Distribution of UVC-induced chromosome aberrations along the X chromosome of TCR deficient and proficient Chinese hamster cell lines

Mutation Research 701 (2010) 98–102

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Distribution of UVC-induced chromosome aberrations along the X chromosome of TCR deficient and proficient Chinese hamster cell lines ˜ a , F. Palitti b W. Martínez-López a,b,∗ , E. Marotta b , M.V. Di Tomaso a,c , L. Méndez-Acuna a b c

Epigenetics and Genomic Instability Laboratory, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay Department of Agrobiology and Agrochemistry, Università degli Studi della Tuscia, Viterbo, Italy Genetics Department, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay

a r t i c l e

i n f o

Article history: Received 9 February 2010 Accepted 10 February 2010 Available online 20 February 2010 Keywords: UVC NER Cockayne’s syndrome B Chromosome breakpoints S-phase BrdUrd labeling

a b s t r a c t Cells with a transcription coupled repair (TCR) deficiency are characterized by a higher sensitivity to UVC irradiation and by an increase in apoptosis and chromosomal aberration frequencies. It has been claimed that the higher frequency of chromosomal aberrations results from the transcription blockage caused by UVC-lesions located in the transcribed strands of the genome. The distribution of chromosome breakpoints in euchromatic and heterochromatic regions of the X chromosome from TCR deficient and proficient Chinese hamster cell lines was studied. Most UVC-induced breakpoints occurred in euchromatic regions of the X chromosome in both cell lines. No increase of UVC-induced breakpoints in the euchromatic region of the UV61 X chromosome was observed, indicating that TCR failure alone cannot be responsible for the increased frequency of chromosomal aberrations. Differential chromatin remodeling in the TCR defective cell line is proposed as a possible mechanism involved in the distribution of UVC-induced breakpoints along the Chinese hamster X chromosome. A similar explanation for the increase of UVC-induced chromosomal aberrations in TCR defective cells is given. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Cyclobutane pyrimidine dimers (CPDs) and pyrimidinepyrimidone 6-4 photoproducts (6-4PP) are the main lesions induced by UVC. About 80% of the total DNA damage are CPDs [1]. Nucleotide excision repair (NER) is one of the major cellular pathways that remove bulky lesions induced by UVC. Such lesions, if either unrepaired or misrepaired, interfere with essential DNA metabolic activities resulting in mutations, chromosomal instability and cell death [2]. Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) show the dramatic consequences of NER deficiencies. Patients with these syndromes are extremely photosensitive and exhibit skeletal as well as neurological abnormalities. CS gene products are components of the transcription coupled repair (TCR) subpathway of NER which is responsible for removing UVC-lesions from the transcribed regions of the genome [3]. CS cells exhibit elevated frequencies of chromosomal aberrations and an enhanced apoptotic potential after UVC irradiation [4]. Since actively transcribing genes constitute only 5–8% of the

∗ Corresponding author at: Epigenetics and Genomic Instability Laboratory, Instituto de Investigaciones Biológicas Clemente Estable, Avenida Italia 3318, CP 11.600, Montevideo, Uruguay. Tel.: +598 2 487 16 21 136; fax: +598 2 487 55 48. E-mail address: [email protected] (W. Martínez-López). 1383-5718/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2010.02.008

eukaryotic genome, lesions repaired by TCR in these regions might be minimal as compared to the remainder of the genome and therefore only a slight increase in apoptosis and chromosomal aberrations in TCR defective cells as compared to normal cells can be expected. Nevertheless, significant increase (3–5 folds) in the induction of both chromosomal aberrations and apoptosis was observed in CS cells upon UVC irradiation [5,6]. AA8 and UV61 are isogenic Chinese hamster cell lines which differ in TCR efficiency. AA8 is the parental cell line, which is proficient in the repair of UVC-induced lesions from the transcribing strand of active genes. UV61 cells (the hamster homologue of human Cockayne syndrome B) originally isolated from AA8 proficiently repair 6-4 PP but are deficient in CPDs removal by the TCR pathway. As TCR of CPDs is the only difference between AA8 and UV61 cell lines, it can be assumed that the increased level of chromosomal aberrations is mainly due to the lack of CPDs removal from the transcribing strand of active genes. Misrepaired CPDs are thought to be responsible for the formation of chromosomal aberrations during S-phase of the cell cycle. Therefore, it is expected that euchromatic chromosome regions of UV61 should be hot spots for the formation of chromosomal aberrations. Chinese hamster X chromosomes have a short euchromatic arm and an almost entirely heterochromatic long arm, which can be easily identify by substitution with 5-bromo-2 -deoxyuridine (BrdUrd) incorporation and indirect anti-BrdUrd immunolabelling during S-

W. Martínez-López et al. / Mutation Research 701 (2010) 98–102

phase of the cell cycle [7–9]. This allows chromosome lesions to be assigned to early or late replicating regions. In order to test if UVC-induced chromosome lesions are clustered in euchromatic regions of UV61 X chromosomes, the distribution of 100 chromosome breakpoints induced by UVC irradiation along the euchromatic and heterochromatic regions of the X chromosome of AA8 and UV61 cells treated during early S-phase of the cell cycle was studied.

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to avoid that PI entered into the nucleus. Since FDA and HO are vital dyes staining the cytoplasm and the nucleus of viable cells, respectively, it was possible to detect normal and early apoptotic cells by observing the characteristic pattern of chromatin fragmentation in the nucleus (HO staining) of cells containing FDA in their cytoplasm. Since PI does not enter into living cells, it was possible to detect necrotic cells as well as late stages of apoptosis (when cell membranes have been degraded) by just observing an entire or fragmented PI stained nucleus, respectively.

2. Material and methods 2.3. Immunodetection of incorporated BrdUrd 2.1. Cell culture and treatments Cells were grown as monolayers in Mc Coy’s 5A culture medium (Gibco) supplemented with 10% foetal bovine serum (Gibco), 200 mM glutamine (Sigma) and antibiotics (100 U/ml penicillin and 125 ␮g/ml dihydrostreptomycin sulphate, Sigma) at 37 ◦ C in a 5% CO2 incubator. Twelve hours before harvesting, exponentially growing cells (2 × 106 ) were irradiated with UVC at 10 J/m2 (AA8) or 4 J/m2 (UV61) and immediately treated for 30 min with 30 ␮M BrdUrd (Sigma). Controls were processed in the same way except for UVC exposures. Following 2 h of exposure to 0.08 ␮g/ml colcemid, mitotic cells were harvested by shake-off, hypotonically treated with 1% sodium citrate for 10 min at 37 ◦ C and fixed twice in methanol–acetic acid (3:1). Cells were dropped onto clean ice-cold slides and air-dried. 2.2. Apoptosis detection by fluorescence staining Exponentially growing AA8 and UV61 cells were irradiated with 10 or 4 J/m2 , respectively, and recovered after 12 h of UVC irradiation. Apoptotic cells were detected by fluorescence microscopy employing a combination of fluorescein di-acetate (FDA, 5 ␮g/ml, Sigma), propidium iodide (PI, 0.5 ␮g/ml, Sigma) and Hoechst 33342 (HO, 1 ␮g/ml, Sigma). In brief, cells were trypsinized, washed once in PBS and resuspended in a mixture of FDA, HO and PI solution. Then, they were kept at 37 ◦ C for 5 min and seeded onto a slide. At least 500 cells were counted in the following 15 min in order

Slides were denatured with 10 mM NaOH in 70% ethanol (1 min), dehydrated in 70%, 90% and 100% ethanol and air-dried. Slides were then incubated in 100 ␮l of 1:100 anti-BrdUrd antibody (Boehringer) in immunological buffer (PBS, 10% BSA, 0.5% Tween 20) for 30 min at 37 ◦ C in a moist chamber. Slides were washed (3×) with PBS and incubated in the dark with 100 ␮l of goat anti-mouse IgG-FITC (Boehringer) for 30 min (37 ◦ C) in a moist chamber. Finally, slides were washed with PBS (3×), dehydrated in 70%, 90% and 100% ethanol (5 min each) and counterstained with 0.35 ␮g/mL PI diluted in antifade (glycerol, 1,4 diazabicyclo [2.2.2] octane, sodium azide and 0.2 M Tris/HCl pH 7.5) and analysed under a fluorescence microscope. 2.4. Chromosomal aberrations and breakpoint assigments on the X chromosome Aberration frequencies induced by UVC irradiation in AA8 and UV61 cells were determined in control and treated metaphases (n = 100 metaphases with 18–22 centromeres per treatment). The following aberrations were recorded: chromatid interchanges, isochromatid–chromatid interchanges, chromatid and isochromatid breaks, interstitial and duplication deletions, intrachanges, dicentrics, translocations and rings [10]. Different doses of UVC irradiation were assayed (10, 11 or 12 J/m2 in AA8 and 4, 5 or 6 J/m2 in UV61) to determine which doses produced similar frequency of chromosomal aberrations in both cell lines. A total of 200 BrdUrd-labeled metaphases were scored per

Fig. 1. BrdUrd immunolabelling (yellow) of the Chinese hamster X chromosome counterstained with propidium iodide (red). (a) ES: early S-phase; (b) LS: late S-phase; (c) UL: unlabeled. Chromosomal aberrations involving the X chromosome from ES metaphases: (d) quadrirradial and (e) trirradial involving the short arm of the X chromosome. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Table 1 Frequencies of chromatid type aberrations induced by UVC (10 or 4 J/m2 ) in early S-phase (12 h recovery time) of AA8 or UV61 cells, respectively. Number of breakpoints were estimated according to Obe and Winkel [13] as follows: dicentric (DIC) = 2; chromatid break (B ) = 1; chromatid interchange (RB ) = 2; chromatid-isochromatid interchange (RB B ) = 3; isochromatid break (B ) = 2; duplication deletion (DD) = 3; interstitial deletion (ID) = 2; chromatid ring (IC) = 2. HDC = heavily damaged cells. Pooled data from three independent experiments. Treatment

AA8 control AA8 10 J/m2 UV61 control UV61 4 J/m2

Percentage of damaged metaphases

4 46 5 44

No. of

No. of

DIC

Aberrations per 100 metaphases RB

RB B

B

B

ID

DD

IC

HDC

Breaks

– – – –

– 12 1 10

– – – –

4 102 4 71

1 6 1 16

– 2 – –

– 2 – 2

– 4 – 2

– – – –

6 156 8 133

Table 2 Frequencies of damaged (D) and undamaged (U) BrdUrd-labeled metaphases. AA8 and UV61 cells were treated with 10 or 4 J/m2 UVC irradiation, respectively. Immediately thereafter cells were pulse-labeled with BrdUrd. After a recovery time of 12 h metaphase spreads were prepared. Based on the BrdUrd incorporation in the X chromosome the cell cycle stage at exposure was determined (200 metaphases per treatment were analysed). Treatment

AA8 10 J/m2 UV61 4 J/m2

Early S-phase

Middle S-phase

Late S-phase

Unlabeled

Metaphases

D

U

D

U

D

U

D

U

52 42

34 48

4 –

2 6

2 –

2 –

– –

2 4

treatment to determine the percentage of damaged or undamaged cells in early, mid or late S-phase. The distribution of 100 chromosome breakpoints induced by UVC in the euchromatic and heterochromatic regions of the X chromosome were analysed in immunolabeled metaphases of AA8 and UV61 cells with an Axiophot microscope (Zeiss) with appropriate filter sets for FITC and PI. 2.5. Statistical analysis Expected frequencies of breakpoints for each X chromosome arm were estimated according to arm length [11]. Since the intercalary secondary constriction in the long arm of the X chromosome is situated in an euchromatic region [12] and can be easily detected with the BrdUrd immunostaining technique (Fig. 1), breakpoints in the secondary constriction of the long arm were considered as breakpoints occurring in the euchromatic region for statistical analysis. In order to test for random or non-random occurrence of breakpoints produced by UVC irradiation in euchromatic or heterochromatic regions of the X chromosome, a 2 -test ( = 1) was used. To compare the amount of induced breakpoints in the euchromatic region of the X chromosome from both cell lines, a two-tailed two-proportion z-test was used. 3. Results Different doses were applied to obtain the same amount of chromosomal aberrations induced by UVC in AA8 and UV61 cells. Results obtained by scoring 100 metaphases from AA8 and UV61 cells exposed to 10 and 4 J/m2 of UVC, respectively, are presented in Table 1. Mainly chromatid type aberrations were observed in both cell lines. The percentages of damaged cells were 46 and 44 for AA8 and UV61 UVC-irradiated cells with only 4 and 5 in controls, respectively. Similar frequencies of chromosomal aberrations and numbers of breakpoints were observed following exposure to 10 J/m2 for AA8 and 4 J/m2 for UV61. In order to determine how many cells were exposed at early or late S-phase after 12 h of recovery following UVC irradiation, cells were pulse labeled for 30 min with BrdUrd after UVC irradiation. Immunodetection of BrdUrd incorporation in the X chromosome enabled treated cells to be assigned precisely to early, middle or final stages of the S-phase. Early S-phase cells showed BrdUrd immunolabeling in the short euchromatic arm. Cells were classified as late S-phase when immunostaining was limited to the long

heterochromatic arm (Fig. 1). The presence of fluorescent labeling in both arms of the X chromosome indicated that the cell was in mid S-phase. As shown in Table 2, BrdUrd-labeled damaged metaphases observed at 12 h recovery time were mainly exposed in early Sphase of both cell lines (Fig. 1). Since UV61 is prone to induce apoptosis after UVC irradiation, the frequencies of apoptosis in AA8 and UV61 were determined to check if the apoptotic process would influence the analysis of the distribution of chromosome breakpoints in the X chromosome. The morphological analysis of apoptoses showed that 10 J/m2 of UVC-induced 1.2 ± 0.3% of apoptotic cells in AA8 and 4 J/m2 of UVCinduced 9.3 ± 1.8% of apoptotic cells in UV61 while controls did not show any significant increase in the frequency of apoptoses. Although an increase in the frequency of apoptoses in UV61 after UVC irradiation in comparison to AA8 was observed, this should not influence chromosome breakpoint distribution since no more than 10% of UV61 cells were affected. To analyse the distribution of UVC-induced chromosome breakpoints in the euchromatic and heterochromatic regions of the X chromosome in AA8 and UV61, 100 breakpoints were assigned to both regions in cells treated during early S-phase of the cell cycle. A non-random distribution in euchromatic and heterochromatic regions of the X chromosomes was found for both cell lines (2 -test, p < 0.001 for AA8 and p < 0.005 for UV61). The amount of breakpoints observed in the euchromatic region of AA8 cells was not statistically different from that in the euchromatic region of UV61 cells (two-proportion z-test, p-value >0.05) (Table 3). 4. Discussion A higher chromosomal instability has been observed in the hamster homologue of Cockayne Syndrome B Chinese hamster cell line UV61 as compared to its parental cell line AA8. This higher sensitivity was attributed to the lack of CSB protein. CSB is part of the TCR subpathway of NER, responsible for removing CPDs from the transcribed strand (see later for detailed description). In this respect, it was proposed that the induction of chromosomal aberrations results from the transcription blockage caused by the CPDs in transcribed strands of the genome, especially because in hamster cells the global genome repair subpathway of NER is not active [5,6]. In the present work, we have shown that similar frequencies of chromosomal aberrations induced by 10 or 4 J/m2 of UVC in AA8 and UV61, respectively, were observed. Chromosome breakpoints

W. Martínez-López et al. / Mutation Research 701 (2010) 98–102 Table 3 Frequency of breakpoints found in the euchromatic (E) and heterochromatic (H) regions of the X chromosome from AA8 and UV61 cell lines exposed to 10 or 4 J/m2 , respectively, during early S-phase. Expected values and the statistical analysis (2 ) of breakpoints distribution along the euchromatic and heterochromatic regions of the X chromosome from AA8 and UV61 cell lines are shown. The p-value obtained with the application of two-tailed two-proportion z-test is shown (P1 = 0.66; P2 = 0.59) / P2 ). (H0: P1 = P2 ; H1: P1 = Breakpoints

Expected values

2 9.80 8.02 17.82*

AA8 AA8 Total

E H

66 34 100

45 55 100

UV61 UV61 Total

E H

59 41 100

45 55 100

Two-proportion z-test

p-Value

* **

4.36 3.56 7.92**

0.3066

p < 0.001. p < 0.005.

were mainly distributed in the more active chromatin regions of the X chromosome in both cell lines. It was claimed that CPDs in vivo are concentrated in transcription factor binding sites, which could lead to hot spots for UVC-induced lesions. Incomplete repair of these sites could lead to mutations [14]. Pfeifer and Riggs [15] showed that photofootprints occur only in regions which are also footprinted by DNase I. Moreover, it was shown that many sequence-specific transcription factors on the active, but not on the inactive human X chromosome, are recognizable by genomic DNase I footprinting. The inactive human X chromosome, which lacks transcription factors at the same sequences, did not show photofootprints [15]. Although it was expected that more breakpoints were concentrated in the euchromatic region of the X chromosome in UV61 than in AA8 due to the lack of TCR, we did not find any statistical difference in the distribution of breakpoints between both cell lines. This result may indicate that TCR failure is not responsible for the increased frequency of chromosomal aberrations observed in UV61 cells. Since DNA accessibility for mutagenic agents and DNA repair proteins is limited in nucleosomes [16,17], different chromatin organization after UVC exposure in AA8 and UV61 cells could influence the distribution of CPDs in eu- and heterochromatic regions as well as their removal by NER system, leading to increased frequencies of chromosomal aberrations in UV61 cells. UVC induces chromatin relaxation dependent on histone acetyltransferase p300 to allow NER to remove UVC-lesions [18]. Furthermore, Yu et al. [19] showed that UVC irradiation triggers a genome-wide histone hyperacetylation at both histones H3 and H4. Acetylated histones could enhance chromatin accessibility either by the loss of negative charges due to acetylation of terminal lysines or by generating docking sites for different kinds of non-histone proteins [20–23]. In this respect, we have demonstrated in Chinese hamster chromosomes that acetylated histone H4 regions are preferred sites for clastogenic agents [24–27]. Alternatively, repair factors themselves could cause chromatin rearrangements. Particularly good candidates for this type of function in the NER system is the transcription coupled repair factor CSB, which is a DNAdependent ATPase of the chromatin remodeling SWI2/SNF2 family, and the TFIIH complex that contains the helicase subunits XP-D and XP-B [28,29]. The CSB protein has a key role as coupling factor to attract histone acetyltransferase p300 and nucleotide excision repair proteins. CSB mutated cells when irradiated with UVC do not attract p300 [30]. Moreover, it has been shown that histone H4 in promoter regions of non-transcribed undamaged genes are underacetylated [31]. Therefore, it can be hypothesized that in CSB mutated cells an imbalance in acetylation levels occurs, which would explain the

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accumulation of UVC DNA lesions in actively transcribed areas. The 5 fold increase in chromosomal aberrations after UVC exposure could be caused by an acetylation imbalance since NER proteins could not properly interact with DNA lesions in a less decondensed chromatin environment [5]. It seems to be clear that nucleosome remodeling has an important role in DNA repair because its failure could strongly hamper damage accessibility. Distribution of chromosome breakpoints may reflect differential chromatin folding following DNA damage insult. However, further experimental evidence will be necessary to confirm the hypothesis of differential chromatin folding in Cockayne syndrome cells as a possible factor that contribute to a higher sensitivity to UVC irradiation. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements This work was partially supported by a Marie Curie Fellowship from the European Community. We are indebted with the PEDECIBA Postgraduate Program from Uruguay. References [1] E. Friedberg, G. Walker, W. Siede, DNA Repair and Mutagenesis, ASM Press, Washington, DC, 1995. [2] J. Boer, J.H.J. Hoeijmakers, Nucleotide excision repair and human syndromes, Carcinogenesis 21 (2000) 453–460. [3] D. Bootsma, K.H. Kraemer, J.E. Cleaver, J.H.J. Hoeijmakers, Nucleotide excision repair syndromes: xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy, in: B. Vogelstein, K.W. Kinzler (Eds.), The Genetic Basis of Human Cancer, McGraw-Hill, New York, 1998, pp. 245–274. [4] A.S. Balajee, L. Proietti De Santis, R.M. Brosh, R. Selzer, V.A. Bohr, Role of the ATPase domain of the Cockayne syndrome group B protein in UV-induced apoptosis, Oncogene 19 (2000) 477–489. [5] L. Proietti De Santis, C. Lorenti-Garcia, A.S. Balajee, G.T. Brea Calvo, L. Bassi, F. Palitti, Transcription coupled repair deficiency results in increased chromosomal aberrations and apoptotic death in the UV-C61 cell line, the Chinese hamster homologue of Cockayne syndrome B, Mutation Research 485 (2001) 121–132. [6] L. Proietti De Santis, C. Lorenti-Garcia, A.S. Balajee, P. Latini, P. Pichierri, O. Nikaido, M. Stefanini, F. Palitti, Transcription coupled repair efficiency determines the cell cycle progression and apoptosis after UV exposure in hamster cells, DNA Repair 28 (2002) 209–223. [7] Y. Xiao, A.T. Natarajan, Heterogeneity of Chinese hamster X chromosomes in X-ray-induced chromosomal aberrations, Int. J. Radiat. Biol. 75 (1999) 419–427. ˜ M.V. Di Tomaso, [8] W. Martínez-López, G.A. Folle, G. Cassina, L. Méndez-Acuna, G. Obe, F. Palitti, Distribution of breakpoints induced by etoposide and X-rays along the CHO X chromosome, Cytogenet. Genome Res. 104 (2004) 182–187. [9] M.V. Di Tomaso, W. Martínez-López, G.A. Folle, F. Palitti, Modulation of chromosome damage localisation by DNA replication timing, Int. J. Radiat. Biol. 82 (2006) 877–886. [10] J.R.K. Savage, Annotation: classification and relationships of induced chromosomal structural changes, J. Med. Genet. 13 (1976) 103–122. ˜ J.R.K. Savage, G. Obe, [11] W. Martínez-López, V. Porro, G.A. Folle, L. Mendez-Acuna, Interchromosomal distribution of gamma ray-induced chromatid aberrations in Chinese hamster ovary (CHO) cells, Genet. Mol. Biol. 23 (2000) 1071–1076. [12] M. Ray, T. Mohandas, Proposed nomenclature for the Chinese hamster chromosomes (Cricetulus griseus), in: J.L. Hamerton (Ed.), Report of the Committee on Chromosome Markers, Cytogen. Cell Genet. 16 (1976) 83–91. [13] G. Obe, E.-U. Winkel, The chromosome breaking activity of restriction endonuclease AluI in CHO cells is independent of the S-phase of the cell cycle, Mutat. Res. 152 (1985) 25–29. [14] G.P. Pfeifer, R. Drouin, A.D. Riggs, G.P. Holmquist, Binding of transcription factors creates hot spots for UV photoproducts in vivo, Mol. Cell. Biol. 12 (1992) 1798–1804. [15] G.P. Pfeifer, A.D. Riggs, Chromatin differences between active and inactive X chromosomes revealed by genomic footprinting of permeabilized cells using DNaseI and ligation-mediated PCR, Genes Dev. 5 (1991) 1102–1113. [16] F. Thoma, Light and dark in chromatin repair: repair of UV-induced DNA lesions by photolyase and nucleotide excision repair, EMBO J. 18 (1999) 6585–6598. [17] F. Thoma, Repair of UV lesions in nucleosomes—intrinsic properties and remodeling, DNA Repair 4 (2005) 855–869. [18] C.P. Rubbi, J. Milner, p53 is a chromatin accessibility factor for nucleotide excision repair of DNA damage, EMBO J. 22 (2003) 975–986.

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