Induction of DNA double-strand breaks by ionizing radiation at the c ...

INT. J. RAD IAT. BIO L

1996,

V O L.

69,

NO .

6, 679 ± 685

Induction of DNA double-strand breaks by ionizing radiation at the c-myc locus com pared with the w hole genom e: a study using pulsed-® eld gel electrophoresis and gene probing A. SAK² *, M. STUSCHKE² , N. STAPPER² , C. STREFFER³

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(Received 14 November 1995; revision received 20 Februar y 1996; accepted 28 February 1996) Abstract. Ionizing radiation-induced double-strand breaks (dsb) in a hum an colon carcinom a-derived cell line C OLO 320H SR were determ ined from the fragm ent size distribution of non-speci® cally labelled D NA and S® I restriction enzym e-digested D NA uniform ly labelled with a c-myc probe. The dose-effect relation for th e induction of DN A dsb was linear with no signi® cant difference betw een slopes for - 9 th e curves in th e whole genom e (7 .2 ± 0 .3 ´ 10 dsb/bp/G y) and in the 130 kbp restriction fragm ents containing c-myc - 9 (6 .5 ± 0 . 5 ´ 10 dsb/bp/G y). The size distribution of th e cmyc fragm ents showed deviations from the random -breakage m odel, indicating heterogeneity of dsb induction at this locus.

single-stran d break (ssb) induction (C hiu an d O leinick 1982, Warters et al. 1987, Bunch et al. 1992, 1995), D N A ± protein crosslink (DPC) form ation (C hiu et al. 1986), and thym ine base dam age production (Warters an d Ch ilders 1982). These results are consistent with the notion th at active or open (i.e. protein free) genom ic regions are m ore accessible to ionizing radiation th an inactive on es. W h eth er the initial num ber of dsb after a test dose is a m ajor determ inant of cellular radiosensitivity of hum an cell lines is controversial at the presen t tim e (P eacock et al. 1992, Ruiz de Alm odovar et al. 1994, Zaffaron i et al. 1994). Stu dies on intragenom ic heterogeneity of dsb induction m ight increase our understan ding of cellular variability in radiosensitivity and m ay have profoun d con sequences for th e assessm ent of the suitability of D N A dam age assays m easuring D NA dsb induction in the overall genom e. The aim of th is study was to exam ine the dsb induction frequen cy in a speci® c region of the genom e in com parison with th e unspeci® c bulk D N A. CO L O 320H SR, a colon carcinom a-derived cell line, w as used for this an alysis because its c-m yc gene is highly am pli® ed with a h aploid num ber of 16 ± 32 for c-m yc (A litalo et al. 1983). T his facilitates the quanti® cation of radiation-in duced breaks in restriction enzym e fragm ents around th e c-m yc locus using Southern hybridization .

1. Introduction DN A double-strand breaks (d sb) are considered to be th e m ost relevant initial even t leading to cell killing by ionizing radiation. The m ajority of work dealing with dsb induction by irradiation h as considered the bulk DN A. Data on induction of dsb in speci® c regions of the gen om e are very scant and lim ited to region s > 1 M bp (LoÈ brich et al. 1994a, b). N o data are available up to now on dsb induction in actively transcribing region s < 200 kbp of the genom e. It has, h ow ever, been show n th at these regions are less com pacted an d proteinated than inactive ones (Weisbrod 1982, Villepon teau et al. 1984) and th erefore exh ibit a greater nuclease accessibility (Weintraub 1976, Weintraub and G roudine 1976). G ene regulatory sequen ces exist in an extended chrom osom al conform ation and are D N ase1-hypersensitive in intact nuclei (P orcher et al. 1991). Intragen om ic heterogeneity has been reported for D N A dam age induction with various dam aging agents (Ram an athan et al. 1976, M etzger et al. 1977, Jahn an d Litm an 1979, Arrand and M urray 1982). For ion izing radiation , intragenom ic heterogen eity h as been reported for

2. M aterials and m ethods 2.1. C ell culture The adenocarcinom a cell line CO LO 320H SR, obtained from Am erican Type Culture Collection (ATTC; Rockville, M D, U SA ), was grown in Eagle’ s m inim al essen tial m edium (M EM ) supplem ented with 10% foetal calf serum , non-essential am ino acids, penicillin/streptom ycin (100 units/m l, all from G ibco-BRL , U K) and 5% Seru-m ax serum

*Auth or for correspondence. ² D epartm ent of Radiotherapy, ³ Institu te of M edical Radiobiology, University of Essen, Germ an y. 0955-3002 /96 $12.00

€

1996 Taylor & Francis Ltd

680

A. Sak et al.

exten der (Sigm a, G erm any) un der an atm osphere of 5% CO 2, 95% air at 37 ÊC. Cell lines were tested routinely for m ycoplasm a using the 6-m ethylpurine desoxyribose m ethod (Boehringer M an nh eim , G erm an y).

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2.2. H ybridization probes The probe phm yc, cloned in plasm id D N A (pBR 322), contains 12 443 bases of which 8082 represent the hum an genom ic c-m yc (D alla Favera et al. 1982) sequen ces. We have cut out the 9 Alu-free 5 part of the probe at th e H indIII/XbaI site, delivering a fragm ent of 3500 bp in size used for hybridization.

2.3. L abelling and irradiation of cells In a ® rst step, cells were uniform ly labelled w ith 14 1.85 kBq [2- C]thym idine/m l (1.92 M Bq/m m ol) for 72 h. A suspension of harvested cells in 0.5% InCert agarose (Biozym FM C) was m ade up in ++ ++ H ank’s buffered salt solution with M g and Ca 7 . containing 1 4 ´ 10 cells per m l. Th e suspension was cast into plug m oulds. A fter solidi® cation , plugs were pushed out and equilibrated in culture m edium for 1 h. Cells were then irradiated at 4 ÊC with 7 M eV electrons from a linear accelerator at a dose rate of 21 G /m in (M evatron77, Siem ens, G erm an y).

2.4. Preparation of genom ic DN A Irradiated plugs were transferred into lysis buffer (100 m M E D TA, 10 m M Tris, 20 m M N aCl, 1 m g/m l proteinase K , 1% (w/v) sodium lauryl sarcosine; pH 8 . 0) and incubated for 24 h at 50 ÊC. Plugs w ere th en washed at least three tim es in TE buffer, cut into 5-m m pieces con taining 3 l g D N A, and subjected to restriction enzym e digestion with S® I (Boehringer M ann heim , recogn ition sequen ce G G CCNN N N /N GG CC) at 50 ÊC overnight.

2.5. Electrophoresis and analysis of the gels Fo r electrophoresis, plugs containing approxim ately 3 l g DN A, either restriction enzym e digested or not, were loaded in wells containing 0 . 8% low endosm osis agarose gel (LE Agarose, Beckm an ), and these sealed w ith the sam e agarose.

E lectrophoresis was per form ed under conditions separatin g either Saccharom yces cerevisiae ch rom osom es (condition 1; 225 ± 2200 kbp) or k -Ladder D N A (condition 2; 48 ± 1000 kbp, both from Biolabs, U K) in a TA FE pulse-® eld electroph oresis system (G en eline I, Beckm an, Germ any). C ondition 1

Condition 2

Stage 1 170 m A constant current 4 s pulse tim es 1 h run, 12 ÊC Stage 2 150 m A constant current 50 s pulse tim es 22 h run, 12 ÊC

170 m A constant current 4 s pulse tim es 1 h run, 12 ÊC 150 m A constant current 25 s pulse tim es 22 h run, 12 ÊC

After running, th e gels were stained with eth idium brom ide (0.5 l g/m l) an d ph otographed un der U V light. Th e gel well and each lane were cut into six segm en ts containing DN A of distinct m olecular weight classes. Agarose slices were m elted in a water bath after addition of 100 l l 1 M H Cl. After cooling, 15 m l Quickszint 2000 scintillation ¯ uid (Z insser Analytics, G erm an y) was added to each vial and radioactivity quanti® ed in a liquid scintillation counter (Beckm an 1801, Beckm an, G erm any). The percentage of D N A p i in a segm ent i of the electrophorezed lan e of an irradiated plug was calculated according to: pi

=

(p im

-

p ic )/ p oc ,

(1)

wh ere p im and p ic are the percen tages of counts per lane in the segm ent i of th e irradiated and control groups respectively, and p oc is th e percentage of the non -m igrating DN A fraction in plugs of the control group. p oc is the th eoretical m axim um of D N A per lane that m ight becom e m obile in the gel after D N A fragm entation by irradiation. The percen tage of m obile D N A after irradiation is the sum of p i over th e six size classes.

2.6. Southern hybridization (Southern 1975) DN A was tran sferred to N ylon m em branes (B oehringer M annh eim ), hybridized with digoxigenine dUTP labelled gen om ic c-myc D NA probes an d detected with a chem olum inescen ce kit from Boehringer M an nh eim . After exposure to an X -ray ® lm (K odac X AR -5), th e signal was scann ed densitom etrically using th e GelScanX L program (P harm acia LKB ). Calibration experim ents showed the hybridization signal to be proportional

Radiation-induced DN A double-strand break s at the c-m yc locus to the num ber of target c-m yc fragm ents. T he fraction of m yc containing D NA of lower m olecular weight than the m ain band was obtained from the scans after a correction according to equation 1.

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2.7. Data analysis Assum ing random breakage (i.e. the probability of a dsb per unit len gth at an y given location is constant over th e entire gen om e and independent of the location of prior breaks), the percen tage of D N A found in fragm ents with £ k base pairs, F £ k , depends on the average n um ber of breaks r per ch rom osom e con sisting of n + 1 base pairs (M ontroll and Sim ha 1940, B loÈ cher 1990) according to: F£ (1

k

=

+

p radm ax

(1

-

-

-

(r x k / n )

.

(2)

For undigested D N A, n was set at 1 . 35 ´ 10 , the m ean n um ber of base pairs per ch rom osom e. The asym ptotic m axim um percentage of m obile D N A after irradiation p radmax w as estim ated from the dose-p ercentage out of well data pairs of un speci® cally labelled D N A (Figure 1), assum ing a linear dependence of th e n um ber of induced dsb on dose. The percentage of D N A in a given size class with fragm ent sizes i betw een l and k base pairs is: 8

=

F£

equation 2 was sim ultan eously ® tted to the observed percen tages of D N A in th e different size classes for all repeated experim en ts at a given radiation dose using least squares m inim isation (Procedure N onlin, SAS Institute 1989). For S® I digested DN A the area A of unbroken c-m yc labelled restriction fragm ents and th e total area, A 0 (b and plus sm ear) w ere m easured. Assum ing a Poisson distribution of th e num ber of dsb per fragm ent but not necessarily uniform distribution of the dsb location over the restriction fragm ents consistin g of 130 kbp, the absolute num ber of dsb per fragm en t w as calculated as: dsb per restriction fragm ent

=-

ln(A / A 0 ).

(4)

Because A 0 is determ ined for each lane equal loading of D NA is n ot required.

p radm ax x

k / n )xr xk / n )xe

Fi

681

k

-

F£ l .

(3)

To estim ate the num ber of breaks (r per ch rom osom e) th e random -breakage m odel according to

Figure 1. Percentage of D NA per lane m igrating out of well ( D ) or of myc labelled D NA ( · ) m igrating out of the restriction enzym e-produced fragm ent band in relation to radiation dose. The lines represent the best curves according to th e random -breakage m odel.

3. Results 3.1. Dsb induction in the whole genom e Figure 1 show s th e percentage of m obile D N A as a fun ction of radiation dose for electroph oresis condition 1 (o pen sym bols). In addition to the data points, th e best ® t to these data according to the random -b reakage m odel is given. T he asym ptotic m axim um percen tage of m obile D N A after irradiation p radmax w as estim ated assum ing linear dependence of th e num ber of induced dsb on dose to be 85 ± 2 .4% , an d the probability of D N A dsb induction per base pair to be - 9 - 1 7 .2 ± 0 .5 ´ 1 0 G y . The fact that p radmax is