A Recurrent 19q11–12 Breakpoint Suggested by Cytogenetic and Fluorescence In Situ Hybridization Analysis of Three Glioblastoma Cell Lines

A Recurrent 19q11–12 Breakpoint Suggested by Cytogenetic and Fluorescence In Situ Hybridization Analysis of Three Glioblastoma Cell Lines Ivana Magnani, Elena Chiariello, Anna Maria Fuhrman Conti, and Gaetano Finocchiaro

ABSTRACT: Loss of genetic material at chromosome 19 is a rather frequent finding in malignant gliomas. Loss of heterozygosity at region 19q13.3 is common in oligodendrogliomas and is also present, together with other genetic alterations on the same chomosome, in glioblastoma multiforme (GBM). Here we describe the results of cytogenetic and fluorescence in situ hybridization analysis on three GBM cell lines in which a series of complex chromosomal rearrangements affecting chromosome 19 were present. These genetic alterations suggest the presence of a common breakpoint at 19q11–12 which may point to the localization of a fragile site and/or to the presence of tumor suppressor gene(s) in the pericentromeric region of chromosome 19. © Elsevier Science Inc., 1999. All rights reserved.

INTRODUCTION In glial neoplasms, the most common of primary human brain tumors, chromosome 19 alterations have been reported in all three histological types, i.e., astrocytomas, oligodendrogliomas, and ependymomas [1–12]. Cytogenetic investigations indicate that these abnormalities may be structural and/or numerical and that they are commonly present in association with other aberrations, apart from one case of oligodendroglioma that showed a t(1;19) (p11;q11) as the only anomaly [7]. The breakpoints in the chromosome rearrangements, in particular, have been found at band 19q13, apparently associated with the loss of regions of the q arm. Numerous chromosome 19 changes affecting the q13 region have also been described in hematologic malignancy and in different solid tumors [13–15]. Various authors have emphasized that, at the molecular level, allelic losses on 19q may point to the presence of a TSG involved in gliomas of different malignancy [16–20]. However, extensive characterization of the chromosome 19 rearrangements detected by cytogenetic approaches has not been reported. The present study describes fluorescence in situ hybridization (FISH) analysis on three glio-

From the Department of Biology and Genetics, School of Medicine University of Milan (I. M., A. M. F .C.), Milan, Italy; and the Laboratory of Neuro-Oncology and Gene Therapy, Istituto Nazionale Neurologico Besta (E. C., G. F.), Milan, Italy. Address reprint requests to: Ivana Magnani, Ph.D., Department of Biology and Genetics, School of Medicine, University of Milan, via Viotti 3, 20131 Milan, Italy. Received June 4, 1998; accepted July 31, 1998. Cancer Genet Cytogenet 110:82–86 (1999)  Elsevier Science Inc., 1999. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

blastoma cell lines, with the aim of defining the karyotype abnormalities and the site of the breakpoints and determining the relationship between cytogenetic abnormalities and genetic losses. MATERIALS AND METHODS Cell lines MI-4, MI-31, and T-60 were obtained from neoplastic fragments of glioblastoma multiforme using methods reported previously [6]. Lines were maintained for more than two years in complete medium (RPMI GIBCO, 10%FCS) at 378C in a 5% CO2 atmosphere. When confluent, they were removed with a cell scraper and split 1:2. Cytogenetic analysis was performed on every three passages and karyotypes were defined according to the ISCN [21]. For chromosomal preparation, cells were collected in a centrifuge tube and the pellet was resuspended in a hypotonic solution (0.8% sodium citrate). The pellet was subsequently washed once in a solution of 5% methanol/3% acetic acid in H2O, once in a solution of methanol, and finally in a solution of methanol/acetic acid (3:1 vol/vol). The pellets were stored at 220 C. The biotin-labeled Whole Chromosome Painting (WCP) probe for chromosome 19 was from Cambio (Cambridge, United Kingdom). The digoxigenin-labeled probes for 19q13.1, chromosome 1 alpha sat (D1Z5), and chromosomes 1/5/19 alpha sat (D1Z7, D5Z2, D19Z3) were from ONCOR (Gaithersburg, MD), and the biotin-labeled 19q arm probe was from LI Biomedical Corporation (Arlington, Virginia). Chromosomal DNA was denatured in 70% formamide/2 3 SSC pH 7 for 2 min and then dehydrated with similar washes in

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ethanol. The hybridization mixtures containing the labeled probes, except for the 19q13.1 probe, were denatured at 708C for 5–10 min. The probes were then applied onto glass slides and covered with a coverslip. The hybridization was performed at 378C for 16 hours in a humid chamber and glass slides were subsequently washed once in 50% formamide, 2 3 SSC pH 7.0 at 438C for 15 min, twice in 2 3 SSC at 378C, and once in PN buffer (134g/l of dibasic sodium phosphate, 9g/l monobasic sodium phosphate, 0.1% NP-40). Each glass slide was overlaid with 60ml of FITC-labeled avidin while the slides were hybridized with 19q13.1, 1 alpha sat, and 1/5/19 alpha sat probes, with 60ml of FITC-labeled digoxigenin and incubated for 20 min at 378C in a humid chamber. After three washes in PN buffer, the signal was amplified by applying 60 ml of anti-avidin or anti-digoxigenin antibodies, followed by a further cycle of signal amplification. Finally, 18ml of a solution containing 0.5 mg/ml of propidium iodide/antifade were applied onto the slide. The fluorescent signal was detected using a Photomicroscope III (Zeiss) with appropriate filters and pictures were taken using 400 ASA film (Kodak). RESULTS AND DISCUSSION Line MI-4 showed a partial G-banded karyotype with 219, der(1)t(1;19)(p10;p10),12mar; line MI-31 displayed two copies of chromosome 19, one having unusual features, 13mar; and line T60 had a partial karyotype defined as 219,der(19)t(19;?),13–4 mar. These three glioblastoma cell lines have progressively doubled the chromosome complement present in the stem line without major variations of the markers initially observed. When examined for FISH at passage XXV, XI, and XV, respectively, cell lines MI-4, MI-31, and T60 only presented one third of near-diploid cells. Cell line MI-4, after painting analysis with a whole chromosome 19 probe, displayed one entire copy of chromosome 19 and chromosome 19 material translocated in two portions of similar length, one at 1p10, giving rise to der(1)t(1;19) previously detected by GTG banding, and one at 9p21, previously detected as add (9)(p21) (6). In addition, two small hybridization signals were located on a marker chromosome (Fig. 1A, a). In an attempt to define the origin of the centromere of der(1)t(1;19)(p10;p10), the specific chromosome 1 alpha satellite probe and a cocktail of 1, 5, and 19 alpha satellite probes were used (specific chromosome 19 alphoid probes are not available). Both these probes revealed an equally intense signal at the der(1) centromere, suggesting that at least part of the chromosome 1 centromere was maintained (data not shown). To check the localization of chromosome 19 arms, we employed the digoxigenin-labeled 19q13.1 band (19 q or p arm probes were not available at the time of this analysis). The segment translocated on chromosome 9p21 was identified as part of the long arm of chromosome 19, including the q13.1 band, implying that the material detected on 1q10 contained the 19p arm and suggesting a possible breakpoint at band 19q11–12. Interestingly, the copy of chromosome 19 that appeared normal on GTG banding

Figure 1 Partial karyotypes of cell lines MI-4, T-60, and MI-31 based on GTG-banding and FISH analysis. (A) and (B) demonstrate in the three cell lines, respectively, the results of chromosomal localization of whole chromosome 19 painting probe (wcp) and 19q13.1 probe. (C) demonstrates in cell line MI-31 the chromosomal localization of 19qarm probe. The chromosomes were counterstained with DAPI (blue).

showed the absence of fluorescence signal at q13.1 band, suggesting the excision of this band, possibly derived from a proximal breakage at 19q11–12 and a distal breakage at band q13.2. Furthermore, a signal with the q13.1 probe

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was localized on the long arm of the derivative chromosome that scored positive with the whole chromosome 19 probe, suggesting the insertion of the 19q13.1 band into the derivative after the deletion on chromosome 19 (Fig. 1A, column b). Chromosome painting on cell line T-60 using the total chromosome 19 probe highlighted that the der(19) originally described as t(19;?) was derived from a t(19;19) and also showed the presence of a small pericentric marker observed in 50% of the near-diploid cells (Fig. 1B, column a). The der(19)t(19;19) translocation, when analyzed by hybridization with the 19q13.1 probe, gave rise to a signal in each arm. Based also on GTG banding, we interpreted der(19) as the result of a breakage involving one of the two chromosomes 19 at band q11–12, followed by the translocation of the portion distal to the breakpoint at the end of the p arm of the chromosome 19 homolog (Fig. 1B, column b). This rearrangement led to a chromosome 19 deleted at

band q11-12 similar to the one involved in the translocation with 1p10 in cell line MI-4. Cell line MI-31 carried two copies of chromosome 19, but one showed an ambiguous profile on GTG-banding. After whole painting analysis, a complex pattern with four hybridization signals was present. One identified a normal copy of chromosome 19, one covered half of a derivative, one was positioned at the telomere of chromosome 22, and the last was present at the centromere of a marker chromosome (Fig. 1C, column a). In MI-31, the specific 19q13.1 probe highlighted one signal on the normal copy of chromosome 19 and one on the derivative, previously identified by the total chromosome 19 probe (Fig. 1C, column b). To understand the arrangement of this derivative, we employed FISH, using a probe for the 19q arm. The results displayed a signal on the q arm of the normal chromosome 19 and different signals both in centromeric and pericentromeric regions of the derivative as well as in the same re-

Table 1 Summary of cytogenetic alterations of chromosome 19 in gliomas Case no.

Diagnosis

D-245 D-250 D-256 D-290 D-299 D-304 D-316 D-340

GBM GBM GBM GBM GBM AMG GBM GBM

D-320 nr nr 37 43 nr 39 33 36 37 40 34 nr 5 MI 85 SJ 56 AW MI-4 MI-32 MI-14 nr T-60 3/T110 26/G227 31/T35 36/T66 nr 2 nr

GBM GBM GBM PA O GBM GBM GBM GBM GBM AA GBM GBM AA AA GBM GBM GBM GBM O GBM AO GBM GBM GBM PXA GBM GBM

Partial Karyotypes 49,XY, . . . 119 47,XY, . . . 119 44,XY, . . . t(9;19)(p13;q13) 45,X,2X, . . . t(1;19)(q21;q13) 46,XY, . . . der(19)t(17,19)(q11;q13) 43,XY, . . . 219 46,XX, . . . der(19)t(10;19)(q11;q11) 66z77,XXYY, . . . 219,219,der(11)t(11q19p)32, der(11)t(11p19q),der(11)t(11;19)(cen;q13) 47,XX, . . . der(19)t(5;10;19)(q15–21;q11–26;q13) 47,XX, . . . der(19)t(19; ?)(q13;?) 79z83,Y, . . . 219,219,der(19)t(19;?)(q13.3;?)32 46, . . . der(19)t(19;?)(q13.1,?),der(19)t(19;?)(p13.3;?) 83z88,XXYY, . . . 219,der(19)t(19;?)(q13.3;?) 39z45,der(X)t(X;?), . . . der(19)t(19;?)(p13;?) 43,X,2X, . . . tdic(19;22)(p13.3;q13) 43,XX, . . . 119 44,XY, . . . der(19)t(9;19)(q13;q13) 47z48,XY, . . . 119 65z75,XY, . . . 119,119 44,XX, . . . der(19)t(12;19)(q13;q13) 82z90, . . . del(19)(q13.1) 80z86,XXXX, . . . 119 48,XY, . . . 119 47,XY, . . . 119 47,XX, . . . der(1)t(1;19)(p10,q10) 47,XX, . . . del(19)(q13.2) 86z89,X,–X, . . . 219,219 45,XY, . . . der(1)t(1;19)(p11,q11) 45z46,XX, . . . 219,der(19)t(19;?) 44z46,XY, . . . 219 44z45,X,2Y, . . . der(19)t(14;19)(q11.2;p13.1) 41z45,XY, . . . der(19)t(14;19)(q13;q13.1) 48z52,XY, . . . der(19)t(1;19)(q21;q13) 46,XY, . . . t(1;11;19)(q24;q23;q13) 44,XX, . . . t(1;19)(q23;q13) . . . t(10;19)(q24;q13)

References Bigner et al. [1]

Jenkins et al. [2]

Lindstrom et al. [3]

Ranson et al. [4] Thiel et al. [5]

Magnani et al. [6]

Magnani et al. [7] Present study Debiec-Rychter et al. [8]

Li et al. [9] Vagner-Capodano et al. [10] Chernova and Cowell. [11]

Abbreviations: GBM, glioblastoma multiforme; AA, anaplastic astrocytoma; AO, anaplastic oligodendroglioma; AMG, anaplastic mixed glioma; PA, pilocytic aastrocytoma; O, oligodendroglioma; PXA, pleomorphic xanthoastrocytoma; nr, not reported.

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Chromosome 19 Translocations in Gliomas gion where the 19q13.1 band was localized. A signal was also present at the centromere of the marker chromosome, scoring positive with the total chromosome 19 probe, probably due to cross hybridization with alpha satellite sequences (Fig. 1C, column c). These findings support the possibility of a breakage at 19q11–12 with translocation of the segment q13.1-qter on the 19 p arm, followed by another translocation of an extra fragment of unknown origin at the end of the q arm. Furthermore, the results suggest deletion of a segment of the p arm that was translocated at the telomere of chromosome 22, as detected by the total chromosome 19 probe. The overall study on these glioblastoma cell lines by FISH with different probes revealed multiple rearrangements of chromosome 19 partially undetected in previous cytogenetic investigations. In particular, the hybridization analysis revealed that a preferred translocation breakpoint appeared in the centromeric-pericentromeric region of 19q. In the MI-4 cell line, we found that both homologs of chromosome 19 had translocations involving band 19q11– q12, but with different partner chromosomes. To our knowledge, we are the first to describe homozygous rearrangements affecting chromosome 19 at band q11–q12 in a case of glioblastoma. In tumors, structural rearrangements of both chromosome homologs are occasionally observed. In particularly, translocations at band 1q42 of both homologs have been described in a patient with pleomorphic xanthoastrocytoma (9). At the molecular level, homozygous deletions in gliomas have been detected on the short arm of chromosome 9, where different tumor suppressor genes are located [22–24]. Breakpoint rearrangements at band 19q11–q12 seem also to be present in the other two cell lines, one causing an intrachromosomal translocation and the other a complex interchromosomal rearrangement. As summarized in Table 1, cytogenetic abnormalities of chromosome 19 have been described in 27 glioblastomas, two oligodendrogliomas, five anaplastic gliomas, and two low-grade astrocytomas. The most commonly occurring breakpoint that affects chromosome 19 has been assigned to the q13 region and, specifically, in two cases to band q13.3, in one to band q13.2, and in three cases to band q13.1. These breakpoints do not coincide with our FISH results, showing a 19q11–q12 recurrent breakpoint. On the other hand, different authors have stressed the difficulty to define by standard cytogenetic analysis the exact breakpoint on chromosome 19, because of the scarce and apparently symmetrical pattern of GTG banding [25–27]. Thus, the differences reported in breakpoint designation may reflect technical difficulties, suggesting that the site of the breakpoint on chromosome 19 should be reviewed, when possible, by FISH techniques and specific probes. The same should be done to ascertain the actual numerical alterations and to unmask cryptic translocations, particularly when unidentified marker chromosomes are present. In our studies, FISH analysis has been facilitated by the use of established cell lines. It has been reported that such cell lines can be obtained from approximately 30–40% of glial tumors and that in these cells a ploidy shift may take place during culture, even if the original markers are re-

tained [12, 28, 29]. In our three cell lines, all chromosome 19 translocations were conserved except for a pericentric marker identified as a deleted chromosome 19 q11–12 that was partially lost in line T-60. In spite of the complexity of chromosome 19 rearrangements, this finding represents the only appreciable loss of genetic material from chromosome 19 that we have found, even though microdeletions at the site of breakage cannot be ruled out. This is in agreement with LOH studies suggesting that total loss of chromosome 19 is an unusual event in glial tumors [16–17], whereas partial genetic deletions are more frequently observed [18]. The presence on chromosome 19 of TSGs relevant to glioma development has been inferred from LOH and cytogenetic studies. However, in spite of the definition of a region of common deletion on band q13.2–q13.4, no specific candidate TSGs have yet been identified [19]. Our data imply that other chromosomal mechanisms may lead to gene inactivation in glioblastoma, such as unbalanced translocations that could mask interstitial deletions affecting putative TSG. In chromosome 9p21 deletions in glioma cell lines [23, 24], in the reciprocal translocation t(10;19) described in one glioblastoma [11], and in our cell lines displaying 19q11-q12 recurrent translocations, the breakpoint might represent a fragile region and/or indicate the position of critical tumor suppressor genes. The characterization of the translocation breakpoint in our cell lines should allow further investigation of the involvement of chromosome 19 alterations in glial tumors.

This work has been partially supported by CNR grant #9504451.CT04 to A.M. F-C and by an AIRC grant to G. F. We thank Dr. Bianca Pollo for help with histological analysis and Ms. Angela Pettinicchio for her collaboration in obtaining tumor samples.

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