Loss of heterozygosity in malignant rat schwannomas chemically induced in hybrids of inbred rat strains with differential tumor susceptibility
Bernd U. Koelsch1,2,
Andrea Kindler-Röhrborn1,2,
Sabine Held1,
Simone Zabel and
Manfred F. Rajewsky1,3
Institute of Cell Biology (Cancer Research), University of Essen Medical School and West German Cancer Center Essen, Hufeland-Strasse 55, D-45122 Essen, Germany
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Abstract
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Rats of the inbred BD strains strongly differ in their susceptibility to the induction of tumors of the central (CNS) and peripheral nervous system (PNS) by N-ethyl-N-nitrosourea (EtNU). Malignant schwannomas induced in (BDIX x BDIV) and (BDIX x BDVI) rat hybrids were analyzed to identify genetic alterations associated with EtNU-induced tumorigenesis in the PNS. EtNU-induced schwannomas exclusively exhibit an A:T®T:A transversion mutation of the neu/Erbb-2 gene located on chromosome 10, with subsequent loss of the wild-type neu/Erbb-2 allele at a post-initiation stage. Targeted allelic deletion mapping previously revealed losses of heterozygosity (LOH) at the distal end of chromosome 10 in a large majority of (BDIX x BDIV) schwannomas. The aims of the present study were (i) to scan the whole genome for further LOHs; (ii) to narrow down the consensus regions of frequently occurring allelic deletions using tumors from different crosses of BD rats; and (iii) to determine the sequence of genetic alterations during schwannoma development. A limited number of (BDIX x BDIV) F1 tumors were initially screened for LOH and microsatellite instability (MI) by amplifying 58 microsatellite markers spanning the whole genome. LOHs on chromosome 5 were detected in 9/17 tumors, with random loss of the parental alleles. Ninety-two schwannomas from different BD rat-crosses were then analyzed to solidify these data and to determine the consensus region of frequent LOHs. The results indicate that LOHs on chromosomes 10 and 5 are required for the development of EtNU-induced malignant schwannomas from immature neu/Erbb-2 mutant glial cells, and that putative tumor suppressor genes are localized on chromosome 10q32.3, corresponding to human chromosome 17q25.3, and the telomeric region of mouse chromosome 11, and on the telomeric quarter of chromosome 5. MI was detected in <0.2% of cases.
Abbreviations: cM, centi Morgan; CNS, central nervous system; EtNU, N-ethyl-N-nitrosourea; LOH, loss of heterozygosity; MI, microsatellite instability; PCR, polymerase chain reaction; PNS, peripheral nervous system; STS, sequence tagged site; EST, expressed sequence tag.
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Introduction
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Rat strain specific, differential susceptibility to the induction of carcinogenesis by N-ethyl-N-nitrosourea (EtNU) in the central (CNS) and peripheral nervous system (PNS) represents a condition inviting the analysis of the influence of genetic background on the process of carcinogenesis. In contrast to rats of the resistant BDIV strain, BDIX as well as BDVI rats exposed to a single dose of EtNU on postnatal day 1 are highly susceptible to the induction of malignant schwannomas in the PNS, preferentially in the trigeminal nerves. A point mutation at nucleotide 2012 of the Erbb-2/neu gene (1) located on chromosome 10q32.1 (2) has been shown to be a very early event in the development of these tumors (3). The wild-type copy of the gene is lost at a subsequent stage of the oncogenic process (3). This could be attributed to the frequently occurring LOHs on chromosome 10. The consensus region of these defines a locus (Dis-1) encompassing 6 cM (centi Morgan) at the telomeric end of chromosome 10, excluding the neu/Erbb-2 gene and harboring putative tumor suppressor gene(s) (4). The aim of the present study was to localize further regions of LOH that are either deleted or amplified during the development of EtNU-induced schwannomas, to narrow down these regions and Dis-I as precisely as possible, and to determine the sequential order of the different genetic alterations. Microsatellite analysis was used to investigate schwannoma development in intercrosses of (BDIV x BDIX) rats with respect to LOH and microsatellite instability (MI). Chromosomal regions affected by LOH were then narrowed down in tumors from (BDIX x BDVI) F1 crosses. In these hybrids, the high susceptibility of the parental strains towards the induction of schwannomas by EtNU results in very high tumor yields. Moreover, BDIX and BDVI rat crosses are more informative at the regions of interest compared with (BDIX x BDIV) hybrids. The hybrid animals used enabled us to determine the chromosomal location and the size of chromosome 5 LOHs more precisely, i.e. to identify smaller regions for the search of candidate genes. In addition, the influence of different genetic backgrounds on the nature of genetic alterations could be investigated.
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Materials and methods
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Rat strains and crosses
Specific pathogen-free animals were bred and maintained at this institute's animal facility. F1 hybrids in both orientations were generated from the inbred strains BDIX and BDIV (5). F1 animals of both orientations were then mated to produce reciprocal F2 intercross progeny. A backcross generation with higher tumor incidence was obtained by crossing F1 rats with the BDIX parental strain (4).
In addition, BDIX rats were mated with rats of the almost equally susceptible BDVI strain in both orientations to generate the most informative F1 rats with high tumor incidence. (BDVI x BDIX) F2 rats were used to establish a high resolution linkage map of the Dis 1 region.
Induction of schwannomas by EtNU
(BDIX x BDIV) F1 and F2 intercross and backcross rats as well as (BDIX x BDVI) F1 animals received a single subcutaneous injection of EtNU (80 µg/g body weight) 24 h postnatally (3). Beginning at 10 weeks after exposure to EtNU, the animals were examined for neurological symptoms twice weekly. Animals displaying cachexia, shortness of breath, paralyses, or behavioral abnormalities, were killed with the use of CO2. Schwannomas were carefully dissected out from normal tissue, snap frozen in liquid N2, and stored at 80°C until further analysis.
Isolation of DNA
Genomic DNA was isolated from tumors as well as from the tail tip of tumor bearing animals as a source of normal DNA, using the `Qia Amp Tissue Kit' (Qiagen, Hilden, Germany), and stored frozen at 20°C.
Erbb-2/neu mutation analysis
The A:T
T:A transversion mutation at nucleotide 2012 of the neu/Erbb-2 gene, diagnostic of EtNU-induced malignant schwannoma of the rat PNS, was detected by amplifying a 129 bp genomic fragment from tumor DNA by the polymerase chain reaction (PCR). With the exception of the use of non-fluorescent primers, the PCR reaction was performed as published previously; subsequent digestion with the restriction enzyme MnlI was followed by electrophoresis and silver staining (6,7). Tumor DNAs displaying >40% of the mutant 104 bp fragment were used for allelic deletion mapping.
Allelic deletion analysis by PCR-amplified microsatellites
To detect LOH, a whole genome scan of schwannomas from BDIV x BDIX F1 and F2 intercross animals was performed. After selecting 58 evenly spaced microsatellite markers polymorphic for both strains (Figure 1
) on the basis of published maps (8), analysis was carried out on 17 F1 and 26 F2 schwannomas.

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Fig. 1. Distribution of genetic markers used in the primary scan to detect LOH and MI in EtNU-induced rat schwannomas. The designations and approximate locations of microsatellite markers are shown on each chromosome.
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All primers were purchased from Research Genetics (Huntsville, AL, USA). PCR amplification of microsatellites using DNA from normal and tumor tissue in parallel, as well as the electrophoretic separation on denaturating polyacrylamide gels with subsequent silver staining have been described in detail elsewhere (4). Genotypes and LOH were directly read from the silver stained gels by two independent individuals. LOH was defined as a pronounced reduction of intensity, or the complete disappearance of one allele, compared with the heterozygous control, while any change in allele size in tumor relative to normal tissue was considered as MI.
In case of confirmed LOHs on chromosome 5 in BDIX x BDIV hybrids, additional microsatellite markers D5Mgh7, D5Mgh21, D5Mgh27, D5Wox3, D5Mco1, D5Rat8, D5Rat10, D5Rat11, D5Rat12, D5Rat24, D5Rat33, D5Rat36, D5Rat37, D5Rat38, D5Rat 45, D5Rat46, D5Rat70, D5Rat84, D5Rat95, D5Rat111, D5Rat115, D5Rat120, and D5Rat126 were used to narrow down the LOH consensus region.
Chromosome 5 allelic deletion mapping in schwannomas of BDIX x BDVI hybrids was performed with the following informative microsatellite markers: D5Wox2, D5Wox8, D5Mgh18, D5Mgh20, D5Rat4, D5Rat13, D5Rat18, D5Rat33, D5Rat37, D5Rat38, D5Rat41, D5Rat42, D5Rat45, D5Rat46, D5Rat52, and D5Rat70.
Moreover (BDVI x BDIX) F1 tumors as well as (F1 x BDIX) backcross tumors were used to minimize the previously detected LOH at the Dis-1 region on chromosome 10, using the markers D10Rat3 and D10Rat2/D10Rat97 to identify tumors with small LOHs.
Construction of a linkage map of the telomeric region of chromosome 10
In the telomeric region of chromosome 10 (D10Rat3-tel) only D10Rat3 and D10Rat2 were polymorphic between BDIX and BDIV rats (4). Therefore, a linkage map for the telomeric region had to be constructed using 355 (BDIX x BDVI) F2 rats heterozygous for nearly all markers of the region. Genotyping data were analyzed with the MAPMAKER 3.0 program to generate a linkage map around the Dis-1 locus.
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Results
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EtNU-induced schwannomas
All schwannomas analyzed in this study displayed at least 40% of neu/Erbb-2 alleles harboring the diagnostic T:A
A:T transversion mutation at nucleotide 2012 (3).
Genomic screening for LOH and MI in EtNU-induced schwannomas
Based on published maps (8), 58 microsatellite markers equally distributed throughout the genome were chosen to provide optimal coverage for each chromosome (Figure 1
). The primary genome wide scan for allelic deletions was performed using DNA from 17 (BDIX x BDIV) F1 schwannomas. Genetic alterations observed in this set of F1 tumors were almost exclusively LOHs. MI was detected in three cases only. In addition to the previously reported LOHs on chromosome 10 (4), frequent LOH (59%) were observed on chromosome 5, always encompassing the whole chromosome. One case of LOH of microsatellite markers D1Mgh12 and D1Mgh13 on chromosome 1q was found. The results of the initial screening are summarized in Table I
. Quantitative differences of allele-specific PCR products were less prominent for LOHs on chromosome 5 compared with the complete loss of one allele in most cases of LOH on chromosome 10 in the same tumors (unpublished data).
Refined allelic deletion mapping on chromosomes 5 and 10
After the primary scan, further microsatellite markers were chosen to confirm the regions of LOH on chromosomes 5 and 10 and to minimize the consensus regions. Fine mapping of the allelic deletions on chromosome 5 was initially performed on 86 malignant schwannomas (17 [BDIX x BDIV] F1, 26 [BDIX x BDIV] F2, and 43 [(BDIX x BDIV) x BDIX] backcross tumors). 72 cases were informative for at least one locus. Forty-seven tumors (65%) displayed LOHs on chromosome 5. There was no significant preference for the loss of one or the other allele (BDIV allele 47% vs BDIX allele 53%). However, none of the schwannomas showed a partial loss of chromosome 5 as required for the more precise location of a putative tumor suppressor gene.
Contrasting with these data, LOH analysis of the 23 BDVI x BDIX F1 tumors resulted in 5 partial losses of chromosome 5 (see Figure 2
for details), with a consensus region (Dis-2) spanning from D5Wox2 to the most telomeric marker D5Mco1 representing the distal quarter of chromosome 5q (Figure 2b
). In total, the LOH frequency in these tumors amounted to 52% (12/23). In 3/12 (25%) of cases the BDVI allele, and in 9/12 (75%) the BDIX allele was lost.

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Fig. 2. Allelic deletion mapping of chromosome 5: (a) eight polymorphic microsatellites analyzed on a silver-stained polyacrylamide sequencing gel. Lane 1, BDVI allele; lane 2, BDIX allele; lanes 3, 5, 7, 9 and 11, wild-type F1 DNA; lanes 4, 6, 8, 10 and 12, respectively, five matched tumor DNAs; (b) schematic drawing of (BDVI x BDIX) F1 tumors exhibiting partial LOHs. Allelic losses are indicated as black boxes (left); the linkage map of the microsatellite markers analyzed is shown on the right side. Gray box: Dis-2 consensus region.
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The region of interest on chromosome 10 spanned ~6 cM from microsatellite marker D10Rat4 to D10Rat2, corresponding to the telomere at band 10q32.3. Forty-three schwannomas that had developed in rats of the (BDIX x BDIV) x BDIX backcross generation (4) were then analyzed with D10Rat2 and D10Rat4. Twenty-two tumors were non-informative for these markers. While 14 schwannomas had lost the BDIV allele, deletions of the BDIX allele were found in five of the tumors. Two tumors showed weak or no loss. None of the tumors displayed an allelic deletion for one of the two markers only. Results obtained for the 23 schwannomas of (BDVI x BDIX) F1 animals were similar: All of the tumors showed simultaneous LOHs for D10Rat4 and the most telomeric marker D10Rat97 (next to D10Rat2), with no allelic preference (52% loss of the BDIV allele vs 48% loss of the BDIX allele). Therefore, none of the tumors analyzed allowed us to further narrow down the Dis-1 locus. Although a total of 109 tumors had been available for analysis, the consensus region for the LOH at the Dis-1 locus therefore still spans about 6 cM.
The high resolution linkage map of the entire Dis-1 locus obtained by genotyping of 355 (BDIX x BDVI) F2 intercross rats (Figure 3
) spans about 4.5 cM when male and female recombinations are combined (sex average map). The map based exclusively on female recombination covers 1 cM only. It contains 12 microsatellite markers from published maps with an average resolution of <0.5 cM and
0.1 cM, respectively, and is presently being used for constructing a contig and an EST/gene map to facilitate the search for candidate genes.

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Fig. 3. Dis-1 linkage map on chromosome 10q32.3. The critical region in the upper map, generated from data from (BDIX x BDIV) F2 rats, spans about 6 cM in length and is flanked by three polymorphic markers only. Based on genotyping (BDIX x BDVI) F2 rats, the lower map spans about 4.5 cM. Dis-1 is covered by 12 informative markers. The location of Dis-1 is indicated on the chromosome 10 ideogram.
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Discussion
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In order to identify additional gene loci involved in the development of EtNU-induced malignant rat schwannomas, 17 (BDIV x BDIX) F1 tumors were initially analyzed with the use of 58 polymorphic microsatellite markers distributed throughout the genome. This represents an average marker distance of about 25 cM in the primary genome scan, limiting the detection power of LOH analysis to gross genetic alterations. Microsatellite analysis alone does not allow us to distinguish between physical loss of genetic material, mitotic recombination, chromosomal aneuploidy, or low level amplification, each of which causes distinct allele ratios in the tumor cells (9). However, additional karyotyping may permit us to differentiate between these mechanisms and could even reveal cytogenetically detectable balanced translocations.
The number of markers used in the primary scan proved to be sufficient to identify all chromosomes with frequent LOHs in the schwannomas. Even one microsatellite marker per chromosome would have led to the same results in this tumor model, as loss of an entire chromosome is a major mechanism to delete genetic material.
Progress in the fine mapping of the previously identified chromosome 10 LOH consensus region Dis-1 at the telomeric tip was hampered by the fact that none of the 66 schwannomas additionally analyzed displayed an LOH smaller than the F1 tumor #17 from the previous study on 43 tumors (4). The minimum consensus region for Dis-1 therefore represents 4.56.0 cM based on linkage studies with (BDIX x BDVI) F2 and (BDIX x BDIV) F2 crosses, respectively (Figure 3
). The average relation between linkage and physical distance, at least in humans, is supposed to be ~1 cM/Mb (10). Nevertheless, there are indications that Dis-1 actually covers a shorter physical distance. While the average recombination frequency in females is higher than in males, the recombination rate in both sexes is elevated near the telomere, with a more prominent increase in males. The linkage map generated by genotyping of the (BDIX x BDVI) F2 generation is based on 83% male recombination. Therefore, the Dis-I locus may in fact be considerably smaller than 4.56.0 Mb. This conclusion is supported by the unequally clustered and statistically improbable distribution of the eight markers placed inside the Dis-1 locus. This distribution is unlikely to represent the real physical distribution, but could reflect major recombination points, so called `hot spots', simulating a nonexistent physical distance, a phenomenon known to occur at male telomeres (11,12). Our assumption that the Dis-1 locus may be smaller than 4.5 Mb is in accordance with recently published data from the human genome project where the homologous region on chromosome 17q25.3 spans ~about 3 Mb.
The observed predominant loss of the BDIV allele on chromosome 10 is not necessarily caused by a rat strain-specific difference concerning a putative tumor suppressor gene at Dis-1. It would equally be consistent with a locus (Mss-1) residing 30 cM away towards the centromere, Mss-1 is simultaneously deleted in most schwannomas and mediates strain-specific susceptibility for the development of trigeminal schwannomas, apparently also displaying tumor suppressor activity (4). Together with the data from the human genome project, the high resolution linkage map constructed, the building of a contig currently in progress, and the resulting STS map, will help identify candidate genes/ESTs at the Dis-1 locus. It is noteworthy that known tumor suppressor genes assigned to rat chromosome 10, such as Nf1, p53, and Brca1 are not located within the Dis-1 consensus region (1315).
As monosomy of chromosome 5 has previously been seen in primary schwannoma cell preparations (unpublished data), LOHs on this chromosome are likely to be due to the loss of genetic material. At least the telomeric quarter of chromosome 5 was affected by LOH in most of the schwannomas analyzed in the present study (Figure 2
). Nevertheless, it is not understood why so far only schwannomas of BDVI x BDIX F1 rats showed partial LOH of chromosome 5. The hypothesis that two tumor suppressor genes located at the ends of a chromosome always result in complete chromosome loss (16) could explain the findings in BDIXxBDIV F1 animals. However taking into account the results from the (BDIX x BDVI) F1 cross, a more complex working hypothesis is needed. In a recent publication by Shao et al. (17) a possible explanation was given for chromosome- and cross-specific mitotic recombination frequency. These authors reported (17) the suppression of mitotic recombination in rodents by weaker sequence homology for a given chromosome. Thus, chromosome 5 sequences in (BDIX x BDVI) F1 rats might possibly be more related than in (BDIXxBDIV) F1 rats, resulting in the observed ~20% and 0% partial LOHs, respectively. It remains for detailed karyotype analyses of primary (BDIX x BDVI) F1 schwannoma cells to clarify whether this difference in partial LOH is due to mitotic recombination or deletion.
In summary, the BD rat model of EtNU-induced schwannomas shows two highly frequent LOHs on chromosomes 5 and 10, with almost no background losses or MI. These genetic alterations are schwannoma-specific, since neither in brain tumors nor in kidney tumors from the same animals LOHs on chromosome 5 or 10 have been detected so far (18).
We assume that the EtNU-induced process of carcinogenesis in the rat PNS is initiated by an activating point mutation in the neu/Erbb-2 proto-oncogene. The selective advantage of the mutant glial precursor cells leads to the formation of a proliferative cell subpopulation at high risk for further genetic alterations. A subsequent mitotic recombination event, at least at the telomeric tip of chromosome 10, results in uniparental disomy, with preferential loss of the allele of the resistant BDIV strain in heterozygous animals. Monosomy of chromosome 10 is rare in rat tumors (19) and was never observed in chromosome spreads from 6 primary schwannoma cell preparations (unpublished data), possibly due to the fact that monosomy does not provide a proliferative advantage for premalignant cells. Later on, LOH of the whole chromosome 5, or at least its telomeric quarter, occurs in most cases through mechanisms which include nondisjunctional chromosome loss. The proposed sequence of LOH events is supported by comparison of the quantitative allelic ratios of chromosomes 5 and 10 in identical tumor samples.
Our recent studies have shown that genes counteracting schwannomagenesis in the resistant BDIV strain might be associated with the recognition and elimination of premalignant cells via apoptopic pathways, possibly involving the immune system (6). More detailed information on gene function, in combination with the localization of candidate genes by LOH as well as linkage analysis, will help identify genes involved in chemical neurooncogenesis in this animal model and hopefully in the human counterpart tumors.
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Notes
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2 Present address: Institute of Neuropathology, University of Bonn, Sigmund-Freud-Strasse, D-53105 Bonn, Germany 
3 To whom correspondence should be addressed Email: rajewsky{at}uni-essen.de 
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Acknowledgments
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We thank Claudia Lechleiter and Thomas Haberland for expert assistance with animal husbandry. This work was supported by the Wilhelm Sander-Stiftung, (grant 94.009.3 to A.K.-R. and M.F.R.), the Dr Mildred Scheel Stiftung für Krebsforschung, (grant W87/92/Ra 5 to M.F.R.), the National Foundation for Cancer Research, USA, through Krebsforschung International e.V. (M.F.R.), and by a fellowship granted to B.U.K. by Krebsforschung International e.V. during part of this study.
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Received December 28, 2001;
revised February 1, 2002;
accepted April 1, 2002.