Structural chromosomal rearrangements in HpaII-treated human lymphocytes

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Mutation Research, 248 (1991) 115-121 © 1991 Elsevier Science Publishers B.V. 0027-5107/91/$03.50 ADONIS 002751079100100Q

MUT 04964

Structural chromosomal rearrangements in HpalI-treated human lymphocytes Bruna Tedeschi, Berardino Porfirio, Daniela Caporossi, Patrizia Vernole and Benedetto Nicoletti Department of Public Health and Cell Biology, H University of Rome "Tor Vergata', via O. Raimondo, 1-00173 Rome (Italy) (Received 30 July 1990) (Revision received 22 October 1990) (Accepted 29 October 1990)

Keywords: Chromosome rearrangement; CG sequences; Fragile sites; HpalI

Summary Restriction endonucleases have been shown to induce chromosome damage in a variety of cultured cells. We recently reported the coincidence between MspI-induced breakage and the location of common fragile sites. We have extended our study to HpaII, which induced a 4.5-fold increase in total breakage compared to controls. It appeared that a major contribution was given by stable chromosome rearrangements, which were present at a 14-fold increased frequency in comparison to the spontaneous levels. Moreover, several chromosome bands were involved in rearrangements in different cultures from different donors. Notably, HpaII-induced breakage occurred in the same bands where breakpoints of constitutional and neoplastic rearrangements are located.

Previous studies concerning the mechanism of the origin of chromosome aberrations (Bryant et al., 1984; Natarajan and Obe, 1984) have demonstrated that restriction endonucleases have clastogenic effects in cultured mammalian cells. We recently reported the non-random distribution of MspI-induced breaks in human lymphocyte chromosomes (Porfirio et al., 1989). Our resuits pointed out that bands preferentially involved in breakage are likely to be coincident with

Correspondence: B. Tedeschi, Department of Public Health and Cell Biology, II University of Rome 'Tor Vergata', via O. Raimondo, 1-00173 Rome (Italy).

bands where common fragile sites (cFS) are located. cFS are ubiquitous in cultured human lymphocytes and are expressed in vitro by several chemicals so that most of them have been considered to be specific targets of mutagens and carcinogens (Yunis et al., 1987). Furthermore, cFS are located in bands which are largely coincident with structural rearrangements in leukemia and cancer (De Braekeleer, 1987; Yunis, 1987). These reports have supported the hypothesis that cFS may be structurally related to specific D N A sequences such as regulatory GC-rich regions, which flank active genes in G-light bands. Indeeed, the positive correlation we found between MspI-sensitive bands and cFS seems to indicate a relationship between

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repeated CpG sequences and chromosome hot spots in human lymphocytes. We extended our study to HpaII to assess the influence of methylation at C C G G sites on the expression of this fragility. It is known that the CpG dinucleotide is less than predicted by base composition in the bulk of vertebrate DNA. Since most of the cytosines are methylated in this position, with the notable exception of C + G-rich islands, the action of HpaII should be expected to be highly targeted. In fact, we found that HpaII induced chromosome breakage in a subset of MspI-sensitive bands. These bands correlated with cFS band location. Furthermore, the large number of structural rearrangements induced by HpaII in our experimental system involved bands frequently rearranged in neoplasia as well as in constitutional chromosome anomalies. Materials and methods

The experimental procedures were essentially the same as used for the study of MspI-induced breakage (Porfirio et al., 1989). In particular, human peripheral blood lymphocytes from 4 male donors were recovered from a Ficoll-Hypaque gradient. Triplicate cultures from each subject were set up at a concentration of 5 × 105 cells/ml medium (RPMI 1640 supplemented with 20% fetal calf serum (FCS), 1% phytohemagglutinin, and antibiotics) in 10-ml plastic tubes and incubated for 72 h at 37°C in a 5% CO 2 atmosphere. 20 h before harvesting, the cells were spun down and the medium was kept as conditioned medium. Lymphocytes were washed with 1 ml of FCS and pelleted. The treatment consisted in adding 120 U HpaII (Boehringer, 10,000 U / m l ) to cells adjusted with FCS in order to keep the final volume of the HpaII solution below 10%. Following 15 rain at 37°C, the reaction was stopped by adding the conditioned medium. Control cultures were mock-treated with HpaII shipping buffer (0.5 m M EDTA, 50 mM KC1, 20 mM Tris-HC1, pH 7.5, 5 mM 2-mercaptoethanol, 50% glycerol). Slides were prepared according to standard procedures and G-banding was produced by acetic saline using Giemsa (GAG; Sumner et al., 1971) for the analysis of the breakage rate and for the

localization of breakpoints. As many metaphases as possible were scored, and those showing chromosome-type aberrations were karyotyped. The aberrations were classified according to the International System for Cytogenetic Nomenclature (ISCN, 1981) and the breakpoints detected in the 8 HpalI-treated cultures were assigned to a 319band haploid karyotype. Poisson distribution was used to estimate the minimal number of events at a given band to be considered as evidence of preferential involvement in breakage by HpaII. Results

In total, 3769 GAG-banded metaphases were scored from the 8 HpaII-treated cultures. Table 1 shows the types and frequencies of chromosomal aberrations observed. Despite intra- and inter-individual variations, the breakage rates were considerably higher in HpaII-treated cultures than in controls. Furthermore, stable chromosomal rearrangements were present at high frequency only in the cultures treated with the enzyme. The occurrence of chromosome-type aberrations is consistent with those cells being in G 1 at the time of the treatment. Representative aberrations are displayed in Fig. 1. Two hundred and twenty-eight breakpoints were localized at a 319-band resolution level after karyotyping (Fig. 2). Breakpoints were involved in reciprocal translocations (9), peri- and para-centric inversions (6), dicentrics (8), rings (1), terminal deletions (38), isochromatid/chromosome breaks (116) and triradials (13). The average number of breaks per band in the sample was 0.7 (228/319). According to the Poisson distribution, any band with 4 or more breaks had a higher than random frequency of breakage ( p < 0.005). The 18 bands satisfying this condition were: lp36, lq12, 3p21, 3q21, 4q12, 5q31, 6p21, 7p22, 9q12, 9q13, 9q22, 11q23, 12p13, 12q24, 17q21, 19q13, 21q11, 22q13. Table 2 shows that all these bands but one (21q11) occurred in different cultures from one or more different donors. Since the possibility exists that breaks at 2 1 q l l are due to cloning from less than 4 independent events, we did not consider this band in further analyses. A cloning contribu-

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Fig. 1. Chromosomal aberrations induced by 120 U HpalI in human lymphocytes. Arrows indicate: in (a), a complex translocation, t(12;14;20Xq22;pll;q13), and chromosome break in 9q13; in (b), a dicentric, dicl2pl3 : : lp36; in (c), a deletion, del(7)(pl3 ---, pter); in (d), uncoiling of 9q12. (e) Multiple aberration metaphase. (f) Heavily damaged metaphase.

tion could be postulated also for the breaks seen in 9q12, but since this band was the site of breakage in different cultures from different donors, it has to be considered HpalI-sensifive.

The 17 HpalI-sensitive bands were matched with the following relevant categories (Table 3): (1) MspI-sensitive bands (Porfirio et al., 1989); (2) very GC-rich bands (Holmquist, 1989); (3) cFS

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Fig. 2. Distribution of the 228 localized breakpoints on the 319-band karyotype diagram, o: localization of the breakage. The number of breaks observed in each band is indicated.

bands (Sutherland and Ledbetter, 1989); (4) protooncogene localization (Trent et al., 1989); (5) breakpoints of structural chromosome changes in neoplasia (Trent et al., 1989); (6) breakpoints in

constitutional chromosome aberrations (Porfirio et al., 1987; Koduru and Chaganti, 1988) and in microdeletion syndromes (Harper et al., 1989). The expected number of bands jointly occupied

119 TABLE 1 CHROMOSOME ABERRATIONS I N D U C E D BY 120 U 20 h)

HpalI IN

H U M A N LYMPHOCYTE CULTURES (RECOVERY TIME

Donor/ culture

Treatment

Number of cells

CTB

CSB

CTE

CSE

Breakage rate a

Percent rearrangements b

la lb lc 2a 2b 2c 3a 3b 3c 4a 4b 4c

HpalI Hpall

370 100 350 395 270 c 100 450 c 340 107 1163 c 681 100

13 3 12 13 19 1 17 7 0 14 7 0

9 3 1 39 18 2 35 12 1 27 11 0

1 0 0 7 2 0 1 0 0 0 0 0

6 3 0 7 2 0 4 6 0 11 3 0

0.097 0.120 0.037 0.203 0.167 0.030 0.138 0.091 0.009 0.054 0.035 0.000

1.9 3.0 0.0 3.5 1.5 0.0 1.1 1.8 0.0 0.9 0.4 0.0

buffer

HpalI HpalI buffer

HpalI Hpall buffer

HpalI HpalI buffer

CTB, chromatid breaks; CSB, isochromatid/chromosome breaks; CTE, chromatid-type exchanges; CSE, chromosome-type exchanges. a CTE and CSE were scored as two-break events. b Each rearrangement was considered as a unit. c One cell had too many breaks to be accurately determined and was not further considered.

by chance alone was calculated according to the formula

Exp = Yi" k/N TABLE 2 O C C U R R E N C E OF BREAKPOINTS IN THE TREATED CULTURES AT 18 SENSITIVE BANDS Breakpoints in bands lp36 lq12 3p21 3q21 4q12 5q31 6p21 7p22 9q12 9q13 9q22 11q23 12p13 12q24 17q21 19q13 21qll 22q13

Within cultures

Between cultures within individuals + + + + + + + + +

HpalI-

Among individuals + + + + + + + + + + + + +

+ + + + +

where Yi is the total number of bands belonging to categories 1-6, k and N being the constant 17 HpaII-sensitive bands and 319 bands of the haploid karyotype, respectively. Indeed, we found a highly significant coincidence with all the categories examined, with p values ranging between 0.005 and 0.001 (Table 3). Discussion The main finding in this report is that HpalI can induce non-random chromosome breakage clustered in G-light bands in human lymphocyte cultures (Fig. 2). Mutagens and carcinogens active on C + G-rich DNA sequences have been shown to break human chromosomes at cFS (Yunis et al., 1987) which are mostly located on G-light bands (Hecht, 1989; Sutherland and Ledbetter, 1989). In our previous work on the clastogenic action of MspI, we found a strong correlation between cFS location and bands prone to breakage by the restriction enzyme (Porfirio et al., 1989). The present results with HpalI confirm the specific action at the cytogenetic level of this isoschizomeric pair indicating that tandemly repeated CCGG sequences are candidate targets for preferential in vivo cleavage.

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TABLE 3 CONCORDANCE OF 17 HpaII-SENSITIVEBANDS WITH 6 CATEGORIESOF BANDS Category

Number of bands

Number of coincident bands Observed Expected

X2

(1) Mspl (2) vGC (3) cFS (4) Oncogenes (5) Neoplasia (6) Constitutional

160 39 87 47 126 74

17 l1 12 8 14 11

8.50 37.72 11.90 12.10 7.95 12.92

8.5 2.1 4.6 2.5 6.7 3.9

< 0.005 < 0.001 < 0.001 < 0.001 < 0.005 < 0.001

MspI, Mspl-sensitive bands; vGC, very GC-rich bands; cFS, common fragile site bands; Oncogenes, protooncogenelocalization: Neoplasia, bands where breakpoints of neoplastic chromosomerearrangements are located; Constitutional, bands where breakpoints of constitutional chromosomerearrangements and microdeletions are located.

In fact, all HpaII-sensitive bands were sites of MspI action under the same treatment (Porfirio et al., 1989). Furthermore, a positive correlation was found with bands known to be very CG-rich (Holmquist, 1989) ( p < 0.001), which further confirms that the sensitivity to HpaII is based on highly repeated C C G G sequences. These sequences are particularly frequent in the unmethylated domains referred to as the CpG islands (Bird, 1986). They are non-randomly distributed in the human genome, rather they have been described to be associated with all the housekeeping genes isolated so far and with many tissue-specific genes ( G a r d i n e r - G a r d n e r and Frommer, 1987). Recently, Mattes et al. (1988) found that 5'-flanking regions of oncogenes and growth factor genes are rich in CG runs. These authors also reported these sequences in the 5'flanking region of the c-Ha-ras gene as preferred sites of in vitro alkylation and hypothesized that induced damage could promote chromosome rearrangements, leading to recombinant genes with oncogenic potential. Structural rearrangements were induced by HpaII at a mean frequency of 1.4%, which is an order of magnitude higher than the spontaneous rate (Anderson et al., 1988; Bender et al., 1988), and were mainly clustered on HpaII-sensitive bands. It is important to note that these bands were positively correlated with bands where structural chromosome rearrangements in neo-

plasia ( p < 0.005) and constitutional chromosome aberrations ( p < 0.001) are located. It has already been reported that chromosome rearrangements are induced by aphidicolin mainly in bands where cFS are located (Yunis et al., 1987; Glover and Stein, 1988) and many cFS have also been found in chromosome bands involved in evolutionary structural rearrangements (Mir6 et al., 1987). We often found, besides the breakage at band 9q12, the uncoiling of 9qh that mimicked true pericentric inversion, in independent HpaIItreated cultures from different donors (Fig. 1). It is well known that pericentric inversions of the heterochromatic region lq12 and mostly 9q12 occur frequently in humans (Kaiser, 1984). The significant clustering of chromosome damage induced by HpaII in these A + T-rich regions seems to support the presence of interspersed CG base pairs as already suggested by Miller (1983) to explain the quenching of quinacrine fluorescence in these regions. The specific HpaII-induced breakage in chromosomes of human lymphocytes strengthens previous hypotheses that cFS are located in regions particularly rich in C + G sequences. Such sequences, as preferential targets of mutagens, seem to be involved in inter- and intra-chromosomal rearrangements eventually leading to recombinant events at the gene level. Therefore, common fragile sites could be the cytogenetic expression of the proneness to breakage of specific CpG-rich re-

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gions. Molecular analysis of DNA sequences involved in breakage would certainly help to clarify these cytogenetic findings.

Note added in proof After submission of our manuscript, we read the paper of Winegar et al. (1990), who reached the same conclusions as we did about the maintenance of the putative specificity and the different effectiveness of the isoschizomer pair MspI/Hpa II in inducing chromosomal aberrations in electrophorated Chinese hamster ovary cells.

Acknowledgements We thank Mr. G. Bonelli and Miss F. Geremia for technical assistance and graphic help. This work was supported in part by grants from the Ministry of Education.

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