed in nuclei of binuclear heterokaryons obtained by fusing fibroblasts from pairs of XP strains. These studies revealed that certain pairs of strains, when fused, had a greater rate of unscheduled DNA synthesis than either s~Tain's unfused mononuclear cells, indicating that such complementing strain pairs had different DNA repair defects [2,3]. These cell fusion studies have demonstrated that there are at le~t five complementation groups (designated A to E) among excision repairdeficient XP fibrobiast strains [4]. Each of these complementation groups is associated with a characteristic rate of unscheduled DNA synthesis [3,4]. Prior to the discovery of XP complementation groups, it had been reported that excision repair
149 at room temperature with ultraviolet light from a germicidal lamp (predominantly 2537 A) (General Electric lamp no. GIST8) at an incident flux of 0.8-2.2 ergs/mm 2/s. Flux was monitored with a Blak-Ray J225 ultravioletintensity meter (Ultra-Violet Products, Inc.) calibrated with malachite green/leukocyanide [13]. The cells were harvested immediately after irradiation and resuspended at each of three concentrations (four dishes per concentration) appropriate to the strain of cellsand the dose of ultraviolet.The medium was replaced with fresh medium after I week and then every 3 or 4 days. ~,fter 14--21 days the cells were fixed with methanol/glacial acetic acid (3 : 1), stained with 0.4% trypan blue in normal saline and examined for colonies (aggregates of at least 15--30 cellsof similar morphology) with a dissecting microscope. Dishes containing more than 80 colonies usually were not included in calculations, for counts in such dishes were inaccurate. Colony-forming efficiency was defined as the number of colonies counted per dish divided by the number of cells plated. The mean colony.forming efficiency is the average of such values obtained from all of the countable replicate dishes at a given ultraviolet dose. The mean colony-forming efficiency ranged from 3.1 to 42%. The colony-forming ability was calculated by dividing the mean colony-forming efficiency of irradiated cellsby the mean colony.forming efficiency of the cells when unirradiated. :qach fibroblast strain was included in 3--5 separate experiments with the exception of X P 1 2 B E which was included in only two experiments. Samples of each fibroblast strain were routinely tested for mycoplasma contamination by culture techniques at Microbiological Associates, Inc., Bethesda, Md. At the conclusion of these experiments, it was reported that X P 7 B E was contaminated w~th mycoplasma, and subsequently the organism was identified as Mycoplasma argininiat Flow Laboratories, Rockville, Md. The effect of this contamination on the post-ultraviolet colony-forming ability of X P 7 B E is not known, but no differences were found between the post-ultraviolet colonyforming ability of this strain and that of XP6BE, another strain from the same complementation group.

Results Fig. 1 shows the post-ultraviolet colony.forming ability of a normal fibreblast strain (contzol donor P), of strain X P 2 B E (complementation group C), and of strain X P # B E (group D). The post-ultravioletcolony-forming abilitiesof the XP strains are markedly lower than that of the normal strain. The XP strains differ from each other~ with the group D strain having a significantly lower post-ultraviolet colony forming ability than the group C strain. At a dose of 40 ergs/mm2 the normal fibroblasts retain at least 70% of their initial colonyforming ability, XP2BE retains less than 3%, and XP7BE retains less than 0.03%. The curve for the normal fibrohlasts has a shoulder between 20 and 30 ergs/mm~, while the curves for XP2BE and XP7BE have extremely small shoul. ders between 0 and 5 ergs/mm 2. The zero-dose extrapolates (broken lines) [14] from the straight.line portions of all three curves intersect the ordinate just below 200% (i.e. they have an extrapolation number just below 2). The post-ultraviolet colony-forming ability of a second normal strain (con-

150

f.0 i ~100 ~

XP2BE (group C)

~,c ..... ,o.... P

,~. °

o c ..... ,D.... •

i ~100 ~t~C~~Do)u' O •pXP12BE (group A)

~ 10!

~ ~ot-

~ 0,i

~ o,i

I

00,Lo

2s

50

~

UV DOSE ~ergs mrn 2 )

~oo

001io

-2~

so

7s

~o

UV DOSE (eTgsturn2}

Fig. 1. Po~t.ultravinlet colony-forming ability of normal and Groups C and D XP flbroblaste plotted as a percentage of the mean colony-formlng efficiency of the uni1~diated cells. The lines were determined bY the method of least squarcs, utilizing all the POints for a givan strain. F o u r points above 100 e r p / m m 2 for Control Donor P are n o t shown, The mean colony-forming efficiency for unl~adiated ceils ranged from 3.1 to 27%. Details in Materials and Methods. UV, ultraviolet. Fig. 2. Post*ultraviotet colony-forming t',bflity of normal and Groups A and D XP fibroblasts plotted as a percentage of the mean colony-forming efficiency of the unix~dla~ed cells, The lines were determined by the method of least squexes~ utilizing all the points for a given strain. Three points above 100 ergs! turn 2 for Control Donor L are not shown. The mean colony-forming efficiency for unirradiated cells ranged from 4,3 to 42%. Details in Materials and Methods. tW, ultraviolet.

trol donor L), strain XP12BE (group A), and XP6BE (group D) are shown in Fig. 2. The curve of control donor L, like that of control donor P (Fig. 1), has a shoulder between 20 and 30 e~'gs/mm 2, and the extrapolation number is also approx. 2. However, the slope of the curve for this strain is somewhat steeper than that of control donor P (Fig. 1). The curve for s~r~in XP6BE is virtually identical with that for the other strain from group D, XP7BE (Fig. 1), with regard both to slope and to extrapolation number. The slope of the curve for strain XP12BE is much steeper than that of control donor L, but is not significantly different from that of strain XP6BE. The Do values and the rates of unschedules DNA synthesis for each of the fibroblasts strains are given in Table I. The Do values are calculated from the straight-line portions of the curves in Figs. 1 and 9. All of the XP ~trains have much smaller Do values than do the normal strains. The group C strain. XP2BE, has a Do value significantly higher than those of the other three XP strains (P < 0.05 by Student's t test), while the Do values for strains XP6BE, XP7BE

ISl TABLE I D O O F P O S T - U L T R A V I O L E T C O L O N Y - F O R M I N G A B | L I T Y F O R N O R M A L A N D XP F I B R O B L A S T S COMPARED WITH R A T E S O F U L T R A V I O L E T - I N D U C E D U N S C H E D U L E S DNA S Y N T H E S I S Fibrob|ast strain

XP complementation ~'oup *

D O of p o s t - u l t ~ v i o l e t c o l b y - f o r m i n g ability * s (ergslmm 2)

Rate of ~ t r a v i o | e t - i ~ , z e d u ~ h e d u l e d DNA I ~ y n t h ~ (% o f no~1~fl)

Control Do n o r P Control Donor L XP2BE X]P6BE XP7BE XP121~E

--

46.7 29.9 8.8 4.0 4.3 6,9

I00 I00 10---20 25--50 25--50 ~2

C D D A

* F r o m Kr aem er e t ai. [ 3 ] , ** Each D O value was obtained from th e straight-line portion of the appropriate curve in Fig~. I o r 2 and represents th e ultraviolet dose required to reduce the colony-forming abflty from any point on the llne t o 37% of t h a t point. T h e D O for strain X P2B E is signifleantly different (P < O . 0 5 b y the St u d en t' s t test) from the D O of the othe r XP strair~ which do n o t differ Mgnifieantly from each other.

and XP12BE are not significantly different from one another. In contrast, as shown in the last column on Table I, we have reported [3] that the rates of unscheduled DNA synthesis for strains XP6BE and XP7BE (the group D strains) are higher than that of either XP2BE or XP12BE. Discussion

The fact that XP fibroblasts have defective repair of ultraviolet-induced DNA damage and diminished post-ultraviolet colony-forming ability (Figs. 1 and 2; Table I) suggests that post-ultraviolet colony-forming ability is dependent, at least in part, on the fibroblasts' capacity for DNA repair. To form a recognizable colony in our assay of post-ultraviolet colony-forming ability, an irradiated cell must survive and multiply at least 4 or 5 times. Thus, in contrast to our studies on unscheduled DNA synthesis, post.ultraviolet colony-forming ability is a measure of the biological effectiveness of such repair. Our stvdy demonstrates that there are significant differences among XP strains with ~egard to post.ultraviolet colony-forming ability. The strain from complementation group C has a greater ability than that of the group A or D strains (Figs, 1 and 2). The post-ultraviolet colony-forming abilities of the group D s*,zains were similar to each other and as low as that of the group A strain. However, the ultraviolet-induced unscheduled DNA synthesis rates of the group D strains, as measured by us (Table I), are much higher than those of the group A or C strains. Thus, at least in the case of these group D strains, the degree of impairment in their rate of ultraviolet-induced unscheduled DNA synthesis does not accurately reflect their degree of impairment in post-ultraviolet colony-forming ability. Day [15] used another measure of the functional effectiveness of DNA repair. He studied host cell reactivation of ultraviolet-irradiated adenovirus 2. In his studies, as in ours, group D strains were fmmd to be more deficient than

152

group C strains and at legist as deficient ~s group A strains. There are several po~;ible explanations for the differences between the re. lative rates of unscheduled DNA synthesis and relative sensitivity to ultraviolet as m~mifested by post.ultraviolet colony-forming ability among the XP strains. Unscheduled DNA synthesis provides no info~,-nation about the accuracy of such synthesis, about the later steps in the excision-repair process, or about other DNA repair processes. The defect in group D stre ins, while permitting a re],tiveiy high rate of unscheduled DNA synthesis, may affect the fidelity of repair, permitting misreplication of bases during ~pair replication. Alternatively, the group D strains might have a severe defect in ~l~ther repair system in addition to their defect in excision repair. Reduced post-replication repair [16] and reduced photoreactivating enzyme [17] have zecently been reported in a number of XP strains. Finally, differences in the relative rates of unscheduled DNA synthesis and those of post-ultraviolet colony-forming ability could result from differences in the culture conditions utilized for the two different assays. Some XP patients have progressive neurological abnormalities in addition to the cutaneous sirens of XP, Such abnormalities result from a chronic degenerative process affecting neurons [1]. All patients with such XP-associated neurologica~ abnormalities are in either Groups A or D [1,4]. Fibroblasts from thes~ groups have the most severe repair deficits as measured by post-ultraviolet colony-forming ability (Figs. 1 and 2; Table I) and by host~ell reactivation of ultraviolet-irradiated virus [15]. XP cells are deficient not only in repair of ultraviolet-inducecl DNA damage but also in the repair of DNA damage caused by certain chemical mutagen6 [8,18,19]. Adequate repair of neuronal DNA receiving damage from exogenous or endogenous chemicals might be required for the maintenance of the metabolic integrity of the nervous system [1]. XPassociated neurological abnormalities may thus be related to severe impairment in the functional effectiveness of DNA repair in the neurons of the central nervous system. Referen~:es I Robbing, J.H., Kraemer, K.H., Lutzner, M.L., Festoff, b.W. mxd Coon, H,G, (1974) Ann. Int. Med. 60, 221--248 2 DeWeerd.!~astelein, E.A., Keijzer, W. and Bootsma, D. (19"]2) Nature 238, 80--83 3 Kraemer, K.H., Coon, H,G., Petlnga, R.A., Bar~tt, S.F., Rahe, A.E. and Robblns, J.n. (1975) Proc. Natl, A©ad. Sci. U,S. 72, 59--63 4 Kraemer, K,H., DeWeeni.Kastelehl, E.A., Robbins, J.H., Barrett, S.F., Petlnga, R.A. and Bootsrna, D. (1975) Murat. Res. 33, 327.--340 5 Cleaver, J,E, (1970) Int. J. Radi~t. Biol. 18,567--565 6 Goldstein, S. (1971) Proc. Soc, Exp. Biol. Med. 137,730--734 7 Takebe, H., Foruyarna, J., Miki, Y. and Don&t, S, (1972) Mut. Re$. 15.98--100 8 Stich, H.F., San, I~.H.C. and Kawnzoe, Y. (1978) Mut. Res. 17,127--137 9 Kmemer, K,H., Barrett, S.F. and Robbins, J.H. (1974) Clin. Res. 22,609A 10 Maher, V.M., Bi~h, N., Otto, J.R. and McCorm|ck, J.J. (1975) J. Natl. Cancer Inst. 64,1287--1294 11 American Type Culture Collection List of Human Skin Fibrobl~s (1974) 3t'd edn., pp. 14~17, Rockvnle. Md. 12 Coon, H.G. and Weiss, M.C. (1969) ,'~toe. Natl. Aead. Sei. U.S. 6 2 , 8 5 2 - 8 5 9 13 Joh~0 H.E, (1969) in Methods ,;n Enzymolo~ (Kustin, K., od,)0 VoL 16, pp. 263--316, Academic Pres~ New York 14 Elkind, M.M. and Whltmo~e, G.F. (1967) The Radiobin|o~ of Cultured Mammalian Cells, p. 39, Gordon and Breaeh~ Science Pub~l~hers, Inc., New York

153

16 Day, IH0 P,~. (1974) ~ Re8. 84,196~"1970 16 l, ehmmn. A.R., KI~-B~I, 8., Axe,t, ¢.F0, P a t ' , ~ m , M.C.o Lohman, P.H.M, D a W ~ ~ , and Bootsms, D° (1975) P:o¢. Nail. A e ~ , S,~i. U.5. ?2, 21~---228 17 Suth~Isnd, B.M., Riee, M. and W q n ~ , ~.K. (1975) P:OC. Nat]. Acad. ~ . ~.5. ".'2, I ~ I . 0 7 18 SUch, H.F.+ ~hln, R.H.C., Miner, J.A. and Mln~, E.C. (1972) N~, New ]~c~o238, 9--10 Zg Rqmn, ~T,D.end 8etlow. P,,B, (1974) Cance~Ree, 84, 8818-8825

E,A.