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Hoechst flow cytometry: implications for Bloom's syndrome
Mutation Research, 238 (1990) 203-207
203
Elsevier MUTREV 02805
Detection of free radical-induced DNA damage with bromodeoxyuridine/Hoechst flow cytometry: implications for Bloom's syndrome Martin Poot, Hugo W. Riidiger 1 and Holger Hoehn Department of Human Genetics, University of Wiirzburg, 8700 Wiirzburg (F.R.G.) and ~ Department of Occupational Medicine, University of Hamburg, Hamburg (F.R. G.)
(Accepted 12 October 1989) Keywords: Oxygenfree radicals; DNA damage; Bloom syndrome; Bromodeoxyuridine/Hoechstflow cytometry
Summary The clinical radiosensitiser bromodeoxyridine (BrdU) was shown to enhance oxygen free radical-mediated growth inhibition. Cells from Bloom's syndrome, a rare autosomal recessive disorder characterized by pre- and post-natal growth deficits, telangiectatic erythema, recurrent respiratory infections and a high incidence of cancer, exhibit in culture a hypersensitivity to BrdU. We analysed disturbed cell kinetics of Bloom's syndrome fibroblasts and permanent B-cell lines with a novel cell kinetic method: B r d U / H o e c h s t flow cytometry. Fibroblasts show a pattern similar to that of normal cells exposed to a breakdown product of lipid peroxides, whereas B-cells exhibit the cell kinetic disturbance provoked by elevated oxygen concentrations in normal cells. In both cell types the cell kinetic pattern was dependent upon the BrdU concentration in the culture medium. These data suggest an elevated endogenous generation of oxygen free radicals in Bloom's syndrome cells, which may relate to the elevated incidence of malignancies in these patients.
Detection of oxygen free radical-induced D N A damage requires complex and cumbersome methods to isolate and to quantify each individual type of altered D N A constituent. A more global approach uses the property of oxygen free radicals to induce an arrest of cells in the G 2 phase of the cell cycle (Balin et al., 1978; Poot et al., 1988). Thus, 35% oxygen, paraquat (an intracellular superoxide-generating c o m p o u n d ) and bleomycin (which attacks the deoxyribose moiety in DNA)
Correspondence: Martin Poot, Department of Human Genetics, Universityof Wiirzburg, Koellikerstrasse2, 8700 Wiirzburg (F.R.G.).
induce a permanent arrest of cells in the G 2 phase of the cell cycle (Poot et al., 1989; Poot and Hoehn, 1989). The extent of this G 2 arrest can be modulated by adding the clinical radiosensitiser b r o m o d e o x y u r i d i n e (BrdU) (Dordjevic and Szybalski, 1969; Szybalski, 1974; Kinsella et al., 1984) to the culture medium (Poot et al., 1989; Poot and Hoehn, 1989). Experiments with the lipophilic model peroxide, cumene peroxide (Sies and Summer, 1975; Poot et al., 1987), and the lipophilic free radical scavenger vitamin E showed that the BrdU-dependent arrest in G 2 is not mediated by lipid peroxidation (Poot et al., 1989; Poot and Hoehn, 1989). Bloom's syndrome (BM) is a rare autosomal recessive disorder involving striking pre- and
0165-1110/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
204
post-natal growth retardation, telangiectatic erythema, recurrent infections of the upper respiratory tract and a high incidence of cancer (Bloom, 1954, 1966; German and Passarge, 1989). Cultured cells from BS patients exhibit an elevated frequency of sister-chromatid exchanges (SCEs) (Latt et al., 1983), the rate of which is dependent upon the amount of BrdU used in the assay (Heartlein et al., 1987; Shiraishi and Ohtsuki, 1987). Genomic instability is also evidenced by the presence of quadriradial figures (German, 1983), an increased prevalence of micronuclei (German, 1983), elevated mutation at the glycophorin A locus (Langlois et al., 1989) and clonal chromosome aberrations (Ray and German, 1983; Hoehn and SaIL 1984). Upon fusion with normal cells the rate of SCEs is normalised in BS nuclei (Bryant et al., 1979; Aldaheff et al., 1980; Shiraishi et al., 1981). Hypotheses to explain the BS phenotype include deficiencies in DNA topoisomerase II (Heartlein et al., 1987), DNA ligase I (Willis and Lindahl, 1987; Chan et al., 1987; Willis et al., 1987) and an elevated generation of superoxide radicals (Nicotera et al., 1989). This oxygen free radical is able to provoke DNA damage, which can be enhanced with BrdU (Poot et al., 1989; Poot and Hoehn, 1989), and is reputed to elicit lipid peroxidation (Freeman and Crapo, 1982).
Deficiency in each of the 2 enzymes involved in DNA replication and the free radical-induced cell lesions will elicit specific and distinct disturbances of cell proliferation. In order to differentiate between the proposed mechanisms underlying the BS phenotype, we analysed the pattern of disturbed cell proliferation in fibroblasts and B-cells from peripheral blood immortalised with Epstein-Barr virus (EBL cells) with a high-resolution cell kinetic assay, BrdU-Hoechst flow cytometry (Rabinovitch, 1983; Rabinovitch et al., 1988). Disturbed cell kinetics in fibroblasts
All fibroblast strains from BS patients used in this study exhibited elevated rates of SCE (results not shown). BS 3 is a strain derived from a patient of one of us (H.W.R.), and the other 2 strains were initially from the Human Genetic Mutant Repository (Camden, N J). Fibroblasts were rendered quiescent by culturing to confluency and subsequent serum starvation. Thereupon, cells were stimulated by replating in minimal essential medium supplemented with 10% foetal bovine serum and 65 /~M each of BrdU and deoxycytidine. After various periods of growth, cells were harvested, stained with a buffer containing 1.2/~g
Control
61oom's
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I
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Fig. 1. Bivariate cytograms of fibroblasts from a healthy subject and a patient with BS cultured during 72 h with BrdU. The abscissa displays 13rdU-quenched Hoechst fluorescence and the ordinate shows unquenched ethidium bromide fluorescence. Due to BrdU quenching of the Hoechst fluorescence the second and third cell cycles after serum stimulation appear progressively shifted to the left from the first cycle. Note the difference in relative abundance of cells in the G 2 cluster of the first cell cycle (arrows).
205 Hoechst 33258 and 2.0 /tg ethidium bromide per ml, and analysed with bivariate flow cytometry. Fig. 1 shows a bivariate cytogram thus obtained from a 72-h culture of fibroblasts from a BS patient and from a healthy individual. Due to BrdU incorporation during the S phase the intensity of Hoechst fluorescence is reduced in these cells and the cluster of cells representing the first G2 phase after serum stimulation is shifted to the left. U p o n mitosis, Hoechst fluorescence intensity is halved and the cluster of the G 1 phase of the second cycle (denoted G~) appears left from the G 0 / G 1 cluster. Hoechst fluorescence of cells traversing a second S phase is again quenched by BrdU incorporation and the G 2 of the second cycle (G~) appears left from the G 2 of the first cycle. Thus, cells can be distinguished according to the number of S phases they have traversed. Cells belonging to the G a, S and G 2 compartments of each cycle are resolved by staining with ethidium bromide, of which the fluorescence intensity remains stoichiometric with D N A content. The striking difference between the bivariate cytograms is the relative number of cells in the G 2 cluster (arrows). Apparently, cells from BS exhibit a difficulty in traversing the G 2 phase, which is
compatible with either a deficiency in D N A ligase I (Willis and Lindahl, 1987; Chan et al., 1987a, b; Willis et al., 1987) or with free radical-induced cell damage (Poot et al., 1987, 1988, 1989; Poot and Hoehn, 1989). Sequential analysis of the type shown in Fig. 1 together with quantitative treatment of the bivariate data gives the minimal and mean durations of each c o m p a r t m e n t of the cell cycles a culture has traversed (Rabinovitch, 1983). Additionally, the fraction of cells permanently arrested in a particular cell-cycle compartment can be computed. Table 1 gives those selected cell kinetic parameters which revealed a difference between cultures of control and BS fibroblasts maintained at 5% (vol/vol) oxygen. Thus, fibroblasts from BS show an elevated permanent arrest in the G 2 phase of the first cycle after serum stimulation and a prolongation of the G 1 compartment of the second cycle. This pattern of disturbed proliferation is akin to that found with control fibroblasts exposed to 10/~M of 4-hydroxynonenal (Table 1), a breakdown product of lipid peroxidation (Poli et al., 1985; Esterbauer et al., 1986). These data are consistent with the hypothesis that BS fibroblasts suffer from lipid peroxidation induced by oxygen free radicals (Nicotera et al., 1989).
TABLE 1 FRACTION OF CELLS ARRESTED IN THE G 2 PHASE OF THE FIRST CYCLE AND MINIMAL DURATION OF THE G 1 COMPARTMENT OF THE SECOND CYCLE AFTER SERUM STIMULATION OF QUIESCENT CELLS FROM CONTROL AND BS FIBROBLAST CULTURES
Disturbed cell kinetics in EBL cells
Cell type
G 2 arrest
G x duration
Control Strain 1 Strain 2
2.4 + 0.1 2.1 +0.3
6.5 + 0.7 7.2 +0.7
14.2 + 0.4 9.6 + 0.5 13.6+_0.2
12.2 + 0.4 12.7 + 0.5 9.8+0.3
BS GM 1492 GM3498B BS 3
Control + 10 #M 4-hydroxynonenal Strain 1 14.0 + 0.6 Strain 2 15.8 + 0.5
14.4 + 0.5 12.4 + 0.5
All data are means and standard deviations as obtained by fitting the cell-cycle distributions from a time sequence of samples out of a single kinetic experiment to the modified cell-cycle kinetic model as described by Rabinovitch (1983).
The alternative hypothesis that cells from BS exhibit a deficiency in D N A ligase I (Willis and Lindahl, 1987; Chan et al., 1987) was based upon analysis of permanent B-cell lines derived by treatment of h u m a n peripheral blood mononuclear cells with E p s t e i n - B a r r virus (EBL cells). Therefore, 2 EBL lines from healthy individuals and from patients with BS were compared (Table 2). The striking difference between control and BS cells was the prolongation of the S phase of the second cycle in BS cells; in contrast to fibroblasts, EBL from BS showed no prolongation of the G~ compartment. The fraction of cells arrested in the G 2 c o m p a r t m e n t of the first cycle was only slightly higher in BS cells relative to control cells, but the fraction arrested in the G 2 phase of the second cycle was clearly elevated in BS cells (Table 2). Apparently, BS cells that incorporated BrdU during 2 S phases show more arrest in the G z com-
206 TABLE 2 MINIMAL DURATIONS OF THE G 1 AND S PHASES OF THE SECOND CELL CYCLE AFTER RELEASE FROM QUIESCENCE OF EBL CELLS FROM BS AND HEALTHY SUBJECTS CULTURED AT 5% OXYGEN (h), AND FRACTION OF CELLS ARRESTED IN THE G 2 PHASE OF THE FIRST AND SECOND CELL CYCLE (% OF TOTAL CELLS) Minimal duration
Arrest fraction
G 1 phase
S phase
First G 2
Second G 2
Control Line WF Line HZ
6.1+0.4 5.8+0.4
7.6+0.2 6.5+0.3
2.35:0.2 1.9+0.4
2.7+0.6 3.6+0.3
BS GM 3404 BD1
6.1+0.5 5.1+0.6
9.0+0.8 9.75:0.6
3.6-t-0.4 3.55:0.3
7.4+0.7 6.0+1.0
All data are means and standard deviations as obtained by fitting the cell-cycle distributions from a time sequence of samples out of a single kinetic experiment to the modified cell-cycle kinetic model as described by Rabinovitch (1983). 20. 18"
FiDroblasts
16. •..,"
GM 3498 B
14' 12" 10,
p a r t m e n t t h a n BS cells that i n c o r p o r a t e d B r d U o n l y once. This result suggests that the extent of G2 arrest d e p e n d s u p o n the i n c o r p o r a t i o n of B r d U into the D N A in a w a y a k i n to the rate of S C E (14). T o test this p o s s i b i l i t y f i b r o b l a s t s a n d E B L cells f r o m h e a l t h y i n d i v i d u a l s a n d f r o m BS patients were c u l t u r e d for 72 h with a c o n c e n t r a t i o n series of B r d U a n d the fraction of cells in G 2 was assessed (Fig. 2). A l l cultures of BS cells d i s p l a y e d a clear B r d U d e p e n d e n c e of the G 2 fraction, whereas c o n t r o l cells d i d not.
Discussion T h e fact t h a t 2 different cell types f r o m BS show 2 distinct types of cell kinetic d i s t u r b a n c e argues a g a i n s t a p u t a t i v e deficiency of a single e n z y m e involved in D N A r e p l i c a t i o n a n d D N A r e p a i r as the cause o f BS. O n the o t h e r hand, an elevated level of o x i d a t i v e stress m i g h t m a n i f e s t itself d i f f e r e n t l y in different cell types. F i b r o blasts, c o n t a i n i n g a high level of lipids, m a y suffer lipid p e r o x i d a t i o n , w h e r e a s E B L are m o r e likely to exhibit signs of D N A d a m a g e . Thus, elevated o x i d a t i v e stress seems a likely e x p l a n a t i o n of the c a n c e r - p r o n e p h e n o t y p e of BS, b u t further research is n e e d e d to test this hypothesis.
Control 8'
Acknowledgements
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W e are i n d e b t e d to Prof. Dr. H. E s t e r b a u e r ( D e p a r t m e n t of Biochemistry, U n i v e r s i t y of Graz, A u s t r i a ) for m a k i n g a s a m p l e of 4 - h y d r o x y n o n enal a v a i l a b l e to us. H e l p f u l discussions a n d sending us a p r e p r i n t o f a m a n u s c r i p t b y Dr. T.M. N i c o t e r a (Roswell P a r k M e m o r i a l Institute, Buffalo, N Y ) is k i n d l y a c k n o w l e d g e d . This w o r k was supported by Deutsche Forschungsgemeinschaft, G r a n t D F G 8 4 9 / 2 - 1 . W e t h a n k Miss Julia K/Shler for p r e p a r a t i o n of the figures.
8'
References
8" 4' 2" O"
. . . . . . . 0
'
I
I
I
20 40 60 80 100 120 140 160 180 200 BrdU concentration
[;JM]
Fig. 2. BrdU dependence of G 2 arrest in fibroblasts and EBL cells. Cells were cultured during 72 h at 5% (vol/vol) oxygen in the continuous presence of a concentration series of BrdU.
Aldaheff, B., M. Velivasakis, I. Pagan-Charry, W.C. Wright and M. Siniscalco (1980) High rate of sister chromatid exchanges of Bloom's syndrome chromosomes is corrected in rodent human somatic cell hybrids, Cytogenet. Cell Genet., 27, 8-23. Balin, A.K., D.B.P. Goodman, H. Rasmussen and V.J. Cristofalo (1978) Oxygen-sensitive stages of the cell cycle of human diploid cells, J. Cell Biol., 78, 390-400.
207 Bloom, D. (1954) Congenital telangiectatic erythema resembling lupus erythematosus in dwarfs. Probable syndrome entity, Am. J. Child Dis., 88, 754-758. Bloom, D. (1966) The syndrome of congenital telangiectatic erythema and stunted growth, J. Pediatr., 68, 103-113. Bryant, E.M., H. Hoehn and G.M. Martin (1979) Normalisation of sister chromatid exchange frequencies in Bloom's syndrome by euploid cell hybridisation, Nature (London), 279, 795-796. Chart, J.Y.H., F.F. Becker, J. German and J.H. Ray (1987) Altered DNA ligase I activity in Bloom's syndrome cells, Nature (London), 325, 357-359. Dordjevic, B., and W. Szybalski (1969) Genetics of human cell lines. III. Incorporation of 5-bromo- and 5-iododeoxyuridine into nucleic acid of human cells and its effects on radiation sensitivity, J. Exp. Med., 112, 509-531. Esterbauer, H., A. Benedetti, J. Lang, R. Fulceri, G. Fauler and M. Comporti (1986) Studies on the mechanism of formation of 4-hydroxynonenal during microsomal lipid peroxidation, Biochim. Biophys. Acta, 876, 154-166. Freeman, B.A., and J.D. Crapo (1982) Biology of disease. Free radicals and tissue injury, Lab. Invest., 47, 412-426. German, J. (1983) Bloom's syndrome. X. The cancer proneness points to chromosome mutation as a crucial event in human neoplasia, in: J. German (Ed.), Chromosome Mutation and Neoplasia, A.R. Liss, New York, pp. 347-357. German, J., and E. Passarge (1989) Bloom's syndrome. XII. Report from the Registry for 1987, Clin. Genet., 35, 57-69. Heartlein, M.W., H. Tsuji and S.A. Latt (1987) 5-Bromodeoxyuridine-dependent increase in sister chromatid exchange formation in Bloom's syndrome is associated with reduction in topoisomerase II activity, Exp. Cell Res., 169, 245-254. Hoehn, H., and D. Salk (1984) Clonal analysis of stable chromosome rearrangements in Bloom's syndrome fibroblasts, Cancer Genet. Cytogenet., 11, 405-415. Kinsella, T.J., J.B. Mitchell, A. Russo, M. Aiken, G. Mortsyn, S.M. Hsu, J. Rowland and E. Glatstein (1984) Continuous intravenous infusion of bromodeoxyuridine as a clinical radiosensitizer, J. Clin. Oncol., 2, 1144-1150. Langlois, R.D., W.L. Bigbee, R.H. Jensen and J. German (1989) Evidence for increased in vivo mutation and somatic recombination in Bloom's syndrome, Proc. Natl. Acad. Sci. (U.S.A.), 86, 670-674. Latt, S.A., R.R. Schreck, C.P. Dougherty, K.M. Gustashaw, L.A. Juergens and T.N. Kaiser (1983) in: J. German (Ed.), Chromosome Mutation and Neoplasia, A.R. Liss, New York, pp. 169-191. Nicotera, T.M., J. Notaro, S. Notaro and A.A. Sandberg (1989) Elevated superoxide dismutase in Bloom syndrome: a genetic condition of oxidative stress, Cancer Res., 49, 5239-5243.
Poli, G., M. Dianzani, K.H. Cheeseman, T.F. Slater, J. Lang and H. Esterbauer (1985) Separation and characterization of the aldehydic products of lipid peroxidation stimulated by carbon tetrachloride or ADP-iron in isolated rat hepatocytes and rat liver microsomal suspensions, Biochem. J., 227, 629-638. Poot, M., and H. Hoehn (1989) A putative free radical mechanism for radiosensitization by bromodeoxyuridine, in: C. Rice-Evans (Ed.), Free Radicals VI, Taylor and Francis, London, in press. Poot, M., A. Verkerk, J.F. Koster, H. Esterbauer and J.F. Jongkind (1987) Influence of cumene hydroperoxide and 4-hydroxynonenai on the glutathione metabolism during in vitro ageing of human skin fibroblasts, Eur. J. Biochem., 167, 287-291. Poot, M., D. Schindler, M. Kubbies, H. Hoehn and P.S. Rabinovitch (1988) Bromodeoxyuridine amplifies the inhibitory effect of oxygen on cell proliferation, Cytometry, 9, 332-338. Poot, M., P.S. Rabinovitch and H. Hoehn (1989) Bromodeoxyuridine amplifies oxygen free radical mediated DNA damage, Biochem. J., 261, 269-271. Rabinovitch, P.S. (1983) Regulation of human fibroblast growth rate by both noncycling cell fraction and transition rate is shown by growth in 5-bromodeoxyuridine followed by Hoechst 33258 flow cytometry, Proc. Natl. Acad. Sci. (U.S.A.), 80, 2951-2955. Rabinovitch, P.S., M. Kubbies, Y.C. Chen, D. Schindler and H. Hoehn (1988) BrdU-Hoechst flow cytometry: a unique tool for quantitative cell cycle analysis, Exp. Cell Res., 174, 309-318. Ray, J.H., and J. German (1983) The cytogenetics of the 'chromosome-breakage syndromes', in: J. German (Ed.), Chromosome Mutation and Neoplasia, A.R. Liss, New York, pp. 135-167. Shiraishi, Y., and Y. Ohtsuki (1987) SCE levels in Bloom-syndrome cells at very low bromodeoxyuridine (BrdU) concentrations: monoclonal anti-BrdU antibody, Mutation Res., 176, 157-164. Shiraishi, Y., S.-I. Matsui and A.A. Sandberg (1981) Normalization by cell fusion of sister chromatid exchange in Bloom syndrome lymphocytes, Science, 212, 820-822. Sies, H., and K.-H. Summer (1975) Hydroperoxide-metabolizing systems in rat liver, Eur. J. Biochem., 57, 503-512. Szybalski, W. (1974) X-ray sensitization by halopyrimidines, Cancer Cliemother. Pep., 58, 539-557. Willis, A.E., and T. Lindahl (1987) DNA ligase I deficiency in Bloom's syndrome, Nature (London), 325, 355-357. Willis, A.E., R. Weksberg, S. Tomlinson and T. Lindahl (1987) Structural alterations of DNA ligase I in Bloom syndrome, Proc. Natl. Acad. Sci. (U.S.A.), 84, 8016-8020.
203
Elsevier MUTREV 02805
Detection of free radical-induced DNA damage with bromodeoxyuridine/Hoechst flow cytometry: implications for Bloom's syndrome Martin Poot, Hugo W. Riidiger 1 and Holger Hoehn Department of Human Genetics, University of Wiirzburg, 8700 Wiirzburg (F.R.G.) and ~ Department of Occupational Medicine, University of Hamburg, Hamburg (F.R. G.)
(Accepted 12 October 1989) Keywords: Oxygenfree radicals; DNA damage; Bloom syndrome; Bromodeoxyuridine/Hoechstflow cytometry
Summary The clinical radiosensitiser bromodeoxyridine (BrdU) was shown to enhance oxygen free radical-mediated growth inhibition. Cells from Bloom's syndrome, a rare autosomal recessive disorder characterized by pre- and post-natal growth deficits, telangiectatic erythema, recurrent respiratory infections and a high incidence of cancer, exhibit in culture a hypersensitivity to BrdU. We analysed disturbed cell kinetics of Bloom's syndrome fibroblasts and permanent B-cell lines with a novel cell kinetic method: B r d U / H o e c h s t flow cytometry. Fibroblasts show a pattern similar to that of normal cells exposed to a breakdown product of lipid peroxides, whereas B-cells exhibit the cell kinetic disturbance provoked by elevated oxygen concentrations in normal cells. In both cell types the cell kinetic pattern was dependent upon the BrdU concentration in the culture medium. These data suggest an elevated endogenous generation of oxygen free radicals in Bloom's syndrome cells, which may relate to the elevated incidence of malignancies in these patients.
Detection of oxygen free radical-induced D N A damage requires complex and cumbersome methods to isolate and to quantify each individual type of altered D N A constituent. A more global approach uses the property of oxygen free radicals to induce an arrest of cells in the G 2 phase of the cell cycle (Balin et al., 1978; Poot et al., 1988). Thus, 35% oxygen, paraquat (an intracellular superoxide-generating c o m p o u n d ) and bleomycin (which attacks the deoxyribose moiety in DNA)
Correspondence: Martin Poot, Department of Human Genetics, Universityof Wiirzburg, Koellikerstrasse2, 8700 Wiirzburg (F.R.G.).
induce a permanent arrest of cells in the G 2 phase of the cell cycle (Poot et al., 1989; Poot and Hoehn, 1989). The extent of this G 2 arrest can be modulated by adding the clinical radiosensitiser b r o m o d e o x y u r i d i n e (BrdU) (Dordjevic and Szybalski, 1969; Szybalski, 1974; Kinsella et al., 1984) to the culture medium (Poot et al., 1989; Poot and Hoehn, 1989). Experiments with the lipophilic model peroxide, cumene peroxide (Sies and Summer, 1975; Poot et al., 1987), and the lipophilic free radical scavenger vitamin E showed that the BrdU-dependent arrest in G 2 is not mediated by lipid peroxidation (Poot et al., 1989; Poot and Hoehn, 1989). Bloom's syndrome (BM) is a rare autosomal recessive disorder involving striking pre- and
0165-1110/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
204
post-natal growth retardation, telangiectatic erythema, recurrent infections of the upper respiratory tract and a high incidence of cancer (Bloom, 1954, 1966; German and Passarge, 1989). Cultured cells from BS patients exhibit an elevated frequency of sister-chromatid exchanges (SCEs) (Latt et al., 1983), the rate of which is dependent upon the amount of BrdU used in the assay (Heartlein et al., 1987; Shiraishi and Ohtsuki, 1987). Genomic instability is also evidenced by the presence of quadriradial figures (German, 1983), an increased prevalence of micronuclei (German, 1983), elevated mutation at the glycophorin A locus (Langlois et al., 1989) and clonal chromosome aberrations (Ray and German, 1983; Hoehn and SaIL 1984). Upon fusion with normal cells the rate of SCEs is normalised in BS nuclei (Bryant et al., 1979; Aldaheff et al., 1980; Shiraishi et al., 1981). Hypotheses to explain the BS phenotype include deficiencies in DNA topoisomerase II (Heartlein et al., 1987), DNA ligase I (Willis and Lindahl, 1987; Chan et al., 1987; Willis et al., 1987) and an elevated generation of superoxide radicals (Nicotera et al., 1989). This oxygen free radical is able to provoke DNA damage, which can be enhanced with BrdU (Poot et al., 1989; Poot and Hoehn, 1989), and is reputed to elicit lipid peroxidation (Freeman and Crapo, 1982).
Deficiency in each of the 2 enzymes involved in DNA replication and the free radical-induced cell lesions will elicit specific and distinct disturbances of cell proliferation. In order to differentiate between the proposed mechanisms underlying the BS phenotype, we analysed the pattern of disturbed cell proliferation in fibroblasts and B-cells from peripheral blood immortalised with Epstein-Barr virus (EBL cells) with a high-resolution cell kinetic assay, BrdU-Hoechst flow cytometry (Rabinovitch, 1983; Rabinovitch et al., 1988). Disturbed cell kinetics in fibroblasts
All fibroblast strains from BS patients used in this study exhibited elevated rates of SCE (results not shown). BS 3 is a strain derived from a patient of one of us (H.W.R.), and the other 2 strains were initially from the Human Genetic Mutant Repository (Camden, N J). Fibroblasts were rendered quiescent by culturing to confluency and subsequent serum starvation. Thereupon, cells were stimulated by replating in minimal essential medium supplemented with 10% foetal bovine serum and 65 /~M each of BrdU and deoxycytidine. After various periods of growth, cells were harvested, stained with a buffer containing 1.2/~g
Control
61oom's
syndrome
I
I
I
~:
G2 f G2".
•.
~ ~
i'
i~ii=~!~.
0~ :~
~ ~'~'~i:
=,:=J:i !' " " S'
t
:
~"
S
::=~:::
~i~
:,:!k;:~.;.S'
:i!i:.~'~-: :::~m=
• :i[~ii~i;Jii!ii:::
:~ii~'~i: ..=.:=...
G0/GI
E
o .o
GI,;,GI''
E
=.
5 laJ I
i
BrdU/hoechst
fluorescence
Fig. 1. Bivariate cytograms of fibroblasts from a healthy subject and a patient with BS cultured during 72 h with BrdU. The abscissa displays 13rdU-quenched Hoechst fluorescence and the ordinate shows unquenched ethidium bromide fluorescence. Due to BrdU quenching of the Hoechst fluorescence the second and third cell cycles after serum stimulation appear progressively shifted to the left from the first cycle. Note the difference in relative abundance of cells in the G 2 cluster of the first cell cycle (arrows).
205 Hoechst 33258 and 2.0 /tg ethidium bromide per ml, and analysed with bivariate flow cytometry. Fig. 1 shows a bivariate cytogram thus obtained from a 72-h culture of fibroblasts from a BS patient and from a healthy individual. Due to BrdU incorporation during the S phase the intensity of Hoechst fluorescence is reduced in these cells and the cluster of cells representing the first G2 phase after serum stimulation is shifted to the left. U p o n mitosis, Hoechst fluorescence intensity is halved and the cluster of the G 1 phase of the second cycle (denoted G~) appears left from the G 0 / G 1 cluster. Hoechst fluorescence of cells traversing a second S phase is again quenched by BrdU incorporation and the G 2 of the second cycle (G~) appears left from the G 2 of the first cycle. Thus, cells can be distinguished according to the number of S phases they have traversed. Cells belonging to the G a, S and G 2 compartments of each cycle are resolved by staining with ethidium bromide, of which the fluorescence intensity remains stoichiometric with D N A content. The striking difference between the bivariate cytograms is the relative number of cells in the G 2 cluster (arrows). Apparently, cells from BS exhibit a difficulty in traversing the G 2 phase, which is
compatible with either a deficiency in D N A ligase I (Willis and Lindahl, 1987; Chan et al., 1987a, b; Willis et al., 1987) or with free radical-induced cell damage (Poot et al., 1987, 1988, 1989; Poot and Hoehn, 1989). Sequential analysis of the type shown in Fig. 1 together with quantitative treatment of the bivariate data gives the minimal and mean durations of each c o m p a r t m e n t of the cell cycles a culture has traversed (Rabinovitch, 1983). Additionally, the fraction of cells permanently arrested in a particular cell-cycle compartment can be computed. Table 1 gives those selected cell kinetic parameters which revealed a difference between cultures of control and BS fibroblasts maintained at 5% (vol/vol) oxygen. Thus, fibroblasts from BS show an elevated permanent arrest in the G 2 phase of the first cycle after serum stimulation and a prolongation of the G 1 compartment of the second cycle. This pattern of disturbed proliferation is akin to that found with control fibroblasts exposed to 10/~M of 4-hydroxynonenal (Table 1), a breakdown product of lipid peroxidation (Poli et al., 1985; Esterbauer et al., 1986). These data are consistent with the hypothesis that BS fibroblasts suffer from lipid peroxidation induced by oxygen free radicals (Nicotera et al., 1989).
TABLE 1 FRACTION OF CELLS ARRESTED IN THE G 2 PHASE OF THE FIRST CYCLE AND MINIMAL DURATION OF THE G 1 COMPARTMENT OF THE SECOND CYCLE AFTER SERUM STIMULATION OF QUIESCENT CELLS FROM CONTROL AND BS FIBROBLAST CULTURES
Disturbed cell kinetics in EBL cells
Cell type
G 2 arrest
G x duration
Control Strain 1 Strain 2
2.4 + 0.1 2.1 +0.3
6.5 + 0.7 7.2 +0.7
14.2 + 0.4 9.6 + 0.5 13.6+_0.2
12.2 + 0.4 12.7 + 0.5 9.8+0.3
BS GM 1492 GM3498B BS 3
Control + 10 #M 4-hydroxynonenal Strain 1 14.0 + 0.6 Strain 2 15.8 + 0.5
14.4 + 0.5 12.4 + 0.5
All data are means and standard deviations as obtained by fitting the cell-cycle distributions from a time sequence of samples out of a single kinetic experiment to the modified cell-cycle kinetic model as described by Rabinovitch (1983).
The alternative hypothesis that cells from BS exhibit a deficiency in D N A ligase I (Willis and Lindahl, 1987; Chan et al., 1987) was based upon analysis of permanent B-cell lines derived by treatment of h u m a n peripheral blood mononuclear cells with E p s t e i n - B a r r virus (EBL cells). Therefore, 2 EBL lines from healthy individuals and from patients with BS were compared (Table 2). The striking difference between control and BS cells was the prolongation of the S phase of the second cycle in BS cells; in contrast to fibroblasts, EBL from BS showed no prolongation of the G~ compartment. The fraction of cells arrested in the G 2 c o m p a r t m e n t of the first cycle was only slightly higher in BS cells relative to control cells, but the fraction arrested in the G 2 phase of the second cycle was clearly elevated in BS cells (Table 2). Apparently, BS cells that incorporated BrdU during 2 S phases show more arrest in the G z com-
206 TABLE 2 MINIMAL DURATIONS OF THE G 1 AND S PHASES OF THE SECOND CELL CYCLE AFTER RELEASE FROM QUIESCENCE OF EBL CELLS FROM BS AND HEALTHY SUBJECTS CULTURED AT 5% OXYGEN (h), AND FRACTION OF CELLS ARRESTED IN THE G 2 PHASE OF THE FIRST AND SECOND CELL CYCLE (% OF TOTAL CELLS) Minimal duration
Arrest fraction
G 1 phase
S phase
First G 2
Second G 2
Control Line WF Line HZ
6.1+0.4 5.8+0.4
7.6+0.2 6.5+0.3
2.35:0.2 1.9+0.4
2.7+0.6 3.6+0.3
BS GM 3404 BD1
6.1+0.5 5.1+0.6
9.0+0.8 9.75:0.6
3.6-t-0.4 3.55:0.3
7.4+0.7 6.0+1.0
All data are means and standard deviations as obtained by fitting the cell-cycle distributions from a time sequence of samples out of a single kinetic experiment to the modified cell-cycle kinetic model as described by Rabinovitch (1983). 20. 18"
FiDroblasts
16. •..,"
GM 3498 B
14' 12" 10,
p a r t m e n t t h a n BS cells that i n c o r p o r a t e d B r d U o n l y once. This result suggests that the extent of G2 arrest d e p e n d s u p o n the i n c o r p o r a t i o n of B r d U into the D N A in a w a y a k i n to the rate of S C E (14). T o test this p o s s i b i l i t y f i b r o b l a s t s a n d E B L cells f r o m h e a l t h y i n d i v i d u a l s a n d f r o m BS patients were c u l t u r e d for 72 h with a c o n c e n t r a t i o n series of B r d U a n d the fraction of cells in G 2 was assessed (Fig. 2). A l l cultures of BS cells d i s p l a y e d a clear B r d U d e p e n d e n c e of the G 2 fraction, whereas c o n t r o l cells d i d not.
Discussion T h e fact t h a t 2 different cell types f r o m BS show 2 distinct types of cell kinetic d i s t u r b a n c e argues a g a i n s t a p u t a t i v e deficiency of a single e n z y m e involved in D N A r e p l i c a t i o n a n d D N A r e p a i r as the cause o f BS. O n the o t h e r hand, an elevated level of o x i d a t i v e stress m i g h t m a n i f e s t itself d i f f e r e n t l y in different cell types. F i b r o blasts, c o n t a i n i n g a high level of lipids, m a y suffer lipid p e r o x i d a t i o n , w h e r e a s E B L are m o r e likely to exhibit signs of D N A d a m a g e . Thus, elevated o x i d a t i v e stress seems a likely e x p l a n a t i o n of the c a n c e r - p r o n e p h e n o t y p e of BS, b u t further research is n e e d e d to test this hypothesis.
Control 8'
Acknowledgements
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W e are i n d e b t e d to Prof. Dr. H. E s t e r b a u e r ( D e p a r t m e n t of Biochemistry, U n i v e r s i t y of Graz, A u s t r i a ) for m a k i n g a s a m p l e of 4 - h y d r o x y n o n enal a v a i l a b l e to us. H e l p f u l discussions a n d sending us a p r e p r i n t o f a m a n u s c r i p t b y Dr. T.M. N i c o t e r a (Roswell P a r k M e m o r i a l Institute, Buffalo, N Y ) is k i n d l y a c k n o w l e d g e d . This w o r k was supported by Deutsche Forschungsgemeinschaft, G r a n t D F G 8 4 9 / 2 - 1 . W e t h a n k Miss Julia K/Shler for p r e p a r a t i o n of the figures.
8'
References
8" 4' 2" O"
. . . . . . . 0
'
I
I
I
20 40 60 80 100 120 140 160 180 200 BrdU concentration
[;JM]
Fig. 2. BrdU dependence of G 2 arrest in fibroblasts and EBL cells. Cells were cultured during 72 h at 5% (vol/vol) oxygen in the continuous presence of a concentration series of BrdU.
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