Chromosomal imbalances and DNA amplifications in SV40 large T antigen-induced primitive neuroectodermal tumor cell lines of the rat

Roland Kappler, Torsten Pietsch1, Sascha Weggen1, Otmar D. Wiestler1 and Harry Scherthan2

Department of Human Biology and Human Genetics, University of Kaiserslautern, E. Schroedinger Straße, D-67653 Kaiserslautern and
1 Department of Neuropathology, University of Bonn Medical Center, S. Freud Straße 25, D-53105 Bonn, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Comparative genomic in situ hybridization analysis of four cell lines derived from SV40 large T antigen-induced primitive neuroectodermal tumors of the rat revealed non-recurrent chromosomal copy number changes and DNA amplifications at chromosomal bands 2q34, 4q43qter and 15q12qter in cell lines TZ102, TZ103 and TZ107, respectively. Semi-quantitative PCR and western blot analysis demonstrated amplification and over-expression of the rat N-ras proto-oncogene in TZ102. Furthermore, all cell lines displayed aneuploid cell populations and variable chromosome numbers as assessed by flow cytometry and cytogenetics. These findings suggest that DNA amplification as well as genomic instability may contribute to the pathogenesis of SV40 large T antigen-induced primitive neuroectodermal tumors of the rat.

Abbreviations: CGH, comparative genomic in situ hybridization; DAPI, 4',6-diamidino-2-phenylindole; DI, DNA index; FCM, flow cytometry; FR, fluorescence ratio; PBS, phosphate-buffered saline; PNET, primitive neuroectodermal tumor; RNO, rat chromosome.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Primitive neuroectodermal tumors (PNETs) comprise a family of morphologically related neoplasms of the central nervous system which share histopathological features and presumably originate from a population of pluripotent neural precursors. In humans, these neoplasms are among the most common brain tumors in childhood (1). A prominent representative in this group is cerebellar medulloblastoma, which was first described in 1925 (2). Cytogenetic studies on this tumor have disclosed a frequent loss of the distal end of chromosome 17p usually associated with the formation of an isochromosome 17q (3). Further aberrations detected either by cytogenetic analysis or loss-of-heterozygosity allelotyping include deletions of chromosomes 5q, 6, 7q, 9, 10, 11, 16q and 22 (38). Recent studies revealed mutations in the human homolog of the Drosophila segment polarity gene patched (9,10), as well as occasional amplification of the proto-oncogenes MYC and MYC-N and the epidermal growth factor receptor gene (1114) in sub-populations of medulloblastomas. However, further analysis is required to determine all the genes that contribute to the pathogenesis of PNETs.

A powerful animal model for PNETs has recently been established using retrovirus-mediated transfer of the SV40 large T antigen into fetal rat brain transplants (15). These tumors exhibit morphological features indistinguishable from human PNETs, including small cell phenotype, formation of neuroblastic rosettes and a potential for both neuronal and glial differentiation. Permanent cell lines established from these tumors either display an immature phenotype with expression of early neuronal markers, spheroid formation and delicate cell processes or show a flat, epithelial-like appearance with expression of both glial and advanced neuronal markers (16). Considering that the cell lines have been passaged only a few times since their outgrowth from the tumor, they represent a useful in vitro model for the analysis of molecular and genomic alterations associated with PNET formation.

Genomic imbalances in solid tumors can be readily detected by comparative genomic in situ hybridization (CGH), which has become an indispensable tool in human and experimental oncology and pathology (reviewed in ref. 17). Recently, CGH has been adapted to the rat genome (18), opening the way to tracing the genomic basis of tumor development and progression in rat model systems of human cancer. CGH is based on the competitive co-hybridization of differentially labeled control and tumor DNA to normal metaphase chromosomes, along which a deviation from a balanced fluorescence intensity ratio indicates chromosomal copy number changes (19,20).

In the present study, four cell lines derived from rat PNETs were examined for genomic alterations using CGH, cytogenetics, flow cytometry (FCM), semi-quantitative PCR and western blot analysis. Our findings suggest that DNA amplification and genomic instability may reflect early events in the pathogenesis of SV40 large T antigen-induced primitive neuroectodermal tumors of the rat.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tumor cell lines
Four cell lines derived from SV40 large T antigen-induced rat PNETs were used (TZ102, TZ103, TZ106 and TZ107; ref. 16). All cell lines were within passages 1 and 5 of isolation, except for TZ106, which was at passage 8.

Cytogenetics
Cell lines were cultured in DMEM (Life Technologies, Gaithersburg, MN) supplemented with 10% fetal calf serum, penicillin, streptomycin and glutamine to near confluency. Chromosome preparations were obtained by arresting cells at metaphase using colcemid treatment (0.2 µg/ml; Serva, Heidelberg, Germany) for 4 h. Thereafter, cells were trypsinized, collected by centrifugation and resuspended in hypotonic solution (0.0375 M KCl). To minimize loss of metaphase cells, the suspension was fixed once in acetic acid/methanol (1:3) and incubated overnight in fixative at –20°C. The suspension was dropped onto clean glass slides after a final wash in fresh fixative. For each cell line at least 50 4',6-diamidino-2-phenylindole (DAPI)-stained metaphases were numerically analyzed.

Flow cytometry
FCM analysis of DNA content was performed as described previously (18). Briefly, collected cells were filtered through a nylon mesh ({emptyset} 70 µm) and fixed in 70% ethanol. Approximately 106 cells were incubated for 10 min at room temperature in 0.5 ml detergent solution (HRA; Partec, Münster, Germany). Staining of nuclear DNA was performed for 30 min by addition of 3 ml DAPI/sulforhodamine 101 staining solution (HRB; Partec). DNA content of 10 000 cells was determined in a PAS III flow cytometer (Partec). Prior to all measurements, the flow cytometer was calibrated with an ethanol-fixed cell suspension of normal rat brain tissue. The degree of DNA content aberration is expressed by the DNA index, which is defined as the ratio of the mode of the relative DNA content of the G0/G1 cells of the sample divided by the mode of the relative DNA measurement of normal diploid G0/G1 reference cells (21).

DNA labeling and CGH
DNA labeling and CGH was performed as described previously (18). Briefly, equal amounts of digoxigenin- and biotin-labeled genomic DNA from normal rat brain tissue and tumor cell lines, respectively, were hybridized to normal metaphases obtained from fetal rat cells. Hybrid molecules were detected with one layer of avidin–FITC (Sigma Chemical Co., St Louis, MO) and rhodamine–anti-digoxigenin Fab fragments (Roche diagnostics, Mannheim, Germany). Slides were mounted and counterstained with antifade solution containing DAPI and 10 µg/ml actinomycin D (both Serva).

Digital imaging and CGH evaluation
Digital gray scale images were generated with a Hamamatsu cooled charge-coupled device camera mounted on a Zeiss Axioscope fluorescence microscope. Images were processed with the ISIS digital image system and ratio profiles were calculated with the CGH package of ISIS software (MetaSystems, Altlußheim, Germany). Chromosomes were identified by inspection of color-inverted digital DAPI images. Ratio profile analysis was based on >10 well-hybridized metaphases. The proximal end of the long arm of rat chromosome 1 was excluded from the analysis due to variable fluorescence ratios in this region (18). Chromosomal alterations were assessed according to ISCN 1995 (22).

Semi-quantitative PCR
DNA from all cell lines was examined by semi-quantitative PCR analysis for amplification of the rat N-ras proto-oncogene, using the ß-globin gene Hbb on rat chromosome (RNO) 1 as an internal control. Oligonucleotide primers were designed according to the published genomic sequences of the respective rat genes (23,24): 5'-TACCACCCCTACCTCCTCAC and 5'-GCTTCAGAGTGCACATACAAAGG for exon 2 of the N-ras gene; 5'-CCAATCTGCTCACACAGG and 5'-CACCTTTCCCCACAGG for exon 1 of the ß-globin gene. PCR amplification was performed with 100 ng purified genomic DNA in a reaction mixture containing 0.3 µM each primer, 0.2 mM each dNTP, 50 mM KCl, 10 mM Tris–HCl, 1.5 mM MgCl2, 2.5 U Taq polymerase (MBI Fermentas, St. Leon-Rot, Germany) in a final volume of 50 µl. The PCR reactions were carried out in a Crocodile II Thermocycler (Appligene Inc., Illkirch, France) for 28 cycles of amplification consisting of 94°C for 1 min, 56°C for 1 min and 72°C for 2 min (first cycle 94°C for 5 min; last cycle 72°C for 10 min). Negative control reactions were carried out with amplification cocktail and deionized water as a substitute of template DNA. The results of the PCR were analyzed by electrophoresis through a 2% agarose gel. Digital images were recorded and analyzed with a computerized image recording system (Cybertech, Berlin, Germany).

Western blot analysis
All cell lines were analyzed for N-ras expression by western blotting. Cells were grown to confluency, detached with Versene (Gibco BRL, Grand Island, NY), washed with phosphate-buffered saline (PBS) and counted. A total of 106 cells were lysed in 100 µl of 20 mM Tris–HCl, pH 7.4, 50 mM NaCl, 1% Nonidet P-40, containing 1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin (Boehringer) and 100 U/ml aprotinin (Calbiochem-Novabiochem, Bad Soden, Germany) for 30 min on ice. Debris was removed by centrifugation for 10 min at 13 000 g and 4°C. Protein concentrations were determined using a DC protein assay (BioRad, Richmond, CA). Total proteins were separated by electrophoresis on 15% SDS–polyacrylamide gels and blotted onto nitrocellulose. After blocking with 5% non-fat dry milk in PBS overnight at 4°C, the filters were incubated with the anti-c-N-ras antibody (clone Ab-1, 10 µg/ml in PBS, 0.1% BSA; Calbiochem-Novabiochem) for 2 h at room temperature. Binding of the primary antibody was detected by alkaline phosphatase–anti-alkaline phosphatase staining. The filters were developed using nitroblue tetrazolium/bromochloroindolyl phosphate substrate.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cytogenetic analysis of four cell lines derived from rat PNETs revealed the modal chromosome number of each tumor cell line (Table IGo), and yielded two distinct groups of cell lines. As compared with the normal rat complement (2n = 42), TZ103 and TZ106 showed near-diploid modal chromosome numbers of 41/42 and 42, whereas TZ102 and TZ107 displayed hyperdiploid values of 47/48 and 46, respectively (Table IGo). The distribution of the chromosome numbers revealed that the former group exhibited, additionally, near-tetraploid cells, which were not apparent in the latter group (not shown). However, a considerable range of chromosome numbers and numerous marker chromosomes of unknown composition were present in all cell lines investigated (not shown).


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Table I. Results of cytogenetics and flow cytometry of rat PNET cell lines
 
To determine the fraction and ploidy grade of the different cell populations in the cell lines, FCM analysis was performed. Cell lines TZ103 and TZ106 displayed diploid and one or two additional aneuploid cell populations, respectively. TZ102 showed a single hyperdiploid cell population, while TZ107 consisted of two aneuploid cell populations (Table IGo and Figure 1Go).



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Fig. 1. Flow histograms displaying DNA values of rat PNET cell lines. FCM analysis of TZ102 revealed a prominent peak of aneuploid cells at channel 119. The flow histogram of TZ103 shows distinct peaks for both diploid and tetraploid cells (channels 100 and 200). TZ106 displays a diploid peak (channel 100), a hyperdiploid peak (channel 158) and a tetraploid peak (channel 200). TZ107 shows two peaks of hyperdiploid cells at channels 118 and 137.

 
CGH analysis was applied to further scrutinize the genomic architecture of the cell lines and revealed an individual, cell line-specific spectrum of chromosomal copy number changes (Table IIGo). In summary, gains were noted for RNO 1, 2, 4–6, 9, 12q, 13q, 14 and 16, while recurrent alterations were not present. A single copy number loss in TZ102 was restricted to RNO2q11. Furthermore, individual amplification sites were detected in cell lines TZ102, TZ103 and TZ107, which involved chromosomal regions RNO2q34, RNO4q43qter and RNO15q12qter, respectively (Figure 2Go). Since appropriate region-specific DNA probes were not available to us, amplifications were not amenable to further FISH analysis.


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Table II. Summary of chromosomal alterations identified by CGH in rat PNET cell lines
 


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Fig. 2. CGH results showing high level copy number gains at RNO2q34 (TZ102), RNO4q42qter (TZ103) and RNO15q12qter (TZ107). The results are displayed as gray scale images of the respective color channels. From left: DAPI (blue channel; DNA counterstain), rhodamine (red channel; control DNA) and FITC (green channel; tumor DNA). High level copy number gains are apparent as white signals in the tumor DNA channel and as a deflection of the fluorescence ratio (FR) profiles to the right. FR profiles are displayed along the respective ideograms and are based on >10 well-hybridized metaphases. The three vertical lines in the center represent FRs of 1 (center line) and the FR thresholds of 0.8 for under-representation (left line) and 1.2 for over-representation (right line) of chromosomal material, respectively. White bars to the right of FR profiles outline regions of gain; the bar to the left indicates loss of chromosomal material at RNO2q11q12 (TZ102).

 
However, semi-quantitative duplex PCR with primers for exon 2 of the rat N-ras gene (mapping to RNO2q34; ref. 25) and exon 1 of the ß-globin gene (control) confirmed an ~6-fold increase in N-ras copy number in TZ102 (Figure 3Go).



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Fig. 3. Semi-quantitative PCR analysis of rat PNET cell lines and normal tissue. The PCR product of rat N-ras exon 2 is seen at 265 bp and the PCR product of exon 1 of the ß-globin gene is seen at 186 bp. Values obtained by video densitometry revealed a 6.26-fold amplification of the genomic N-ras fragment as compared with the ß-globin control in cell line TZ102. The ratios N-ras:ß-globin are given below the respective lanes. A ratio of 1.6 is consistent with a low copy gain of RNO2 in TZ107 (Table IIGo).

 
To clarify whether N-ras amplification was associated with over-expression, western blot analysis was applied. It was found that TZ102 displayed a strongly increased expression of the 21 kDa N-ras protein as compared with the other cell lines (Figure 4Go).



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Fig. 4. (Top) Western blot analysis of N-ras expression in rat PNET cell lines. Expression of the 21 kDa N-ras protein was detected in all four cell lines (arrow). Consistent with the amplification of the N-ras gene, cell line TZ102 showed strong overexpression of N-ras as compared with TZ103, TZ106 and TZ107. (Bottom) Corresponding Coomassie stain. For cell lines TZ102 and TZ103, 15 µg of total protein were loaded, whereas 30 µg were loaded for cell lines TZ106 and TZ107.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
CGH analysis, cytogenetics and FCM of four established PNET cell lines of the rat revealed specific chromosomal and genomic anomalies. In TZ102, FCM analysis detected a single aneuploid cell population. Cytogenetics disclosed the presence of numerous marker chromosomes in all metaphase cells analyzed, while CGH revealed the gain of six large chromosomal regions in this cell line. These results suggest that chromosomal instability may account for the variation of chromosome numbers in this line. A diploid and a small tetraploid cell population were detected by FCM in TZ103, while CGH disclosed a single regional high level copy number gain. Variable chromosome numbers were revealed by cytogenetics, suggesting genomic instability of this cell line as well. In TZ107, the major fraction of cells displayed a DNA index (DI) of 1.19, which suggests the gain of three or four large chromosomes in this cell population. This gain is compatible with a modal chromosome number of 46 and the CGH results, which displayed a copy number gain of three large chromosomes. Given that CGH reveals only chromosomal imbalances if aberrant cells make up at least 50% in a sample (26,18), the aberrations disclosed by this technique in TZ107 will only represent the genomic composition of the DI 1.19 cells.

In cell line TZ106, cytogenetics again showed highly variable chromosome numbers and CGH revealed a low copy number gain of RNO 1 material as the sole genomic aberration. TZ106 consisted predominantly of tetraploid cells but also contained a diploid and a near-triploid cell population. Since the latter two cell populations were again below the detection limit of CGH, the RNO 1 gain will most likely reflect the genomic imbalance of the tetraploid cell population (18,26). In cervical carcinoma progression, tetraploidization is considered to be an early event (2729). Since tetraploid cells were abundant in TZ106 and CGH displayed a gain of RNO 1 as the sole genomic aberration, it may be speculated that tetraploidization occurred early during formation of this PNET cell line and that the variable chromosome numbers as well as the lower DIs of the minor cell populations result from an erosion of the initially tetraploid tumor genome.

In summary, the results from cytogenetic and FCM analysis suggest a considerable genomic and/or chromosomal instability in the PNET tumor cell lines investigated. These effects are possibly mediated by a deterioration of cell cycle control due to large T antigen expression. Such a mechanism appears consistent with the observation that the SV40 large T antigen binds to and inactivates gene products involved in surveillance of genome integrity and cell cycle progression, such as the tumor suppressor protein p53 and the retinoblastoma protein (3032).

CGH analysis of all four established large T antigen-induced PNET cell lines disclosed individual, non-recurrent copy number alterations, predominantly low copy gains of large chromosomal segments. This contrasts with the situation in human PNETs, where the recurrent loss of 17p has been demonstrated by CGH and cytogenetics (3,33,34). Furthermore, loss of RNO 10, which shares a great deal of homology with human 17p (35), was not apparent in rat PNET cells. The most surprising finding of this study was the detection of cell line-specific, regional high level copy number gains/amplifications in three of the rat PNET cell lines. Only TZ106, which expresses a truncated form of the large T antigen (16), failed to show an amplification site.

In human PNETs, amplification of the proto-oncogenes MYC and rarely MYC-N, as well as the epidermal growth factor receptor gene, has been observed in cell lines derived from medulloblastomas and in a few primary tumors (1114). Interestingly, the N-ras proto-oncogene, which has been implicated in the formation of a variety of human tumors (36), maps to RNO2q34 (25), the region amplified in TZ102. Semi-quantitative PCR and western blot analysis confirmed the amplification and over-expression of the N-ras rat proto-oncogene in TZ102. Hence, it may be speculated that these events may have contributed to the neoplastic transformation of this PNET. In human PNETs, however, N-RAS amplifications have not been detected to date (12). On the other hand, activating mutations in N-RAS were found in three of 32 cases (37). Thus, further investigations are required to unravel whether activation of N-ras/N-RAS plays an important role in a subgroup of SV40 large T antigen-induced rat PNETs and possibly also in human PNETs. The other two amplifications identified in our investigation involved regions RNO4q43qter and RNO15q12qter. Since these regions of the rat genome are poorly mapped, subsequent studies will have to identify candidate genes at these sites.

It has been shown that the rat PNET cell lines investigated share considerable homologies in cell morphology and expression of marker molecules with human PNETs (16). The present investigation, however, showed that the four rat PNET cell lines display considerable genomic instability and non-recurrent chromosomal alterations which fail to mark syntenic segments known to be involved in human PNET formation (see above). Since it is known that SV40 transformation induces a variety of chromosomal changes (38,39), it is likely that the chromosomal alteration spectrum of the PNET cell lines investigated relates to large T antigen expression in cells of the fetal rat brain.


    Acknowledgments
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (nos Sche 350/7-1 to H.S. and SFB400-C2 to T.P. and O.D.W.).


    Notes
 
2 To whom correspondence should be addressed Email: scherth{at}rhrk.uni-kl.de Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 18, 1999; revised April 28, 1999; accepted April 30, 1999.