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Werner's syndrome lymphoblastoid cells are hypersensitive to topoisomerase II inhibitors in the G2 phase of the cell cycle
Mutation Research 459 Ž2000. 123–133 www.elsevier.comrlocaterdnarepair Community address: www.elsevier.comrlocatermutres
Werner’s syndrome lymphoblastoid cells are hypersensitive to topoisomerase II inhibitors in the G2 phase of the cell cycle P. Pichierri a
a,1
, A. Franchitto
a,2
, P. Mosesso a , L. Proietti de Santis a , A.S. Balajee b, F. Palitti a,)
Dipartimento di Agrobiologia ed Agrochimica, UniÕersita` della Tuscia, Via S. Camillo de Lellis s.n.c., 01100 Viterbo, Italy b Laboratory of Molecular Genetics, National Institute on Aging, NIH, Baltimore, MD USA Received 2 June 1999; received in revised form 22 September 1999; accepted 22 November 1999
Abstract Werner’s syndrome ŽWS. is a rare autosomal recessive human disorder and the patients exhibit many symptoms of accelerated ageing in their early adulthood. The gene ŽWRN . responsible for WS has been biochemically characterised as a 3X –5X helicase and is homologous to a number of RecQ superfamily of helicases. The yeast SGS1 helicase is considered as a human WRN homologue and SGS1 physically interacts with topoisomerases II and III. In view of this, it has been hypothesised that the WRN gene may also interact with topoisomerases II and III. The purpose of this study is to determine whether the loss of function of WRN protein alters the sensitivity of WS cells to agents that block the action of topoisomerase II. This study deals with the comparison of the chromosomal damage induced by the two anti-topoisomerase II drugs, VP-16 and amsacrine, in both G1 and G2 phases of the cell cycle, in lymphoblastoid cells from WS patients and from a healthy donor. Our results show that the WS cell lines are hypersensitive to chromosome damage induced by VP-16 and amsacrine only in the G2 phase of the cell cycle. No difference either in the yield of the induced aberrations or SCEs was found after treatment of cells at G1 stage. These data might suggest that in WS cells, because of the mutation of the WRN protein, the inhibition of topoisomerase II activity results in a higher rate of misrepair, probably due to some compromised G2 phase processes involving the WRN protein. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Werner’s syndrome; Topoisomerase II inhibitors; Recombination and DNA repair; Chromosomal aberrations
1. Introduction Werner’s syndrome ŽWS. is an autosomal recessive disorder and the patients exhibit several features that are suggestive of premature ageing in early ) Corresponding author. Tel.: q39-0761-357206; fax: q390761-357242; e-mail: [email protected] 1 The two authors contributed equally to this work. 2 The two authors contributed equally to this work.
adulthood w1x. Some of the clinical features include bilateral cataracts, diabetes mellitus, osteoporosis, arteriosclerosis and trophic ulcers of the legs. The WS patients have an enhanced risk of developing various neoplasms including different types of carcinomas and sarcomas w2–4x. As might be expected from the premature ageing phenotype, fibroblast cells derived from WS patients have a reduced proliferative capacity in vitro w5x. The Werner syndrome gene ŽWRN . has been cloned w6x, it encodes a 1432 amino
0921-8777r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 8 7 7 7 Ž 9 9 . 0 0 0 6 5 - 8
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acid protein, comprising a central region that is homologous to many members of the RecQ family of DNA helicases. The WRN protein has been recently demonstrated to be a helicase exhibiting DNA unwinding activity w7,8x. One of the hallmarks of WS patients is the genomic instability observed by the spontaneous chromosome anomalies and large deletions in many genes w1x. The increase in spontaneous frequency of the chromosome breaks w9x suggests that WS is a classical case of chromosome breakage syndrome like Ataxia telangiectasia ŽAT. and Bloom syndrome ŽBLM.. The lymphoblast and fibroblast cells of WS patients show another unique feature called ‘‘ variegated translocation mosaicism’’ ŽVTM.. VTM involves the expansion of different structural chromosome rearrangements in different independent clones of the cell line from the same individual w10x.
Recent studies indicate a possible interaction between helicases and topoisomerases, which may work together in many aspects of DNA metabolism including progression of replication forks, segregation of newly replicated chromosomes, disruption of nucleosome structure, DNA supercoiling, recombination and repair w11x. The SGS1 protein of Saccharomyces cereÕisiae is a member of the RecQ helicase family, w12x and is homologous to WRN gene w13,14x. SGS1 has been shown to interact physically with topoisomerases II and III w14x and this interaction in yeast may represent an important clue with regard to function of WRN helicase in mammalian cells. Yeast strains lacking SGS1p are characterised by high levels of chromosome breakage and missegregation in both mitosis and meiosis w14x. Furthermore, sgs1 mutants also show shortened life span in culture like WS fibroblast cell lines w15x. Some of these similari-
Fig. 1. Experimental schemes.
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ties observed between WS cells and sgs1 mutants suggest that the interaction between helicases and topoisomerases may be critical for chromosomal integrity. Therefore, it is reasonable to determine whether the loss of interaction between WRN helicase and topoisomerases, owing to mutation in the WRN gene, alters the sensitivity of WS cells to agents that block the action of topoisomerase activities.
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The two inhibitors of topoisomerase II, etoposide ŽVP-16. and 4X-Ž9-acridinylamino. methanesulfonm-anisidide Žamsacrine., stabilise the DNA–topoisomerase ‘‘cleavable complex’’ resulting in DNA strand breaks w16x, induction of chromosomal aberrations w17,18x, sister chromatid exchanges and cell death w19x. Although no defects in topoisomerase II activity were found in WS cell lines w20x, the possibility of an interaction between WRN and topoiso-
Table 1 Induction of chromosomal aberrations in SNW646, KO375 and DJG ŽWS. cells by treatments with VP-16 or amsacrine for 3 h Treatmentsa
VP-16 3 h Colcemid
3 h 0.13 mM
3 h 0.25 mM
3 h 0.5 mM
3 h 1 mM
Amsacrine 3 h Colcemid
3 h 0.25 mM
3 h 0.5 mM
3 h 1 mM
3 h 2.5 mM
MI Ž%.
Cells scored
Abnormal cells Ž%.
Total aberrationsb
Aberrationsrcell Ž"S.D..
Unlabelled
Labelled
Unlabelled
Unlabelled
Unlabelled
11.8 9.4 10 5.4 7.4 7 5 6.3 5.8 4.2 3.7 4 1.8 2.2 2
88 86 85 100 100 100 100 100 100 100 100 100 100 100 100
12 14 15 0 0 0 0 0 0 0 0 0 0 0 0
5 2.5 2.3 10 23 20 16 27 25 16 39q 40q 28 44q 48q
2 2 2 14 38 35 17 43 40 20 77 80 40 120 120
0.02 " 0.16 0.02 " 0.16 0.02 " 0.16 0.14 " 0.46 0.38 " 0.68U 0.35 " 0.60U 0.17 " 0.44 0.43 " 0.80U 0.40 " 0.75U 0.20 " 0.58 0.77 " 1.27U 0.80 " 1.15U 0.40 " 0.83 1.20 " 4.00U 1.20 " 3.50U
11.8 9.4 10 5.5 5.2 5 5 4 4.5 5 5 4 3.8 1.4 1.7
88 86 85 100 100 100 100 100 100 100 100 100 100 100 100
12 14 15 0 0 0 0 0 0 0 0 0 0 0 0
5 2.5 2.3 16 16 15 33 62q 60q 36 72q 70q 54 93q 95
2 2 2 14 26 25 60 140 150 70 200 185 100 370 350
0.02 " 0.16 0.02 " 0.16 0.02 " 0.16 0.14 " 0.46 0.26 " 0.58 0.25 " 0.50 0.60 " 1.08 1.40 " 1.63U 1.50 " 1.50U 0.70 " 1.37 2.00 " 2.15U 1.85 " 2.00U 1.00 " 1.19 3.70 " 1.95U 3.50 " 2.00U
a For each experimental point, the values of the SNW646, KO375, and DJG cell lines are reported in the first, second, and third rows, respectively, of each entry. b Aberrations were chromatid breaks and chromatid exchanges. q Statistically significant p - 0.05 ŽFisher’s test.. U Statistically significant p - 0.05 ŽStudent’s t-test..
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Table 2 Induction of chromosomal aberrations in SNW64, KO375 and DJG ŽWS. cells by treatment with VP-16 and amsacrine for 3 h at different recovery times Treatments (VP-16)a 3 h colcemid
3 h 0.25 mM
3 h 0.5 mM
3 h mediumq 3 h colcemid
3 h 0.25 mM q 3 h colcemid
3 h 0.5 mM q 3 h colcemid
6 h mediumq 3 h colcemid
3 h 0.25 mM q 3 h medium q3 h colcemid 3 h 0.5 mM q 3 h medium q3 h colcemid Amsacrine a 3 h colcemid
3 h 0.5 mM
3 h 1 mM
3 h mediumq 3 h colcemid
3 h 0.5 mM q 3 h colcemid
3 h 1 mM q 3 h colcemid
6 h mediumq 3 h colcemid
MI Ž%.
Cells scoredb
Abnormal cells Ž%.
Total aberrations c
Aberrationsrcell Ž"S.D..
Unlabelled
Unlabelled
Unlabelled
Labelled
Unlabelled
8 7 6.5 3.5 4.5 4 2.8 3.7 3.5 7 7 6.5 1 0.8 1 0.5 0.5 0.5 8.5 7.9 8 5 4.5 4 3.2 3 3
90 92 91 100 100 100 100 100 100 75 80 79 100 100 100 100 100 100 10 15 14 50 56 55 76 70 70
10 8 9 0 0 0 0 0 0 25 20 21 0 0 0 0 0 0 90 85 86 50 44 45 24 30 30
0 4 4 15 25 24 16 35q 35q 2 3 3 24 28 25 30 36 34 0 0 0 17 9.6 9 18 11 11
0 4 4 17 42 40 20 70 68 3 4 4 28 32 30 35 50 50 0 0 0 10 10 10 11 20 20
0 0.04 " 0.13 0.04 " 0.10 0.17 " 0.44 0.42 " 0.72U 0.40 " 0.70U 0.20 " 0.48 0.70 " 0.96U 0.68 " 0.90U 0.03 " 0.10 0.04 " 0.14 0.04 " 0.12 0.28 " 0.60 0.32 " 0.57 0.30 " 0.55 0.35 " 0.70 0.50 " 0.70 0.50 " 0.65 0 0 0 0.10 " 0.25 0.10 " 0.25 0.10 " 0.25 0.11 " 0.36 0.20 " 0.45 0.20 " 0.40
8.5 7.8 6.5 4.0 4.5 4.5 2.5 3.5 3 7.5 7 6.5 0.5 0.5 0.5 0.2 0.1 0.1 8 8 8
87 88 91 100 100 100 100 100 100 74 76 79 50d 50d 50d 50d 50d 50d 4 10 14
13 12 9 0 0 0 0 0 0 26 24 21 0 0 0 0 0 0 96 90 86
0 0 4 40 60q 58q 50 68q 70q 0 0 3 42 50 50 40 58q 55q 0 0 0
0 0 4 60 120 120 70 170 160 0 0 4 60 80 80 60 100 100 0 0 0
0 0 0.04 " 0.10 0.60 " 1.12 1.20 " 1.25U 1.20 " 1.20U 0.70 " 1.00 1.70 " 1.50U 1.60 " 1.45U 0 0 0.04 " 0.12 0.60 " 1.15 0.80 " 1.20 0.80 " 1.15 0.60 " 1.20 1.00 " 1.20U 1.00 " 1.20U 0 0 0
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Table 2 Žcontinued. Treatments
MI Ž%.
3 h 0.5 mM q 3 h medium q3 h colcemid
0 0 0 0 0 0 9 8.5 8 5 4.8 4.5 0 0 0
Cells scoredb Unlabelled
3 h 1 mM q 3 h medium q3 h colcemid 9 h mediumq 3 h colcemid
3 h 0.5 mM q 6 h medium q3 h colcemid 3 h 1 mM q 6 h medium q3 h colcemid
0 0 0 80 85 85
Labelled
50 50 50 20 15 15
Abnormal cells Ž%.
Total aberrations c
Aberrationsrcell Ž"S.D..
Unlabelled
Unlabelled
Unlabelled
ND ND ND 50 60 60
ND ND ND 0.50 " 0.65 0.60 " 0.80 0.60 " 0.75
No mitosis No mitosis No mitosis No mitosis No mitosis No mitosis ND ND ND 25 21 21 No mitosis No mitosis No mitosis
a For each experimental point, the values of the SNW646, KO375, and DJG cell lines are reported in the first, second, and third rows, respectively, of each entry. b When the number of unlabelled cells was less than 100, an extra scoring was made to amend it to 100. c The aberrations were chromatid breaks and chromatid exchanges. d There are few mitotic cells in the sample; the values of total aberrations are referred to 100 metaphases. q Statistically significant p - 0.05 ŽFisher’s test.. U Statistically significant p - 0.05 ŽStudent’s t-test..
merase II might make WS cells more sensitive to treatments interfering with the activity of DNA topoisomerase II. In this study, we tested this hypothesis by comparing the relative sensitivity of chromosomal damage induced by VP-16 and amsacrine in both normal and WS lymphoblastoid cell lines which might give clues with regard to the functional interaction between WRN helicase and topoisomerase II.
performed with cells in logarithmic phase of growth. They were seeded at 2 = 10 6 cells per 25 cm2 flask, from a single pool of cells, and treated 24 h later. Under our experimental conditions, the SNW646, KO375 and DJG cell lines showed a modal number of chromosome of 46; low percentages of hypoploid or hyperploid metaphases were found in KO375 cells. 2.2. Chemicals
2. Materials and methods 2.1. Cell culture The EBV-transformed normal ŽSNW646. and WS ŽKO375 and DJG. lymphoblast cell lines, were originally obtained from Dr. G.M. Martin ŽSeattle.. The cells were cultured in RPMI 1640 medium, supplemented with 10% heat-inactivated foetal bovine serum and 2% L-glutamine, incubated at 378C in a 5% CO 2 atmosphere Ž100% humidity nominal.. All cultures were maintained in logarithmic growth at a density 5 = 10 5 cellsrml. All the experiments were
Etoposide ŽVP-16., amsacrine, colcemid and 5bromo-2X-deoxyuridine ŽBrdUrd. were purchased from Sigma-Aldrich. VP-16 and amsacrine were dissolved in DMSO at the appropriate concentrations for treatments and used freshly. 2.3. G1 or S treatments For the G1 or S treatments, cells were exposed to VP-16 Ž0.5 and 1 mM. or amsacrine Ž1 and 2 mM. for 2 h. The cells were washed twice in PBS and the complete medium containing BrdUrd Ž3 mgrml. was
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added. The cells were allowed to grow for 21 or 24 h for analysis of metaphase chromosomes, which underwent one cell division after the treatment ŽM1.. Cells were harvested after the addition of colcemid Ž0.2 mgrml. for the last 3 h ŽFig. 1c.. The slides were prepared according to the standard procedures and were stained by fluorescence plus Giemsa technique w21x. One hundred metaphase cells in first mitosis ŽM1. were analysed for each experimental point and all classes of chromatid-type or chromosome-type aberrations were recorded. Mitotic indices ŽMI. were determined from 1000 cells and the proportion of cells, which underwent two mitotic divisions ŽM2., was determined from 100 metaphases.
warmed hypotonic solution KCl Ž0.075 M. for 18 min and fixed in methanolracetic acid Ž3:1 vrv. solution. Air dry preparations were made and slides were stained by fluorescence plus Giemsa procedure w21x. A total of 100 metaphases were examined to determine the proportion of cells that underwent one, two or more divisions. The proliferation index ŽPI. was calculated as percentage of metaphases, which underwent two cell divisions ŽM2.. For each sample a total of 50 metaphases containing 46 " 1 chromosomes were examined to determine the SCE frequency per cell. 2.6. Immmunodetection of the BrdUrd incorporation
Exponentially growing cells were exposed to VP16 or amsacrine at different concentrations together with BrdUrd Ž30 mgrml. and colcemid Ž0.2 mgrml., which remained until harvesting, 3 h later ŽFig. 1a.. The final concentration of inhibitors in treated cultures was: 0.13, 0.25, 0.5 or 1 mM VP-16, and 0.25, 0.5, 1 or 2.5 mM amsacrine. Doses were chosen based on our pilot experiments. Alternatively the two cell lines were exposed to 0.25, 0.5 mM VP-16 or 0.5, 1 mM amsacrine for 3 h and cells collected after different recovery times adding colcemid for the last 3 h ŽFig. 1b.. Cytological microscope slides prepared according to standard procedures were processed using immunocytogenetic technique with anti-BrdUrd antibodies in order to score only G2 cells Žunlabelled.. One hundred metaphase cells were analysed for each test point and all classes of chromatidtype aberrations or chromosome-type aberrations were classified, recording the BrdUrd labelling status of the damaged cells. Gaps have been omitted from the total aberration yield. We defined gaps as discontinuities wider than the chromatid width, in which the distal segment was dislodged with respect to the axis of the chromatid. MIs were determined from 1000 cells.
The slides were processed using immunocytogenetic techniques with ant-BrdUrd antibodies conjugated with fluorescein isothiocyanate ŽFITC. to allow the precise analysis of metaphases which were treated in the G2 phase Žunlabelled metaphases. or in the S phase Žlabelled metaphases.. The slides were denatured for 1 min in 10 mM NaOHr70% ethanol, dehydrated in a 70%, 90% and 100% ethanol series and air-dried. The slides were then incubated in a moist chamber for 30 min with 100 mlrslide of mouse anti-BrdUrd antibody ŽBoheringer-Mannheim; 1:100 dilution. in immunological buffer ŽPBS, 0.5% BSA, 0.5% Tween 20. under a 24 = 50 coverslip. The slides were then washed three times with PBS and subsequently incubated with 100 mlrslide of goat anti-mouse IgG-FITC antibody ŽBoheringerMannheim; 5:100 dilution. in immunological buffer for 30 min. After three washes in PBS and dehydration in ethanol, the slides were embedded with Vectashield mounting medium ŽVector Labs. containing 0.3 mgrml propidium iodide ŽPI.. BrdUrd-labelling of mitotic cells was evaluated using a Zeiss ŽAxiophot. fluorescence microscope equipped with single and dual banded pass filter for FITC and PI, and a CCD camera ŽPhotometrix. operated by IPlab spectrum software. Images were captured using the CCD camera and analysed.
2.5. SCE analysis
2.7. Statistical analysis
After 15 h incubation with 3 mgrml BrdUrd, the inhibitors were added to the cultures for 27 h at different concentrations followed by colcemid for 3 h before harvesting ŽFig. 1d.. The cells were collected by centrifugation, resuspended in a pre-
Differences in the yield of aberrations were evaluated by Student’s test, comparing the total of chromosomal aberrations observed between untreated and treated cells. The value of abnormal cells was evaluated using the Fisher’s test. The differences in SCEs
2.4. G2 treatments
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induction between the normal and the WS cell lines were evaluated by Student’s test.
3. Results 3.1. Chromosomal effects of a G2 treatment with the inhibitors of DNA topoisomerase II, VP-16 or amsacrine Table 1 summarises the fraction of mitotic cells ŽMI., the percentage of abnormal cells and the total
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number of aberrations in G2 cells Žunlabelled. after 3 h of treatment with topoisomerase II inhibitors. The observed reduction of the MI was dose-dependent in both cell lines for VP-16. However, the reduction of the MI in cells treated with amsacrine remained constant from 0.25 to 1 mM and increased only at the higher dose. Both VP-16 and amsacrine induced significantly higher levels of abnormal cells and total aberrations in the WS cells compared to the normal line SNW646 at any dose level assayed. The increase of the total aberrations ranged from ; 2.7 to ; 3 times, with
Table 3 Induction of chromosomal aberrations in SNW646 and KO375 ŽWS. cells by treatments with VP-16 or amsacrine in the G1 or S phase of the cell cycle Cells were harvested with colcemid added in the last 3 h. Aberrations were scored only in M1 cells. Treatmentsa VP-16 23 h medium 26 h medium 2 h 0.5 mM q 21 h medium 2 h 0.5 mM q 24 h medium 2 h 1 mM q 21 h medium 2 h 1 mM q 24 h medium
Amsacrine 23 h medium 26 h medium 2 h 1 mM q 21 h medium 2 h 1 mM q 24 h medium 2 h 2 mM q 21 h medium 2 h 2 mM q 24 h medium
MI Ž%.
M2 Ž%.
Abnormal cells Ž%.
Total aberrationsb
Aberrationsrcell Ž"S.D..
Total dicentrics
Dicentricsr cell
7 6 7 7.6 4.6 4 5.6 6 3.7 3.2 6.4 6
20 20 27 24 2 3 7 9 0 0 4 5
2 4 2 4 20 20 27 23 30 23 26 27
2 4 2 4 23 23 28 25 40 33 50 33
0.02 " 0.14 0.04 " 0.20 0.02 " 0.12 0.04 " 0.18 0.23 " 0.41 0.23 " 0.51 0.28 " 0.40 0.25 " 0.45 0.40 " 0.65 0.33 " 0.51 0.50 " 0.80 0.33 " 0.51U
0 0 0 0 23 15 7 7 30 30 30 30
0 0 0 0 0.23 0.15 0.07 0.07 0.30 0.30 0.30 0.30
7 6 7 7.6 3 3.2 5.9 6 2.5 3 4 3.4
20 20 27 24 0 0 5 6 0 0 2 3
2 4 2 4 34 30 20 16 50 51 46 46
2 4 2 4 40 43 28 30 70 65 65 54
0.02 " 0.14 0.04 " 0.20 0.02 " 0.12 0.04 " 0.18 0.40 " 0.54 0.43 " 0.50 0.28 " 0.40 0.30 " 0.55 0.70 " 0.80 0.65 " 0.71 0.65 " 0.80 0.54 " 0.80
0 0 0 0 20 20 20 20 60 55 50 44
0 0 0 0 0.20 0.20 0.20 0.20 0.60 0.55 0.52 0.44
a For each experimental point, the values of the SNW646 and KO375 cell lines were reported in the first and second rows, respectively, of each entry. b Aberrations scored were mainly chromosome breaks and dicentrics; each dicentric was assigned one double minute, which was not included in the aberration counts. U Statistically significant p - 0.05 ŽStudent’s t-test..
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respect to the yield observed in the control cell line, for VP-16 and ; 1.9 to ; 3.7 times for amsacrine. When cells were exposed to VP-16 or amsacrine at the two intermediate dose levels and allowed to recover in drug free medium for various times, a drop in the MI was detected in the first 3 h of recovery in the normal and the WS cell lines and for both the two drugs. The VP-16-induced mitotic delay was found to be 3 h and that induced by amsacrine was 6 h for the lower dose and 9 h for the higher one. The observed increase in the MI was mainly due to the contribution of G2 cells Žunlabelled metaphases. presumably arrested at the G2 checkpoint. Cells which recover from the mitotic delay were less damaged as compared to those immediately harvested. The differences in the induction of chromatid-type aberrations between the cell lines tended to disappear due to a decrease of aberrations in WS cells which already passed mitosis. The incidence of aberrations in WS cells dropped to ; 1.14 and ; 1.42, with respect to the control cell line, for VP-16 after 3-h recovery at dose level of 0.25 and 0.50 mM, respectively, and ; 1.0 and ; 1.8 after 6-h recovery at the same dose levels. For amsacrine, the incidence of aberrations in WS cells dropped to ; 1.3 and ; 1.6 at dose levels of 0.5 and 1.0 mM, respectively, after 3-h recovery and 1.2 after 9-h recovery at 0.5 mM dose-level ŽTable 2.. 3.2. Chromosomal effects of a G1 treatment with the inhibitors of DNA topoisomerase II, VP-16 and amsacrine and SCE analysis SNW646, KO375 and DJG were separately exposed to VP-16 Ž0.5 and 1.0 mM. and amsacrine Ž1.0 or 2.0 mM. for 2 h and the cells were harvested after 21 or 24 h of treatment. Table 3 summarises the MI, frequencies of M2 cells, the percentage of abnormal cells, the total number of chromosomal aberrations as well as the frequencies of the dicentrics. Only the data for KO375 cells are presented because KO375 and DJG behave in a similar manner. Contrary to the results obtained at G2 stage, all the parameters studied were found to be essentially similar for both agents. A higher induction of chromosomal damage was observed only for amsacrine at the later sampling time in both cell lines ŽTable 3.. No statistical differences ŽStudent t-test, p ) 0.05.
Table 4 SCE induction by VP-16 or amsacrine treatments in SNW646 and KO375 cells Cells were harvested with colcemid added in the last 3 h. Treatmentsa
MI Ž%. M2 Ž%. SCEsrcell Ž"S.D..
VP-16 27 h MediumqBrdUrd 10.4 9.4 27 h 0.01 mMqBrdUrd 7.5 7.74 27 h 0.02 mMqBrdUrd 5.1 5.1 27 h 0.05 mMqBrdUrd 3 2.8
72 74 65 63 50 51 36 36
4.5"1.82 4.3"1.19 6.9"3.00 7.6"3.05 9.0"3.42 9.2"3.47 9.0"3.50 11.0"4.62U
Amsacrine 27 h MediumqBrdUrd 10.4 9.4 27 h 0.02 mMqBrdUrd 4 4.2 27 h 0.05 mMqBrdUrd 3 2.3 27 h 0.1 mMqBrdUrd 4 3.2
72 74 50 50 36 31 5 3
4.5"1.82 4.3"1.19 9.9"3.85 8.6"4.00 12.4"4.00 13.2"4.41 20.2"5.20 21.7"5.61
a
For each experimental point, the values of the SNW646 and KO375 cell lines were reported in the first and second rows, respectively, of each entry. U Statistically significant p- 0.05 ŽStudent’s t-test..
were observed for the induction of SCEs between the SNW646 and KO375 cells either for VP-16 or amsacrine treatment, even if a dose-dependent SCE induction was observed for each cell line ŽTable 4.. These results indicate that the WS cells are hypersensitive to topoisomerase II inhibitors only at the G2 stage of the cell cycle.
4. Discussion In this study, we demonstrate that the lymphoblastoid cells from two different WS patients are hypersensitive to treatment with topoisomerase II inhibitors. The increased sensitivity of WS cells was observed only at the G2 phase of the cell cycle, as there was no difference in the induction of chromosomal damage between normal and WS cells at G1 or S phase. This G2 sensitivity appeared to be higher and almost only present in cells collected during the 3 h treatment with both topoisomerase II inhibitors.
P. Pichierri et al.r Mutation Research 459 (2000) 123–133
This G2 sensitivity was lost in cells harvested at different recovery times after VP-16 or amsacrine treatment. These results indicate that there is a critical time point in the cell cycle, which sensitises the WS cells towards increased DNA damage after treatment with topoisomerase II inhibitors. These findings further suggest the presence of some specific G2 phase repair process that could be impaired in WS cells, leading to higher induction of chromosomal damage after treatment with topoisomerase II inhibitors. Recent studies have shown specific interaction between SGS1 Žyeast homologue of human WRNp. and the topoisomerases and that this interaction may be critical for chromosome segregation and integrity w13,22x. The finding of increased chromosome missegregation in both mitosis and meiosis in yeast sgs1 mutants, similar to WS cells, raises the possibility that the WRN gene products may also interact with topoisomerases. Although WS cells do not show enhanced sensitivity to agents like bleomycin and X-rays, that predominantly cause DNA double strand breaks ŽDSBs., the increase in nonhomologous recombination in WS cells tend to suggest some kind of deficiency in the repair of DSBs and the error prone ligation activity, leading to large genomic deletions w23x. It has been proposed recently that topoisomerase II together with SGS1 might control hyper-recombination w24x. In view of the role of topoisomerase II in recombination, it is reasonable to assume that the enhanced sensitivity of WS to topoisomerase II inhibitors may be due to altered recombination activities. WS cells have been shown to be sensitive to both topoisomerase I and II inhibitors w25,26x. A recent study has shown that the WS cells are more prone to undergo apoptosis following treatment with camptothecin, a topoisomerase I inhibitor w27x. In the present study, we demonstrate that the WS cells are hypersensitive to topoisomerase II inhibitors in the G2 stage, but not in G1 or S phase. This enhanced sensitivity to topoisomerase inhibitors appear to be mediated primarily by the WRN gene mutationŽs. rather than by secondary effect, as the murine embryonic stem ŽES. cells, carrying a deletion mutation in the WRN helicase domain, are also sensitive to topoisomerase inhibitors w28x. It is possible that the WRN gene is involved in the recombination repair
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pathway through its interaction with topoisomerase II. It is not clear why the WS cells show enhanced sensitivity to topoisomerase II inhibitors only in the G2 stage. Inhibition of topoisomerase II activity induced by VP-16 or amsacrine might enhance misrepair of DSBs thereby affecting a G2 dependent recombinational process w29x. Aratani et al. w30x reported that treatments with VP-16 or amsacrine effectively enhance the illegitimate mitotic recombination through the inhibition of the topoisomerase II activity. In WS cells, the inhibition of the topoisomerase II activity, together with loss of WRN gene function, may result in a higher rate of misrepair and increased chromosomal damage. Once the inhibition of the topoisomerase II activity is removed by washing off the inhibitors from the cultures, WS cells restored a normal capacity to repair DSBs. This might explain the abrogation of the higher G2 sensitivity to the two inhibitors induction of chromosomal damage observed in cells harvested after the recovery times. Another likely explanation for the enhanced sensitivity is that the topoisomerase inhibitors cause a higher level of DSBs in WS cells because of a defect in the topoisomerase II activity itself or in chromatin structure. However, these possibilities appear less probable because of the similar sensitivity of WS cells to the two anti-topoisomerase II drugs in the G1 phase, as well as to X-ray-induced chromosomal damage reported elsewhere w31x. The G2 sensitivity of the WS cells to VP-16 or amsacrine may not be attributable to a defect in the G2 checkpoint response, as no clear differences in the mitotic inhibition, caused by the two agents, was detected in the WS cell line as compared to the normal cells. Additionally, data reported in literature support the hypothesis that in WS cells the G2 checkpoint response to DNA damage is normal w20,31x. On the basis of our observations, we speculate that the WRN helicase, together with topoisomerases, may mediate an effective recombinational repair pathway, operating in the G2 phase of the cell cycle, prior to the onset of mitosis. Disruption of the interaction between WRN helicase and topoisomerases may predispose the cells to hypersensitivity due to increased illegitimate recombination, resulting from the error prone joining of DNA strand breaks
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induced by topoisomerase II inhibition. However, the precise mechanism responsible for enhancing the sensitivity of WS cells to topoisomerase inhibitors is not clearly understood and requires further detailed investigations.
w10x
Acknowledgements
w11x
The skilful technical collaboration of Mr. A. Schinoppi is highly appreciated. The financial assistance for this work from MURST grants and EC contract No. F14P-CT95-0001 is greatly acknowledged. The authors wish to thank Dr. J. Crawley for her helpful review of the English of this manuscript. This paper is dedicated to Prof. Gian Tommaso Scarascia Mugnozza on the occasion of his retirement.
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Werner’s syndrome lymphoblastoid cells are hypersensitive to topoisomerase II inhibitors in the G2 phase of the cell cycle P. Pichierri a
a,1
, A. Franchitto
a,2
, P. Mosesso a , L. Proietti de Santis a , A.S. Balajee b, F. Palitti a,)
Dipartimento di Agrobiologia ed Agrochimica, UniÕersita` della Tuscia, Via S. Camillo de Lellis s.n.c., 01100 Viterbo, Italy b Laboratory of Molecular Genetics, National Institute on Aging, NIH, Baltimore, MD USA Received 2 June 1999; received in revised form 22 September 1999; accepted 22 November 1999
Abstract Werner’s syndrome ŽWS. is a rare autosomal recessive human disorder and the patients exhibit many symptoms of accelerated ageing in their early adulthood. The gene ŽWRN . responsible for WS has been biochemically characterised as a 3X –5X helicase and is homologous to a number of RecQ superfamily of helicases. The yeast SGS1 helicase is considered as a human WRN homologue and SGS1 physically interacts with topoisomerases II and III. In view of this, it has been hypothesised that the WRN gene may also interact with topoisomerases II and III. The purpose of this study is to determine whether the loss of function of WRN protein alters the sensitivity of WS cells to agents that block the action of topoisomerase II. This study deals with the comparison of the chromosomal damage induced by the two anti-topoisomerase II drugs, VP-16 and amsacrine, in both G1 and G2 phases of the cell cycle, in lymphoblastoid cells from WS patients and from a healthy donor. Our results show that the WS cell lines are hypersensitive to chromosome damage induced by VP-16 and amsacrine only in the G2 phase of the cell cycle. No difference either in the yield of the induced aberrations or SCEs was found after treatment of cells at G1 stage. These data might suggest that in WS cells, because of the mutation of the WRN protein, the inhibition of topoisomerase II activity results in a higher rate of misrepair, probably due to some compromised G2 phase processes involving the WRN protein. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Werner’s syndrome; Topoisomerase II inhibitors; Recombination and DNA repair; Chromosomal aberrations
1. Introduction Werner’s syndrome ŽWS. is an autosomal recessive disorder and the patients exhibit several features that are suggestive of premature ageing in early ) Corresponding author. Tel.: q39-0761-357206; fax: q390761-357242; e-mail: [email protected] 1 The two authors contributed equally to this work. 2 The two authors contributed equally to this work.
adulthood w1x. Some of the clinical features include bilateral cataracts, diabetes mellitus, osteoporosis, arteriosclerosis and trophic ulcers of the legs. The WS patients have an enhanced risk of developing various neoplasms including different types of carcinomas and sarcomas w2–4x. As might be expected from the premature ageing phenotype, fibroblast cells derived from WS patients have a reduced proliferative capacity in vitro w5x. The Werner syndrome gene ŽWRN . has been cloned w6x, it encodes a 1432 amino
0921-8777r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 8 7 7 7 Ž 9 9 . 0 0 0 6 5 - 8
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acid protein, comprising a central region that is homologous to many members of the RecQ family of DNA helicases. The WRN protein has been recently demonstrated to be a helicase exhibiting DNA unwinding activity w7,8x. One of the hallmarks of WS patients is the genomic instability observed by the spontaneous chromosome anomalies and large deletions in many genes w1x. The increase in spontaneous frequency of the chromosome breaks w9x suggests that WS is a classical case of chromosome breakage syndrome like Ataxia telangiectasia ŽAT. and Bloom syndrome ŽBLM.. The lymphoblast and fibroblast cells of WS patients show another unique feature called ‘‘ variegated translocation mosaicism’’ ŽVTM.. VTM involves the expansion of different structural chromosome rearrangements in different independent clones of the cell line from the same individual w10x.
Recent studies indicate a possible interaction between helicases and topoisomerases, which may work together in many aspects of DNA metabolism including progression of replication forks, segregation of newly replicated chromosomes, disruption of nucleosome structure, DNA supercoiling, recombination and repair w11x. The SGS1 protein of Saccharomyces cereÕisiae is a member of the RecQ helicase family, w12x and is homologous to WRN gene w13,14x. SGS1 has been shown to interact physically with topoisomerases II and III w14x and this interaction in yeast may represent an important clue with regard to function of WRN helicase in mammalian cells. Yeast strains lacking SGS1p are characterised by high levels of chromosome breakage and missegregation in both mitosis and meiosis w14x. Furthermore, sgs1 mutants also show shortened life span in culture like WS fibroblast cell lines w15x. Some of these similari-
Fig. 1. Experimental schemes.
P. Pichierri et al.r Mutation Research 459 (2000) 123–133
ties observed between WS cells and sgs1 mutants suggest that the interaction between helicases and topoisomerases may be critical for chromosomal integrity. Therefore, it is reasonable to determine whether the loss of interaction between WRN helicase and topoisomerases, owing to mutation in the WRN gene, alters the sensitivity of WS cells to agents that block the action of topoisomerase activities.
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The two inhibitors of topoisomerase II, etoposide ŽVP-16. and 4X-Ž9-acridinylamino. methanesulfonm-anisidide Žamsacrine., stabilise the DNA–topoisomerase ‘‘cleavable complex’’ resulting in DNA strand breaks w16x, induction of chromosomal aberrations w17,18x, sister chromatid exchanges and cell death w19x. Although no defects in topoisomerase II activity were found in WS cell lines w20x, the possibility of an interaction between WRN and topoiso-
Table 1 Induction of chromosomal aberrations in SNW646, KO375 and DJG ŽWS. cells by treatments with VP-16 or amsacrine for 3 h Treatmentsa
VP-16 3 h Colcemid
3 h 0.13 mM
3 h 0.25 mM
3 h 0.5 mM
3 h 1 mM
Amsacrine 3 h Colcemid
3 h 0.25 mM
3 h 0.5 mM
3 h 1 mM
3 h 2.5 mM
MI Ž%.
Cells scored
Abnormal cells Ž%.
Total aberrationsb
Aberrationsrcell Ž"S.D..
Unlabelled
Labelled
Unlabelled
Unlabelled
Unlabelled
11.8 9.4 10 5.4 7.4 7 5 6.3 5.8 4.2 3.7 4 1.8 2.2 2
88 86 85 100 100 100 100 100 100 100 100 100 100 100 100
12 14 15 0 0 0 0 0 0 0 0 0 0 0 0
5 2.5 2.3 10 23 20 16 27 25 16 39q 40q 28 44q 48q
2 2 2 14 38 35 17 43 40 20 77 80 40 120 120
0.02 " 0.16 0.02 " 0.16 0.02 " 0.16 0.14 " 0.46 0.38 " 0.68U 0.35 " 0.60U 0.17 " 0.44 0.43 " 0.80U 0.40 " 0.75U 0.20 " 0.58 0.77 " 1.27U 0.80 " 1.15U 0.40 " 0.83 1.20 " 4.00U 1.20 " 3.50U
11.8 9.4 10 5.5 5.2 5 5 4 4.5 5 5 4 3.8 1.4 1.7
88 86 85 100 100 100 100 100 100 100 100 100 100 100 100
12 14 15 0 0 0 0 0 0 0 0 0 0 0 0
5 2.5 2.3 16 16 15 33 62q 60q 36 72q 70q 54 93q 95
2 2 2 14 26 25 60 140 150 70 200 185 100 370 350
0.02 " 0.16 0.02 " 0.16 0.02 " 0.16 0.14 " 0.46 0.26 " 0.58 0.25 " 0.50 0.60 " 1.08 1.40 " 1.63U 1.50 " 1.50U 0.70 " 1.37 2.00 " 2.15U 1.85 " 2.00U 1.00 " 1.19 3.70 " 1.95U 3.50 " 2.00U
a For each experimental point, the values of the SNW646, KO375, and DJG cell lines are reported in the first, second, and third rows, respectively, of each entry. b Aberrations were chromatid breaks and chromatid exchanges. q Statistically significant p - 0.05 ŽFisher’s test.. U Statistically significant p - 0.05 ŽStudent’s t-test..
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Table 2 Induction of chromosomal aberrations in SNW64, KO375 and DJG ŽWS. cells by treatment with VP-16 and amsacrine for 3 h at different recovery times Treatments (VP-16)a 3 h colcemid
3 h 0.25 mM
3 h 0.5 mM
3 h mediumq 3 h colcemid
3 h 0.25 mM q 3 h colcemid
3 h 0.5 mM q 3 h colcemid
6 h mediumq 3 h colcemid
3 h 0.25 mM q 3 h medium q3 h colcemid 3 h 0.5 mM q 3 h medium q3 h colcemid Amsacrine a 3 h colcemid
3 h 0.5 mM
3 h 1 mM
3 h mediumq 3 h colcemid
3 h 0.5 mM q 3 h colcemid
3 h 1 mM q 3 h colcemid
6 h mediumq 3 h colcemid
MI Ž%.
Cells scoredb
Abnormal cells Ž%.
Total aberrations c
Aberrationsrcell Ž"S.D..
Unlabelled
Unlabelled
Unlabelled
Labelled
Unlabelled
8 7 6.5 3.5 4.5 4 2.8 3.7 3.5 7 7 6.5 1 0.8 1 0.5 0.5 0.5 8.5 7.9 8 5 4.5 4 3.2 3 3
90 92 91 100 100 100 100 100 100 75 80 79 100 100 100 100 100 100 10 15 14 50 56 55 76 70 70
10 8 9 0 0 0 0 0 0 25 20 21 0 0 0 0 0 0 90 85 86 50 44 45 24 30 30
0 4 4 15 25 24 16 35q 35q 2 3 3 24 28 25 30 36 34 0 0 0 17 9.6 9 18 11 11
0 4 4 17 42 40 20 70 68 3 4 4 28 32 30 35 50 50 0 0 0 10 10 10 11 20 20
0 0.04 " 0.13 0.04 " 0.10 0.17 " 0.44 0.42 " 0.72U 0.40 " 0.70U 0.20 " 0.48 0.70 " 0.96U 0.68 " 0.90U 0.03 " 0.10 0.04 " 0.14 0.04 " 0.12 0.28 " 0.60 0.32 " 0.57 0.30 " 0.55 0.35 " 0.70 0.50 " 0.70 0.50 " 0.65 0 0 0 0.10 " 0.25 0.10 " 0.25 0.10 " 0.25 0.11 " 0.36 0.20 " 0.45 0.20 " 0.40
8.5 7.8 6.5 4.0 4.5 4.5 2.5 3.5 3 7.5 7 6.5 0.5 0.5 0.5 0.2 0.1 0.1 8 8 8
87 88 91 100 100 100 100 100 100 74 76 79 50d 50d 50d 50d 50d 50d 4 10 14
13 12 9 0 0 0 0 0 0 26 24 21 0 0 0 0 0 0 96 90 86
0 0 4 40 60q 58q 50 68q 70q 0 0 3 42 50 50 40 58q 55q 0 0 0
0 0 4 60 120 120 70 170 160 0 0 4 60 80 80 60 100 100 0 0 0
0 0 0.04 " 0.10 0.60 " 1.12 1.20 " 1.25U 1.20 " 1.20U 0.70 " 1.00 1.70 " 1.50U 1.60 " 1.45U 0 0 0.04 " 0.12 0.60 " 1.15 0.80 " 1.20 0.80 " 1.15 0.60 " 1.20 1.00 " 1.20U 1.00 " 1.20U 0 0 0
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Table 2 Žcontinued. Treatments
MI Ž%.
3 h 0.5 mM q 3 h medium q3 h colcemid
0 0 0 0 0 0 9 8.5 8 5 4.8 4.5 0 0 0
Cells scoredb Unlabelled
3 h 1 mM q 3 h medium q3 h colcemid 9 h mediumq 3 h colcemid
3 h 0.5 mM q 6 h medium q3 h colcemid 3 h 1 mM q 6 h medium q3 h colcemid
0 0 0 80 85 85
Labelled
50 50 50 20 15 15
Abnormal cells Ž%.
Total aberrations c
Aberrationsrcell Ž"S.D..
Unlabelled
Unlabelled
Unlabelled
ND ND ND 50 60 60
ND ND ND 0.50 " 0.65 0.60 " 0.80 0.60 " 0.75
No mitosis No mitosis No mitosis No mitosis No mitosis No mitosis ND ND ND 25 21 21 No mitosis No mitosis No mitosis
a For each experimental point, the values of the SNW646, KO375, and DJG cell lines are reported in the first, second, and third rows, respectively, of each entry. b When the number of unlabelled cells was less than 100, an extra scoring was made to amend it to 100. c The aberrations were chromatid breaks and chromatid exchanges. d There are few mitotic cells in the sample; the values of total aberrations are referred to 100 metaphases. q Statistically significant p - 0.05 ŽFisher’s test.. U Statistically significant p - 0.05 ŽStudent’s t-test..
merase II might make WS cells more sensitive to treatments interfering with the activity of DNA topoisomerase II. In this study, we tested this hypothesis by comparing the relative sensitivity of chromosomal damage induced by VP-16 and amsacrine in both normal and WS lymphoblastoid cell lines which might give clues with regard to the functional interaction between WRN helicase and topoisomerase II.
performed with cells in logarithmic phase of growth. They were seeded at 2 = 10 6 cells per 25 cm2 flask, from a single pool of cells, and treated 24 h later. Under our experimental conditions, the SNW646, KO375 and DJG cell lines showed a modal number of chromosome of 46; low percentages of hypoploid or hyperploid metaphases were found in KO375 cells. 2.2. Chemicals
2. Materials and methods 2.1. Cell culture The EBV-transformed normal ŽSNW646. and WS ŽKO375 and DJG. lymphoblast cell lines, were originally obtained from Dr. G.M. Martin ŽSeattle.. The cells were cultured in RPMI 1640 medium, supplemented with 10% heat-inactivated foetal bovine serum and 2% L-glutamine, incubated at 378C in a 5% CO 2 atmosphere Ž100% humidity nominal.. All cultures were maintained in logarithmic growth at a density 5 = 10 5 cellsrml. All the experiments were
Etoposide ŽVP-16., amsacrine, colcemid and 5bromo-2X-deoxyuridine ŽBrdUrd. were purchased from Sigma-Aldrich. VP-16 and amsacrine were dissolved in DMSO at the appropriate concentrations for treatments and used freshly. 2.3. G1 or S treatments For the G1 or S treatments, cells were exposed to VP-16 Ž0.5 and 1 mM. or amsacrine Ž1 and 2 mM. for 2 h. The cells were washed twice in PBS and the complete medium containing BrdUrd Ž3 mgrml. was
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added. The cells were allowed to grow for 21 or 24 h for analysis of metaphase chromosomes, which underwent one cell division after the treatment ŽM1.. Cells were harvested after the addition of colcemid Ž0.2 mgrml. for the last 3 h ŽFig. 1c.. The slides were prepared according to the standard procedures and were stained by fluorescence plus Giemsa technique w21x. One hundred metaphase cells in first mitosis ŽM1. were analysed for each experimental point and all classes of chromatid-type or chromosome-type aberrations were recorded. Mitotic indices ŽMI. were determined from 1000 cells and the proportion of cells, which underwent two mitotic divisions ŽM2., was determined from 100 metaphases.
warmed hypotonic solution KCl Ž0.075 M. for 18 min and fixed in methanolracetic acid Ž3:1 vrv. solution. Air dry preparations were made and slides were stained by fluorescence plus Giemsa procedure w21x. A total of 100 metaphases were examined to determine the proportion of cells that underwent one, two or more divisions. The proliferation index ŽPI. was calculated as percentage of metaphases, which underwent two cell divisions ŽM2.. For each sample a total of 50 metaphases containing 46 " 1 chromosomes were examined to determine the SCE frequency per cell. 2.6. Immmunodetection of the BrdUrd incorporation
Exponentially growing cells were exposed to VP16 or amsacrine at different concentrations together with BrdUrd Ž30 mgrml. and colcemid Ž0.2 mgrml., which remained until harvesting, 3 h later ŽFig. 1a.. The final concentration of inhibitors in treated cultures was: 0.13, 0.25, 0.5 or 1 mM VP-16, and 0.25, 0.5, 1 or 2.5 mM amsacrine. Doses were chosen based on our pilot experiments. Alternatively the two cell lines were exposed to 0.25, 0.5 mM VP-16 or 0.5, 1 mM amsacrine for 3 h and cells collected after different recovery times adding colcemid for the last 3 h ŽFig. 1b.. Cytological microscope slides prepared according to standard procedures were processed using immunocytogenetic technique with anti-BrdUrd antibodies in order to score only G2 cells Žunlabelled.. One hundred metaphase cells were analysed for each test point and all classes of chromatidtype aberrations or chromosome-type aberrations were classified, recording the BrdUrd labelling status of the damaged cells. Gaps have been omitted from the total aberration yield. We defined gaps as discontinuities wider than the chromatid width, in which the distal segment was dislodged with respect to the axis of the chromatid. MIs were determined from 1000 cells.
The slides were processed using immunocytogenetic techniques with ant-BrdUrd antibodies conjugated with fluorescein isothiocyanate ŽFITC. to allow the precise analysis of metaphases which were treated in the G2 phase Žunlabelled metaphases. or in the S phase Žlabelled metaphases.. The slides were denatured for 1 min in 10 mM NaOHr70% ethanol, dehydrated in a 70%, 90% and 100% ethanol series and air-dried. The slides were then incubated in a moist chamber for 30 min with 100 mlrslide of mouse anti-BrdUrd antibody ŽBoheringer-Mannheim; 1:100 dilution. in immunological buffer ŽPBS, 0.5% BSA, 0.5% Tween 20. under a 24 = 50 coverslip. The slides were then washed three times with PBS and subsequently incubated with 100 mlrslide of goat anti-mouse IgG-FITC antibody ŽBoheringerMannheim; 5:100 dilution. in immunological buffer for 30 min. After three washes in PBS and dehydration in ethanol, the slides were embedded with Vectashield mounting medium ŽVector Labs. containing 0.3 mgrml propidium iodide ŽPI.. BrdUrd-labelling of mitotic cells was evaluated using a Zeiss ŽAxiophot. fluorescence microscope equipped with single and dual banded pass filter for FITC and PI, and a CCD camera ŽPhotometrix. operated by IPlab spectrum software. Images were captured using the CCD camera and analysed.
2.5. SCE analysis
2.7. Statistical analysis
After 15 h incubation with 3 mgrml BrdUrd, the inhibitors were added to the cultures for 27 h at different concentrations followed by colcemid for 3 h before harvesting ŽFig. 1d.. The cells were collected by centrifugation, resuspended in a pre-
Differences in the yield of aberrations were evaluated by Student’s test, comparing the total of chromosomal aberrations observed between untreated and treated cells. The value of abnormal cells was evaluated using the Fisher’s test. The differences in SCEs
2.4. G2 treatments
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induction between the normal and the WS cell lines were evaluated by Student’s test.
3. Results 3.1. Chromosomal effects of a G2 treatment with the inhibitors of DNA topoisomerase II, VP-16 or amsacrine Table 1 summarises the fraction of mitotic cells ŽMI., the percentage of abnormal cells and the total
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number of aberrations in G2 cells Žunlabelled. after 3 h of treatment with topoisomerase II inhibitors. The observed reduction of the MI was dose-dependent in both cell lines for VP-16. However, the reduction of the MI in cells treated with amsacrine remained constant from 0.25 to 1 mM and increased only at the higher dose. Both VP-16 and amsacrine induced significantly higher levels of abnormal cells and total aberrations in the WS cells compared to the normal line SNW646 at any dose level assayed. The increase of the total aberrations ranged from ; 2.7 to ; 3 times, with
Table 3 Induction of chromosomal aberrations in SNW646 and KO375 ŽWS. cells by treatments with VP-16 or amsacrine in the G1 or S phase of the cell cycle Cells were harvested with colcemid added in the last 3 h. Aberrations were scored only in M1 cells. Treatmentsa VP-16 23 h medium 26 h medium 2 h 0.5 mM q 21 h medium 2 h 0.5 mM q 24 h medium 2 h 1 mM q 21 h medium 2 h 1 mM q 24 h medium
Amsacrine 23 h medium 26 h medium 2 h 1 mM q 21 h medium 2 h 1 mM q 24 h medium 2 h 2 mM q 21 h medium 2 h 2 mM q 24 h medium
MI Ž%.
M2 Ž%.
Abnormal cells Ž%.
Total aberrationsb
Aberrationsrcell Ž"S.D..
Total dicentrics
Dicentricsr cell
7 6 7 7.6 4.6 4 5.6 6 3.7 3.2 6.4 6
20 20 27 24 2 3 7 9 0 0 4 5
2 4 2 4 20 20 27 23 30 23 26 27
2 4 2 4 23 23 28 25 40 33 50 33
0.02 " 0.14 0.04 " 0.20 0.02 " 0.12 0.04 " 0.18 0.23 " 0.41 0.23 " 0.51 0.28 " 0.40 0.25 " 0.45 0.40 " 0.65 0.33 " 0.51 0.50 " 0.80 0.33 " 0.51U
0 0 0 0 23 15 7 7 30 30 30 30
0 0 0 0 0.23 0.15 0.07 0.07 0.30 0.30 0.30 0.30
7 6 7 7.6 3 3.2 5.9 6 2.5 3 4 3.4
20 20 27 24 0 0 5 6 0 0 2 3
2 4 2 4 34 30 20 16 50 51 46 46
2 4 2 4 40 43 28 30 70 65 65 54
0.02 " 0.14 0.04 " 0.20 0.02 " 0.12 0.04 " 0.18 0.40 " 0.54 0.43 " 0.50 0.28 " 0.40 0.30 " 0.55 0.70 " 0.80 0.65 " 0.71 0.65 " 0.80 0.54 " 0.80
0 0 0 0 20 20 20 20 60 55 50 44
0 0 0 0 0.20 0.20 0.20 0.20 0.60 0.55 0.52 0.44
a For each experimental point, the values of the SNW646 and KO375 cell lines were reported in the first and second rows, respectively, of each entry. b Aberrations scored were mainly chromosome breaks and dicentrics; each dicentric was assigned one double minute, which was not included in the aberration counts. U Statistically significant p - 0.05 ŽStudent’s t-test..
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respect to the yield observed in the control cell line, for VP-16 and ; 1.9 to ; 3.7 times for amsacrine. When cells were exposed to VP-16 or amsacrine at the two intermediate dose levels and allowed to recover in drug free medium for various times, a drop in the MI was detected in the first 3 h of recovery in the normal and the WS cell lines and for both the two drugs. The VP-16-induced mitotic delay was found to be 3 h and that induced by amsacrine was 6 h for the lower dose and 9 h for the higher one. The observed increase in the MI was mainly due to the contribution of G2 cells Žunlabelled metaphases. presumably arrested at the G2 checkpoint. Cells which recover from the mitotic delay were less damaged as compared to those immediately harvested. The differences in the induction of chromatid-type aberrations between the cell lines tended to disappear due to a decrease of aberrations in WS cells which already passed mitosis. The incidence of aberrations in WS cells dropped to ; 1.14 and ; 1.42, with respect to the control cell line, for VP-16 after 3-h recovery at dose level of 0.25 and 0.50 mM, respectively, and ; 1.0 and ; 1.8 after 6-h recovery at the same dose levels. For amsacrine, the incidence of aberrations in WS cells dropped to ; 1.3 and ; 1.6 at dose levels of 0.5 and 1.0 mM, respectively, after 3-h recovery and 1.2 after 9-h recovery at 0.5 mM dose-level ŽTable 2.. 3.2. Chromosomal effects of a G1 treatment with the inhibitors of DNA topoisomerase II, VP-16 and amsacrine and SCE analysis SNW646, KO375 and DJG were separately exposed to VP-16 Ž0.5 and 1.0 mM. and amsacrine Ž1.0 or 2.0 mM. for 2 h and the cells were harvested after 21 or 24 h of treatment. Table 3 summarises the MI, frequencies of M2 cells, the percentage of abnormal cells, the total number of chromosomal aberrations as well as the frequencies of the dicentrics. Only the data for KO375 cells are presented because KO375 and DJG behave in a similar manner. Contrary to the results obtained at G2 stage, all the parameters studied were found to be essentially similar for both agents. A higher induction of chromosomal damage was observed only for amsacrine at the later sampling time in both cell lines ŽTable 3.. No statistical differences ŽStudent t-test, p ) 0.05.
Table 4 SCE induction by VP-16 or amsacrine treatments in SNW646 and KO375 cells Cells were harvested with colcemid added in the last 3 h. Treatmentsa
MI Ž%. M2 Ž%. SCEsrcell Ž"S.D..
VP-16 27 h MediumqBrdUrd 10.4 9.4 27 h 0.01 mMqBrdUrd 7.5 7.74 27 h 0.02 mMqBrdUrd 5.1 5.1 27 h 0.05 mMqBrdUrd 3 2.8
72 74 65 63 50 51 36 36
4.5"1.82 4.3"1.19 6.9"3.00 7.6"3.05 9.0"3.42 9.2"3.47 9.0"3.50 11.0"4.62U
Amsacrine 27 h MediumqBrdUrd 10.4 9.4 27 h 0.02 mMqBrdUrd 4 4.2 27 h 0.05 mMqBrdUrd 3 2.3 27 h 0.1 mMqBrdUrd 4 3.2
72 74 50 50 36 31 5 3
4.5"1.82 4.3"1.19 9.9"3.85 8.6"4.00 12.4"4.00 13.2"4.41 20.2"5.20 21.7"5.61
a
For each experimental point, the values of the SNW646 and KO375 cell lines were reported in the first and second rows, respectively, of each entry. U Statistically significant p- 0.05 ŽStudent’s t-test..
were observed for the induction of SCEs between the SNW646 and KO375 cells either for VP-16 or amsacrine treatment, even if a dose-dependent SCE induction was observed for each cell line ŽTable 4.. These results indicate that the WS cells are hypersensitive to topoisomerase II inhibitors only at the G2 stage of the cell cycle.
4. Discussion In this study, we demonstrate that the lymphoblastoid cells from two different WS patients are hypersensitive to treatment with topoisomerase II inhibitors. The increased sensitivity of WS cells was observed only at the G2 phase of the cell cycle, as there was no difference in the induction of chromosomal damage between normal and WS cells at G1 or S phase. This G2 sensitivity appeared to be higher and almost only present in cells collected during the 3 h treatment with both topoisomerase II inhibitors.
P. Pichierri et al.r Mutation Research 459 (2000) 123–133
This G2 sensitivity was lost in cells harvested at different recovery times after VP-16 or amsacrine treatment. These results indicate that there is a critical time point in the cell cycle, which sensitises the WS cells towards increased DNA damage after treatment with topoisomerase II inhibitors. These findings further suggest the presence of some specific G2 phase repair process that could be impaired in WS cells, leading to higher induction of chromosomal damage after treatment with topoisomerase II inhibitors. Recent studies have shown specific interaction between SGS1 Žyeast homologue of human WRNp. and the topoisomerases and that this interaction may be critical for chromosome segregation and integrity w13,22x. The finding of increased chromosome missegregation in both mitosis and meiosis in yeast sgs1 mutants, similar to WS cells, raises the possibility that the WRN gene products may also interact with topoisomerases. Although WS cells do not show enhanced sensitivity to agents like bleomycin and X-rays, that predominantly cause DNA double strand breaks ŽDSBs., the increase in nonhomologous recombination in WS cells tend to suggest some kind of deficiency in the repair of DSBs and the error prone ligation activity, leading to large genomic deletions w23x. It has been proposed recently that topoisomerase II together with SGS1 might control hyper-recombination w24x. In view of the role of topoisomerase II in recombination, it is reasonable to assume that the enhanced sensitivity of WS to topoisomerase II inhibitors may be due to altered recombination activities. WS cells have been shown to be sensitive to both topoisomerase I and II inhibitors w25,26x. A recent study has shown that the WS cells are more prone to undergo apoptosis following treatment with camptothecin, a topoisomerase I inhibitor w27x. In the present study, we demonstrate that the WS cells are hypersensitive to topoisomerase II inhibitors in the G2 stage, but not in G1 or S phase. This enhanced sensitivity to topoisomerase inhibitors appear to be mediated primarily by the WRN gene mutationŽs. rather than by secondary effect, as the murine embryonic stem ŽES. cells, carrying a deletion mutation in the WRN helicase domain, are also sensitive to topoisomerase inhibitors w28x. It is possible that the WRN gene is involved in the recombination repair
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pathway through its interaction with topoisomerase II. It is not clear why the WS cells show enhanced sensitivity to topoisomerase II inhibitors only in the G2 stage. Inhibition of topoisomerase II activity induced by VP-16 or amsacrine might enhance misrepair of DSBs thereby affecting a G2 dependent recombinational process w29x. Aratani et al. w30x reported that treatments with VP-16 or amsacrine effectively enhance the illegitimate mitotic recombination through the inhibition of the topoisomerase II activity. In WS cells, the inhibition of the topoisomerase II activity, together with loss of WRN gene function, may result in a higher rate of misrepair and increased chromosomal damage. Once the inhibition of the topoisomerase II activity is removed by washing off the inhibitors from the cultures, WS cells restored a normal capacity to repair DSBs. This might explain the abrogation of the higher G2 sensitivity to the two inhibitors induction of chromosomal damage observed in cells harvested after the recovery times. Another likely explanation for the enhanced sensitivity is that the topoisomerase inhibitors cause a higher level of DSBs in WS cells because of a defect in the topoisomerase II activity itself or in chromatin structure. However, these possibilities appear less probable because of the similar sensitivity of WS cells to the two anti-topoisomerase II drugs in the G1 phase, as well as to X-ray-induced chromosomal damage reported elsewhere w31x. The G2 sensitivity of the WS cells to VP-16 or amsacrine may not be attributable to a defect in the G2 checkpoint response, as no clear differences in the mitotic inhibition, caused by the two agents, was detected in the WS cell line as compared to the normal cells. Additionally, data reported in literature support the hypothesis that in WS cells the G2 checkpoint response to DNA damage is normal w20,31x. On the basis of our observations, we speculate that the WRN helicase, together with topoisomerases, may mediate an effective recombinational repair pathway, operating in the G2 phase of the cell cycle, prior to the onset of mitosis. Disruption of the interaction between WRN helicase and topoisomerases may predispose the cells to hypersensitivity due to increased illegitimate recombination, resulting from the error prone joining of DNA strand breaks
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induced by topoisomerase II inhibition. However, the precise mechanism responsible for enhancing the sensitivity of WS cells to topoisomerase inhibitors is not clearly understood and requires further detailed investigations.
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Acknowledgements
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The skilful technical collaboration of Mr. A. Schinoppi is highly appreciated. The financial assistance for this work from MURST grants and EC contract No. F14P-CT95-0001 is greatly acknowledged. The authors wish to thank Dr. J. Crawley for her helpful review of the English of this manuscript. This paper is dedicated to Prof. Gian Tommaso Scarascia Mugnozza on the occasion of his retirement.
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