DNA repair dependent NAD+ metabolism is impaired in cells from ...

The EMBO Journal Vol.2 No.3 pp.303 -307, 1983

DNA repair dependent NAD + metabolism is impaired in patients with Fanconi's anemia H.Klocker, B.Auer, M.Hirsch-Kauffmann, H.Altmannt, H.J.Burtscher and M.Schweiger* Institut fur Biocheniie (Nat. Fak.), Universitat Innsbruck, A-6020 Innsbruck, Austria Communicated by M.Schweiger Received on 25 October 1982; revised on 24th January 1983

In vitro cultivated fibroblasts derived either from patients with Fanconi's anemia (FA) or from healthy probands were analyzed for their DNA repair-dependent NAD+ metabolism. No difference in NAD+ pools was found. NAD+ consumption after cell damage by u.v. irradiation was, however, significantly reduced in FA cells. Several FA cell lines had a lowered ability to transfer ADP-ribose to acid-precipitable material. Additionally, a decreased activity of NAD: protein ADP-ribosyltransferase was found for three FA cell lines. Our data indicate, that FA is accompanied by a defective NAD+ metabolism during DNA repair. Key words: DNA repair/NAD+/Fanconi's anemia/ADP-

ribosylation Introduction Fanconi's anemia (FA) belongs to a group of hereditary repair deficiencies (reparatoses) which are accompanied by chromosomal instability, elevated incidence of cancer and often by mental retardation and anatomical abnormalities (Fanconi, 1927; Schroeder et at., 1964; Prindull et al., 1975). Several causes for FA have been discussed including defects of excision repair exonuclease (Poon et al., 1974), cross-link repair (Sasaki and Tonomura, 1973; Sasaki, 1975; Auerbach and Wolman, 1976,1978), nucleotide metabolism (Frazelle et al., 1980), intracellular distribution of topoisomerase I (Wunder et al., 1981; however, see also Auer et al., 1982) and oxygen metabolism (Nordenson, 1977; Joenje et al., 1981). U.v. repair is impaired in fibroblasts from patients with FA (Hirsch-Kauffmann et al., 1978; Schwaiger et al., 1982). U.v.-induced thymine dimers are excised from DNA with the same rates in FA and normal fibroblasts (Hirsch-Kauffmann, 1978; Klocker, unpublished data) and DNA repair synthesis is not reduced in FA fibroblasts (Burtscher, 1979). However, nucleoid recondensation after u.v. irradiation is retarded in FA fibroblasts, indicating a delay of the rejoining step (Schwaiger et al., 1982) and reduced ligation capacity was found in homogenates of fibroblasts derived from a patient with FA (Hirsch-Kauffmann et al., 1978). Nicotinamide adenine dinucleotide (NAD + )-dependent ADP-ribosylation plays a role in DNA repair (Miller, 1975a; Hilz and Stone, 1976; Mullins et al., 1977; Berger et al., 1978; Benjamin and Gill, 1980; Berger and Sikorski, 1980; Durkacz et al., 1980; Edwards and Taylor, 1980; Purnell et al., 1980; Durkacz et al., 1981b; McCurry and Jacobson, 1981; Miwa et al., 1981; Althaus et al., 1982). The cellular content of NAD + is reduced during DNA repair, and restored to a nor1Permanent address: Austrian Research Centre Seibersdorf, A-1082 Wien, Austria. *To whom reprint requests should be sent. IRL Press Limited, Oxford, England.

cells

from

mal level during recovery of the cells (Schein and Loftus, 1968; Chang, 1972; McCurry and Jacobson, 1981). Indirect (Klocker et al., 1982) as well as direct (Creissen and Shall, 1982) evidence indicates, that this NAD+ consumption is involved in activation of DNA ligase. This prompted us to investigate NAD+ metabolism in cells derived from patients with FA. Here we report that, compared with normal fibroblasts, cells from FA patients showed a reduced NAD+ consumption after u.v. irradiation, although there is no difference in NAD + levels in these cells. In addition, we found a decrease of ADP-ribosylation capacity in permeabilized fibroblasts of some of the FA strains. ADP-ribosyltransferase (ADPRT) activity is also diminished in nuclear extracts of these cells. Results

NAD+ contents of normal and FA fibroblasts Nucleotides were extracted with perchloric acid and analysed by h.p.l.c. on an anion-exchange column. 106 cells of fibroblast strain contained between 2.59 and 9.74 nmol NAD+ (Table I). The mean values were 5.42 nmol for normal and 6.39 nmol for FA fibroblasts with standard deviations of 2.08 and 1.68, respectively. Since the relationship of NAD + to cell number does not take into account the variable cell sizes of individual cell strains, it is not a useful parameter for calculating NAD+ concentrations in cells. Therefore, the entire adenine nucleotide pool, which was measured simultaneously, was chosen as an internal reference. Every Table I. NAD+ contents of normal and FA fibroblasts

NADI sd. nmol/106 cells

n NADI NADI + AMP + ADP + ATP

Beau

8.12 6.56 + 0.20 5.26 0.79 4.57 i 1.55 2.59 ± 0.39

0.28 0.26 0.24 0.21 0.23

MEAN:

5.42 + 2.08

0.24 + 0.03

FA: FISG FLH F71 1424 FI FB FLP FLB

4.07 5.91 5.22 7.32 5.80 9.74 5.84 6.74

MEAN:

6.33 + 1.68

Strain CONTROLS: Dop Hebu Hekl Hika

0.70 0.89 0.14 0.26 0.75 0.20 0.18 i 0.54

+ + + + + +

0.26 0.24 0.22 0.24 0.20 0.24 0.21 0.25

1 4 3 4 3

2 3 4 2 2 3 2 2

0.23 + 0.02

Cells were transferred to cell culture dishes (diameter = 6 cm) with a density of 2 x 105 cells/plate. After 3 days the medium was renewed. On the fourth day after transfer, nucleotides were extracted and determined as described in Materials and methods.

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repair time (hi -

repair ime (hi

Fig. 1. Decrease of NAD+ during u.v.-repair. Cells were grown to confluency on cell culture dishes (diameter = 6 cm) and arrested with medium containing 107o FCS only. The plates were irradiated with 18 J/m2 and 36 J/m2, respectively, and incubated for repair with medium containing 1% FCS. At the times indicated, u.v.-irradiated plates and unirradiated control plates were extracted with 0.4 M perchloric acid. The acidic extract was neutralized, NADI determined by h.p.l.c. analysis and calculated as a percentage of unirradiated controls. Each point is the mean value of two independent samples. A: 18 J/m2, B: 36 A Dop (pass. 13); * * Kraha (pass. 11); * J/m2. Controls: A * Hekl (pass. 11). FA: A-.....A F71 (pass. 9); O-----O 1424 (pass. 19); EL-.-.-. O FI (pass.6).

change in the cellular NAD + concentration should change the ratio of NAD+ to total adenine nucleotides. Indeed, this relationship remained nearly constant in all cell strains tested. The ratio of NAD + to the sum of all adenine nucleotides (ATP + ADP + AMP + NAD+) was 0.24 0.03 in normal cells and 0.23 0.02 in FA cells (Table I). There was no apparent difference between NAD+ levels in normal and in FA fibroblasts. Decrease of NAD+ during u. v. repair After irradiation of fibroblasts with a u.v. dose of 18 J/m2 the cellular contents of NAD+ decreased to -70Wo and returned to normal levels within 30 h (Figure IA). The minima of the curves show that NAD+ consumption is completed after 15 -25 h. It is interesting, that nucleoid recondensation after u.v. irradiation (Schwaiger et al., 1982) follows the same time course, suggesting a connection between these two processes. A dose of 36 J/m2 reduced NAD+ to 30-50o (Figure iB). This u.v. dose was too high for complete recovery. As expected, cells derived from various healthy probands showed some individual variability. The same u.v. dose resulted in a depression of NAD + to 300/o in Dop fibroblasts (after 15 h) but only to 55% in Hekl cells. In FA fibroblasts NAD+ was significantly less decreased by both u.v. doses. A dose of 18 J/m2 had only a slight effect. The higher u.v. dose (36 J/mn, which caused reduction of NAD+ in normal cells to -40%, reduced NAD+ to -70% (Figure 1A,B). Dose-dependent decrease of NAD+ The decrease in cellular NAD+ levels was dose-dependent (Figure 2A,B). Seven hours after u.v. irradiation control cells had NAD+ contents of 50% at u.v. doses of 61 (Hekl), 80 (Hebu) and 85 J/m2 (Dop), respectively. The six FA strains analysed (Fl, FLH, FLB, FLP, F71, 1424) showed significantly less reduction in their NAD+ contents with increasing u.v. doses than did normal cells. At 96 J/m2, the 304

__ UV-dose

(Jm-21

Hg. 2. Decrease of NADI with increasing u.v. doses. Cells were grown on cell culture dishes, irradiated with the u.v. doses indicated, incubated at 37°C for 7 h and then harvested. Samples were prepared as described in Materials and methods. NADI was determined by h.p.l.c. analysis and calculated as a percentage of unirradiated controls. A: control fibroblasts: A A Dop (pass. 28); * 0* Hebu (pass. 28); * U* Held (pass. 33). B: FA fibroblasts: 0------0 1424 (pass. 20);0-- ) FLB (pass. 17); 0-0 FLH (pass. 14); x-..-..-x FLP (pass. 19); OI-.-.-O] Fl (pass. 31); A.A----- F71 (pass. 17).

highest u. v. dose applied, NAD + was lowered to 60-800% in FA cells, compared with 30 -40!7o in normal cells. Neither in normal nor in FA fibroblasts do the NAD + decreases show dose saturation up to 96 J/m2. Higher u.v. doses were not used because cells do not survive them. In con-

NAD+ metabolism in Fanconi's anemia Table II. Incorporation of [3H]NAD+ into acid-precipitable material of permeable cells (pmol/106 cells h)

Strain

NAD+ X s.d. pmol/106 cells h

CONTROLS: Hebu Hekl Dop Beau Hika

124.8 137.1 116.6 100.2 101.2

MEAN:

118.0 + 17.6

FA: FLP FLB Fl FLH 1424 F71 FB FISG

115.8 119.2 107.5 98.1 73.0 82.0 67.2 52.0

7 Cf controls

5 6 4 6 5

± 13.3 : 10.9 + 5.5 i 24.7 + 17.3

+ 8.7

+ + + + + + ±

6.2 17.8 5.8 9.1 12.3 1.1 11.0

n

98 101 91 83 62 69 57 44

4 2 6 3 6 6 4 4

0

C

c r-

Fig. 3. ADPRT activity in nuclear extracts. Preparation of nuclear extracts and enzyme assays were performed as described in Materials and methods. The values for each experiment were depicted. For each strain mean and standard deviation are given.

trast to NAD + consumption, excision of the main u.v. lesion, thymine dimers, is saturated beyond 27 J/m2 (Klocker et al., 1982). This indicates that increased NAD+ consumption during u.v. repair is not triggered by excision of thymine

dimers.

Incorporation of [3IH]NAD+ in permeabilized cells The absence of a decrease of NAD + in FA cells during u.v. repair could be a consequence of reduced activity of the NAD + -consuming enzyme ADPRT. Therefore we measured ADP-ribosylation in permeabilized cells as well as in nuclear extracts. Fibroblasts were permeabilized by hypotonic shock in the cold and incubated with [3H]NAD+. Incorporation of [3H]NAD+ into acid-precipitable material was reduced to 44-69%o of normal cells in fibroblasts from four FA patients (FB, F71, 1424, FISG). Cells from four other FA patients (FLP, FLB, FJ, FLH) showed no reduction of [3H]NAD+ incorporation (Table II). ADPRT activity in nuclear ectracts The activity of ADPRT was determined in nuclear extracts

of five different strains of normal fibroblasts and seven strains of FA fibroblasts. The mean value of ADP-ribosyltransferase activity in normal fibroblasts was 1.08 units/,g nuclear protein (standard deviation 0.39, variance of the mean 0.06, p = 0.01). Three FA strains (1424, FB, FLB) showed a significant reduced enzyme activity (0.42 0.19, 0.37 + 0.18 and 0.56 + 0.25, respectively). Only slightly reduced values were found for three other FA strains: FLP (0.92 E 0.28), F71 (0.83 4 0.58) and FI (0.84 L 0.38). FISG cells had essentially normal ADPRT activity (1.10 0.57 units//Lg nuclear protein). Discussion NAD + appears to play an essential role in excision repair. NAD+-dependent ADP-ribosylation is stimulated by single strand breaks in DNA (Miller, 1975b; Hilz and Stone, 1976; Hayaishi and Ueda, 1977; Mullins et al., 1977; Purnell et al., 1980; Cohen and Berger, 1981). During repair, this stimulation leads to a decrease of cellular NAD + (Berger et al., 1978; Benjamin and Gill, 1980; Halldorson et al., 1978). The NAD + consumption by ADP-ribosylation is neither required for thymine dimer excision (Klocker et al., 1983) nor for DNA repair synthesis (Sims et al., 1982; Durkacz et al., 1981a). Therefore, the last step of excision repair (ligation) probably involves ADP-ribosylation and NAD + was reported to be utilized for activation of DNA ligase (Creissen and Shall, 1982). In fibroblasts derived from patients with FA, a defect in the ligation step was found (Hirsch-Kauffmann et al., 1978; Schwaiger et al., 1982). This could be the result of a defective DNA ligase or a defect of the regulation of DNA ligase by ADP-ribosylation. Since NAD + is a substrate for ADPRT, a decreased NAD+ level would explain the defect in FA cells. However, in all FA cell strains tested, normal levels of NAD + were found. This contrasts with results from Berger et al. (1982), who reported a significant reduction of NAD+ levels in FA fibroblasts. An explanation could be that Berger's FA strains with low NAD+ levels belong to a complementation group, which is not present in our set of FA strains. The NAD + concentrations reported by Berger et al. (1982) were remarkably low: that of control fibroblasts were about one fifth of the values obtained in our experiments and that of FA fibroblasts about one tenth only. Berger et al. used an indirect cycling assay for NAD + and related the NAD + values to cell numbers. Our direct h.p.l.c. method allows the simultaneous determination of NAD + and other nucleotides, which can be used as internal references. The ratio of NAD + to the sum of all adenine nucleotides (NAD+ + AMP + ADP + ATP) is a suitable parameter for intracellular NAD + concentrations. Relating NAD+ to cell number leads to considerable variations of NAD+ values (see Table I), probably due to varying cell sizes. We controlled our h.p.l.c. technique by measuring several samples additionally with the enzymatic method. We obtained almost the same values as with h.p.l.c. (20% lower). Considering the conformity of the two methods, we have no explanation for the discrepancy between our data and that of Berger. Comparison of the decrease of NAD+ in u.v.-irradiated and unirradiated cells revealed diminished NAD+ consumption in all FA fibroblasts strains during DNA repair. This indicates a defect in the NAD+ netabolism in FA fibroblasts. In normal cells NAD+ is consumed by increased ADP305

H.Klocker et al.

ribosylation during DNA repair (for a review, see Mandel et al., 1982). The reduced NAD+ consumption in FA fibroblasts can be due to a defective ADPRT or a shortage of ADP-ribose acceptors. The ADPRT defect can be caused by an alteration of either the active site or the recognition sites for substrate and ADP-ribose acceptors or by an impaired activation by DNA lesions. In strains 1424 and FB, reduced ADPRT activities in permeabilized fibroblasts as well as in nuclear extracts were found. Most likely their ADPRTs have defective active sites or substrate recognition sites. The normal ADPRT activities in strains FJ and FLP in both assays indicate an impaired stimulation of the enzyme by DNA lesions. The remaining strains (FLB, F71, FISG) show differing ADPRT activities in permeabilized cells and in nuclear extracts. The diverse manifestations of the enzyme defect can be due to different assay conditions, such as pH, ionic strength, substrate concentration and activation by partially digested DNA. Thus, the defects of these strains cannot be classified unambiguously. The ADP-ribosylation activity of FISG cells is of special interest. In this case, increased chromosomal instability was found by prenatal diagnosis (Hirsch-Kauffmann and Schweiger, 1981). This abnormality was confirmed in fibroblasts and lymphocytes after birth. Chromosomal aberrations are a characteristic feature of FA and some other repair diseases (Arlett and Lehmann, 1978). FISG cells have low ADP-ribosylation activity in permeabilized cells (Table II). Since reduced ADP-ribosylation activities were found in FA cells, as indicated in this report, and considering the cytogenetical results, FISG cells should have the FA defect. However, the child does not have clinical symptoms as yet. This is no surprise since skeletal abnormalities do not appear in all FA cases and manifestation of the characteristic pancytopenia usually occurs later in childhood. In summary, our results demonstrate that NAD + metabolism during repair is impaired in FA. This defect possibly affects the activation of ligase by ADP-ribosylation, and thereby causes the repair deficiency in FA. Materials and methods Cultivation of cells Primary human fibroblast cells were grown in minimal essential medium (MEM) supplemented with 10% fetal calf serum (FCS) in an atmosphere containing 5% CO2 in air. Cell cultures were routinely tested for mycoplasma contamination by determining the enzyme adenosinophosphorylase (Uitendaal et al., 1978). Fibroblast strains Control fibroblasts (Hika, Beau, Kraha, Dop, Hebu and Hekl) were established from skin biopsies of healthy probands. FA fibroblasts had the following origins: FLP, FI, FLB and FLH were derived from skin biopsies of an 11-year-old boy and from three girls, 12, 13 and 19 years of age, respectively. FLP and FLB are brother and sister. FA characterisation based on our cytogenetical data and the clinical diagnosis of the Department of Pediatrics, University of Innsbruck and the Children's Hospital, Linz, Austria. F71 was kindly supplied from the Department of Human Genetics, Hadassah University, Jerusalem. FB was derived from a skin biopsy kindly supplied by Dr. Stuck, Berlin. 1424 was derived from a skin biopsy of a 10-year-old boy with cytologically and clinically verified FA. These fibroblasts were biochemically characterized by Hirsch-Kauffmann et al. (1978). FISG was derived from a skin biopsy of a 6-month-old boy. In this case increased chromosomal instability was discovered during the course of routine prenatal testing in a family with unknown hereditary disposition. After birth, chromosomal instability was again verified although the boy showed no clinical symptoms (HirschKauffmann and Schweiger, 1981). U.v. irradiation of cells For u.v. irradiation, cells were grown on plastic cell culture dishes. The

306

medium was removed, cells were irradiated with a low pressure mercury germicidal lamp (Hanau 15/44, maximum emmission at 254 nm) ata flow rate of 1.2 J/m2/s. Subsequently the cells were fed with prewarmed medium and incubated for repair. Extraction of nucleotides from fibroblasts Nucleotides were extracted with 0.4 M perchloric acid, neutralisation was achieved according to Khym (1975). The medium was removed from the cell culture plate (diameter = 6 cm), and the cell monolayer was extracted with 0.6 ml 0.4 M perchloric acid in the cold. The extract was neutralized by adding 0.65 ml of a solution of trioctylamine in 1,1,2-trichlorotrifluoroethane (3.6 g trioctylamine + 10 ml trichlorotrifluoroethane) and vigorous shaking. After centrifugation in an Eppendorf centrifuge, the aqueous phase at the top was collected and stored at - 20°C. Determination of nucleotides by h.p.l.c. analysis Nucleotides were analysed according to Doppler (1982). Nucleotide extracts were diluted 3-5 times with water. 0.25 ml thereof was applied to a h.p.l.c. anion exchange column (Nucleosil 5 SB, Machery-Nagel, 50 x 2 mm). Nucleotides were eluted successively by a salt and pH gradient and determined by u.v. absorption at 254 nm. The starting buffer A was 15 mM potassium phosphate, 2% acetonitrile pH 3.40. A 0-70% linear gradient with buffer B (30 mM potassium phosphate, 0.5 M potassium chloride, 2% actonitrile, pH 3.50) was used for elution. The column was washed with 10007o buffer B and re-equilibrated with buffer A for the next run. The flow rate was 0.7 or 0.8 ml per min. One run lasted 46 min. To determine NADI alone, a shorter program (25 min) was used. The sample was applied to the column with buffer A. After elution of the NADI peak, the column was washed with buffer B and re-equilibrated with buffer A. Retention times for nucleotides in minutes: NAD+ = 3.8; AMP = 6.7; UDP-glucose = 14.5; CDP = 15.2; UDP = 15.9; ADP = 17.5; GDP = 20.7; UTP = 22.3; ATP = 25.2; GTP = 28.9. ADP-ribosylation in permeable cells Cells were transferred from cell culture flasks to dishes (diameter = 6 cm; 3 x 105 cells/plate). After 15-20 h at 37°C, the cells were permeabilized by dithiothreitol (DTT) buffer and cold treatment. For permeabilization, the medium was removed, cells were washed with prewarmed phosphate buffered saline (PBS) and 0.6 ml ice cold DTT buffer (20 mM Tris-HCI, 150 mM sucrose, 4 mM MgCl2, 1 mM EGTA, 6 mM DTT, pH 7.80) were added. The dishes were placed in a refrigerator for 30 min. 0.3 ml of NADI buffer (100 mM Tris-HCI, 20 mM MgCl2, 150 AM [H]NAD+ (NEN) sp. act. = 18.2 Ci/mol, pH 7.8) were added and the dishes were incubated at 37°C for 1 h. At the end of the incubation period the buffer was removed and the cells were scraped off with a rubber policeman into cold PBS. Macromolecules were precipitated with two volumes of 10% trichloroacetic acid (TCA). After 15-30 min on ice, the samples were filtered through glass fibre filters (Schleicher and Schull, diameter = 2.5 cm), washed five times with 5% TCA + 1% pyrophosphate and twice with methanol/ether, dried and counted in a liquid scintillation counter. In parallel, two plates from the same batch were used to determine the cell number. The cells were washed once with PBS, trypsinized, scraped off with a rubber policeman and counted with a microcell counter (TOA CC-108). Preparation of nuclear extracts Primary fibroblast cultures were grown to a density of 0.5-1 x I(} cells/culture dish (9 cm in diameter), washed with PBS, trypsinized and harvested by scraping off with a rubber policeman. The cells were collected by centrifugation at 200 g for 5 min, washed again with PBS and resuspended in 0.2 ml of ice-cold hypotonic buffer A (10 mM Tris-HCI, 10 mM NaCl, 1 mM EDTA, pH 7.4). All the following operations were carried out at 4°C. After swelling for 15 min, the cells were disrupted by adding Triton X-100 to a concentration of 0.25%. The nuclei were spun down at 1000 g for 8 min and washed once with buffer B (10 mM Tris-HCI, 10 mM NaCl, 1.5 mM MgCl2, pH 7.5). Finally, the nuclear pellet was extracted by a 60 min incubation with 0.07 ml buffer C (20 mM Tris-HCl, 500 mM KCI, 2 mM 2-mercaptoethanol, 1.5 mM MgCl2, pH 8.0). The nuclear extracts were cleared by centrifugation at 10 000 g for 10 min, and the supernatant immediately subjected to the ADPRT assay. Protein concentrations were determined according to Bradford (1976). ADPRT assay Enzyme activity was assayed by the incorporation of radioactivity from NADI, labelled in the adenine part of the molecule, into chromatin. The assay was performed in a 100 yd incubation mixture containing: 40 mM TrisHCI pH 8.7, 50 mM NaF, 30 mM MgCl2, 10 mM DTT, 4 /g histone (2A, Sigma), 2 ug activated DNA (Loeb, 1969), (NH)SO4 to 35% saturation, 10 $M NAD+ (sp.act. = 100 Ci/mol) and 5-20 Al nuclear extract. After 60 min incubation at 37°C, 80 i1 of the mixture were put on a glass fibre filter (2.5 cm diameter) and the macromolecules were precipitated by immersion of

NAD+ metabolism in Fanconi's anemia the filters into a 5°0% TCA liWo sodium pyrophosphate (PP) solution for 5 min. The filter discs were then washed 10 times with 5°% TCA 1% PP, three times with methanol/ether and dried. Radioactivity was counted in a liquid scintillation counter. One unit of enzyme activity is defined as incorporation of 1 pmol NAD+ into acid-insoluble material per hour.

Acknowledgements This work was supported by Deutsche Forschungsgemeinschaft (Projekt Schw. 98/4-1) and by the Fonds zur Forderung der wissenschaftlichen Forschung (Projekt 4241).

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