Murine Pancreatic Tumor Cell Line TD2 Bears the Characteristic Pattern of Genetic Changes with Two Independently Amplified Gene Loci

Murine pancreatic tumor cell line TD2 bears the characteristic pattern of genetic changes with two independently ampliﬁed gene loci

Bettina Schreiner1, Florian R Greten2,3, Dorotthe M Baur4, Alexander A Fingerle4, Ulrich Zechner4, Christian Bo¨ hm5, Michael Schmid5, Horst Hameister1 and Roland M Schmid3,4,*

1Department of Human Genetics, University of Ulm, D-89069 Ulm, Germany; 2Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0636, USA; 3Department of Internal Medicine II, Technical University of Munich, D-81675 Munich, Germany; 4Department of Internal Medicine I, University of Ulm, D-89069 Ulm, Germany; 5Institute of Human Genetics, University of Wu¨rzburg, D-97074 Wu¨rzburg, Germany

TGFa/p53 þ /À transgenic mice represent a genetically that lead to final tumor development (Hruban et al., engineered mouse model for pancreatic adenocarcinoma. 2000; Bardeesy et al., 2001). To be able to investigate the The tumors develop a characteristic pattern of secondary different steps of genetic alterations occuring during genetic changes. From one of these tumors, the permanent cancer progression, a transgenic mouse tumor model cell line TD2 was established. Here, we describe in detail was established, in which TGFa is expressed under the the genetic changes by molecular–cytogenetic techniques. regulation of the elastase promoter. This ensures TGFa The original tumor-specific CGH profile has been retained overexpression restricted to pancreatic acinar cells unchanged. The most characteristic aberration pattern (Sandgren et al., 1990, 1991). It has already been shown bears chromosome 11. Egfr, localized on proximal that in this tumor model, the Ras/ERK pathway is chromosome 11, is amplified two to three times and leads constitutively active (Wagner et al., 2001). After a to an easily identifiable, stable marker chromosome with a latency period for more than 1 year, this leads to large amplification unit, which is present in each pancreatic tumor development. When crossed into a metaphase. The wild-type p53 gene on distal chromosome background of p53 þ /À heterozygous mice, the latency 11 is lost. The p16Ink4a locus on chromosome 4 is for tumor induction is reduced in a manner which turns hypermethylated. For c-Myc a 15-fold amplification, this tumor model feasible for laboratory investigation. present in a 1.65 Mb amplification unit, is detected on The tumors bear similar genetic changes, as were chromosome 15. Transition between presence in the form observed in human pancreatic cancer, which recom- of several double minutes, DMs, or a single homoge- mends this system as an in vivo model for pancreatic neously staining region, HSR, was observed for c-Myc. cancer (Wagner et al., 2001). Molecular–cytogenetic analysis of both amplification In this study, we describe the genomic changes in the units show that Egfr amplification and c-Myc amplifica- cell line TD2 originating from this genetically engineered tion represent two alternative modes by which genes get tumor model. A pattern of specific chromosomal aberra- amplified in tumor cells. The expression level of the tions that can be found in the original tumor tissue respective genes was proven by Northern blot analysis. (Schreiner et al., submitted) was detected. These changes The cell line TD2 represents a valuable in vitro model for include gain of proximal chromosome 11, encompassing pancreatic adenocarcinoma. epidermal growth factor receptor (Egfr), combined with Oncogene (2003) 22, 6802–6809. doi:10.1038/sj.onc.1206836 deletion of the distal part of the same chromosome, encompassing the wild-type p53 locus and hypermethy- Keywords: murine pancreatic tumor cell line; c-Myc lation of the p16Ink4a locus. Further recurrent genetic and Egfr amplification; p53 deletion; p16Ink4a hyper- changes were observed as gain of part of chromosome methylation 15, where c-Myc has been mapped, or – alternatively – ONCOGENOMICS loss of distal chromosome 14 including the Rb1 locus. Thus, the cell line TD2 and the mouse tumor progres- Introduction sion model represent powerful tools for future studies of pancreatic tumor growth in vitro and in vivo. Cancer of the pancreas is one of the most common cancer entities in human. Owing to the silent course of cancer development in the peritoneal cavity, this tumor Results gets diagnosed only at far advanced stages. This has hampered description of the cascade of genetic lesions Cytogenetic studies

*Correspondence: RM Schmid, Chromosome analysis of the TD2 cell line was E-mail: [email protected] performed at passage 23 and 103. No striking differ- Received 11 April 2003; revised 2 June 2003; accepted 2 June 2003 ences were noticed. The chromosome number ranged c-Myc and Egfr amplification in the pancreatic cell line TD2 B Schreiner et al 6803 between 42 and 80. Of all the analysed metaphases, 87% confirmed. In the original tumor sample, an additional were hypertriploid to hypotetraploid, containing 65–77 loss of distal chromosome 11 including the p53 locus is chromosomes. In all metaphases, one large marker observed. In contrast to these findings, the CGH profile chromosome with distinct bands in the proximal half of distal MMU 11 of TD2 cells does not reach the cutoff and a varying number of several small marker chromo- level (Figure 2a). However, the loss of the wild-type p53 somes of uncertain origin were observed (Figure 1). allele on MMU 11 in TD2 cells was proven by independent PCR analysis (not shown). Analysis by spectral karyotypinq (SKY) Calibration of DNA copy changes by real-time PCR Spectral karyotyping (SKY) was performed at passage 20. One representative karyotype is shown in Figure 1, To define more exactly the length of the amplification consisting of an inverted DAPI-banded karyotype unit and the degree of amplification, calibration was together with the classified images in pseudocoloration. performed by real-time PCR (Figure 2b). For chromo- The chromosomal aberration pattern consists mostly of some 11, the following genes mapping to the proximal numerical aberrations. All chromosomes are represented 50 Mb of MMU 11 were tested: Egfr (9 cM; 16.8 Mb); three to four times except X and Y chromosomes, and reticuloendotheliosis oncogene, c-Rel (13 cM; 23.8 Mb); chromosome 17, which is present in five copies. Besides serine/threonine kinase 10, Stk10 (16 cM; 35.6 Mb); and several small marker chromosomes of varying number FMS-like tyrosine kinase 4, Flt4 (25 cM; 50.0 Mb). and undefined origin, one additional large marker Except Flt4, all analysed genes showed a relative chromosome is found in all metaphases. The proximal amplification of 2.2–3.0 times. Flt4, which maps at part of this marker chromosome is of chromosome 11 50 Mb, is not included in the amplification unit. There- origin and consists of three conspicuous dark-staining fore, the amplification unit extends at least 35.6 Mb but bands as already noticed in G-banded metaphases. The at most 49 Mb. distal part of the marker derives from chromosome 5. From MMU 15, the genes myelocytomatosis onco- Conventional banding and SKY analysis revealed this gene, c-Myc (32.0 cM; 62.4 Mb), adenylate cyclase 8, marker chromosome together with the small unidentifi- Adcy8 (37.5 cM; 65.1 Mb) and protein tyrosine kinase 2, able marker chromosomes as the only structurally Ptk2 (42.0 cM; 73.7 Mb) were chosen. The copy number rearranged chromosomes. of the genes Adcy8 and Ptk2 is not affected in this cell line, but for c-Myc 15–16 times amplification was CGH analysis detected (Figure 2b). To allow comparison of the cell line with the original Direct fluorescence in situ hybridization (FISH) analysis tumor sample, CGH analysis was performed with DNA with gene-specific probes extracted from the cell line at passage 25. Though the mean chromosome number indicates a highly aberrant Real-time PCR and CGH verified the amplification of karyotype, only two gains which surpassed the cutoff the c-Myc locus, although this has not been observed by level of 1.25 are detected (Figure 2a). Almost the whole conventional cytogenetic and SKY analysis (Figure 1). chromosome 17 and the proximal part of chromosome To detect the localization of the amplified c-Myc gene, 11 are amplified. For MMU 15, a very specific CGH fluorescence in situ hybridization (FISH) with c-Myc- profile is noticed with gain of chromosomal material at specific BAC probes (RPCI23-235H07, RPCI23-98D8, the beginning of the second half of the chromosome. RPCI23-55F11) was performed. Many strong signals Compared to the original tumor sample of that cell outside the chromosomes were observed (Figure 3a). line (not shown) the aberration pattern is widely These signals indicate the presence of double minutes

Figure 1 Karyogram of a representative metaphase spread of cell line TD2 (passage 20) hybridized for SKY. Inverted DAPI stained and classiﬁed chromosomes in pseudocoloration are shown. Marker chromosomes are indicated by M

Oncogene c-Myc and Egfr amplification in the pancreatic cell line TD2 B Schreiner et al 6804 (DMs) which are too small to get detected by conven- with HSRs it is too weak to get detected simultaneously tional DAPI banding. Another type of c-Myc amplifica- with the brilliant HSR signal. Therefore, a partial tion is observed by hybridization of the same BAC metaphase of TD2 is shown in Figure 3c. probes to metaphases prepared from higher passage The integration of the HSR does not occur on MMU cultures. Metaphases with a homogeneous staining 15 which harbors the endogenous c-Myc locus, but on region (HSR) of c-Myc DNA existed in parallel with another chromosome. By use of whole-chromosome metaphases, in which c-Myc was present as DMs painting probes, WCPs, the HSR-bearing chromosome (Figure 3b). DM appeared in about 46% of all was revealed as chromosome 6 in most cases. As is metaphases and a HSR is noticed in 54%. shown in Figure 4a, b, d and f, the integration of the The original FISH signal from the endogenous c-Myc HSR on chromosome 6 occurred at variable positions. locus on MMU 15 is also detectable, but in metaphases In a minority of metaphases, the HSR is even integrated in further, hitherto unknown chromosomes (Figure 4c, e). The use of the WCP 6 probe further made clear that some chromosomal material of chromosome 6 is included in the DMs and is found interspersed in some HSRs (Figure 4b, c, g). The analysis of the amplification unit on MMU 15 by real-time PCR revealed that genes mapping next to c- Myc are not amplified. To determine the length of the amplification unit, overlapping BACs from a contig of MMU 15D2-3 of the Ensembl Mouse Genome Server, which includes c-Myc-specific BACs, were hybridized to TD2 metaphases (Figure 5). According to these FISH analyses, the amplification unit spans about 1.65 Mb from 61.65 to 63.3 Mb. All BACs mapping between RPCI23-386K4 and RPCI23-23L17 were amplified together with the c-Myc locus. No difference with respect to the length of the amplification unit on DMs or HSR was observed. Also, the length of the amplification unit from proximal chromosome 11 was defined by analysis with chromosome-specific BAC probes. Probes mapping from the centromere down to 50 Mb were hybridized Figure 2 CGH analysis and real-time PCR of cell line TD2. (a) CGH: only chromosomes with conspicuous profiles are shown. on metaphase spreads. Signals on the marker chromo- Green lines on the left of the chromosome profile represent losses, some were detected from BACs RPCI23-282P12 (4 Mb) red lines on the right indicate gains of chromosomal material. to RPCI23-273P11 (36.1 Mb) including Egfr, c-Rel and For each chromosomal profile, at least 20 chromosomes Stk10. All of these BAC probes revealed the same were calculated. (b) Quantitation by real-time PCR. The relative amplification rates of the tested genes of proximal chromosome 11 number of repetitions on the same marker chromosome (dark gray) and chromosome 15 (light gray) are shown. The Gcnt2 (Figure 6). On elongated chromosomes, five signals gene was taken as internal reference were observed. Flt4-specific probes revealed signals only

Figure 3 Hybridization of c-Myc-speciﬁc BACs (RPCI23-98D8; RPCI23-55F11; RPCI23-235H7) on metaphase spreads from cell line TD2. The c-Myc locus is ampliﬁed in the form of DMs (a), or an HSR chromosome (b). In (c) the endogenous c-Myc locus is shown in a partial metaphase

Oncogene c-Myc and Egfr ampliﬁcation in the pancreatic cell line TD2 B Schreiner et al 6805

Figure 4 Most frequent chromosomal markers in the TD2 cell line containing c-Myc ampliﬁcation. Top column: schematic illustration of the different markers. The six most frequent marker chromosomes (a–f) and DM chromosomes (g) are shown. Green: chromosomal material from chromosome 6; Red: c-Myc-HSR. Middle column: FISH with WCP 6 (green signal) and c-Myc-speciﬁc BAC-clones (red signal). Of all metaphases, 66% with HSR marker chromosomes contain marker A, 13% marker B or C and 21% contain marker D, E, F or some more seldom variants. Also, in DMs, some residual DNA from chromosome 6 is detectable (g)

Figure 5 BACs from library RPCI23 or RPCI24 were chosen from contig 15006 (Ensembl mouse genome Server V. 7.3a.1) and aligned from centromere to telomere. BAC clones positive for c-Myc are indicated in red. BAC clones shown in green and the c-Myc- specific BAC clones (red) are included in the amplification unit on DMs and the HSR. BACs shown in blue are not included in the amplification unit

on the normal chromosome 11, but not on the marker respective Northern blot is shown in Figure 7a–e. Only a chromosome. Therefore, Flt4 at 50.0 Mb is not part of basic expression level is detected for c-Rel (Figure 7d). the amplification unit. As shown in Figure 6 and Egfr is expressed at a low level, 1.8 times more than in illustrated in the accompanying scheme, five large NIH3T3 cells (Figure 7c), and 3.5 times when compared amplification units in inverted order are integrated in with wild-type pancreas (not shown). c-Myc is over- tandem in the marker chromosome. expressed three times compared to NIH3T3 cells (Figure 7a). The p16INK4a locus has been suggested to play a specific Expression studies role during progression of pancreatic adenocarcinomas The genes found amplified, Egfr and c-Rel from MMU (Caldas et al., 1994). However, CGH and LOH analyses 11 and c-Myc from MMU 15, were analysed for their in TD2 cells were unconspicuous for this locus on state of gene expression by Northern blot analysis. A chromosome 4. Analysis of the methylation status of the

Oncogene c-Myc and Egfr ampliﬁcation in the pancreatic cell line TD2 B Schreiner et al 6806

Figure 6 SKY analysis and FISH of the typical marker chromosome of cell line TD2. (a) SKY: with pseudo-coloration, the different origin of chromosomal material of this marker could be classified. (b) Inverted DAPI staining image. (c) FISH with specific BAC probes for c-Rel (RPCI23-207N19; green) and Stk10 (RPCI23-47J10; red) together with a schematic drawing. Arrows on the right side of the scheme illustrate the head-to-head and tail-to-tail orientation of the amplification unit

p16Ink4a promoter region using a methylation-specific proximal chromosome 11 including the TGFa receptor PCR assay showed hypermethylation of this sequence Egfr, combined with deletion of distal chromosome 11 (Figure 7f), as was already observed in the original including the p53 locus and hypermethylation of the tumor sample. Also, some unmethylated p16Ink4a pro- p16Ink4a locus on chromosome 4. This specific chromo- moter sequences were detectable by a nested PCR somal aberration pattern is found complemented with procedure specific for the unmethylated promoter amplification of the c-Myc locus on chromosome 15. sequence (Figure 7f). To prove the successful conversion From one of the original tumors, the cell line TD2 was of cytosin to uracil residues after bisulfite treatment, a established (Greten et al., 2002). primer pair for the unmodified sequence was used and As proven by the combined molecular–cytogenetic no amplification product obtained (data not shown). analysis, the original pattern of chromosomal changes has been preserved (Figure 2). According to PCR p53 Electromobility shift assay (EMSA) analysis also the wild-type locus is deleted, though the CGH profile did not reach the cutoff level for the In the large amplification unit of MMU 11, Egfr and deletion of distal chromosome 11. In a minority of the also c-Rel is included. Therefore, the possible effects of original tumor samples, LOH for the p16Ink4a locus was c-Rel amplification on NF-kB-binding activity were present; in other tumors, p16Ink4a was inactivated by studied by performing EMSA. Nuclear extracts of TD2 promoter methylation (Schreiner et al., submitted). In cells (passage 105) were incubated with a 32P-labeled the TD2 cell line, the regulatory 50 region of the p16Ink4a oligonucleotide containing the sequence of the NF-kB locus is hypermethylated, though upon thorough recognition site. A strong binding activity was revealed analysis some unmethylated p16Ink4a alleles were also (Figure 7g). To determine to which NF-kB proteins the found (Figure 7f). p53 and p16Ink4a deletion/inactivation binding activity is due, supershift assays were performed are combined in the same tumor cell line. This cell line using antibodies against all members of the NF-kB reflects all genetic alterations observed in the primary transcription factor family: RelA, NF-kB1, NF-kB2, c- tumor. Rel and RelB. A supershift band was observed for RelA It was surprising to observe mostly numerical and NF-kB1, but not for c-Rel (Figure 7g). chromosomal changes. Without regard to the unidentifi- able small marker chromosomes (Figure 1), only one aberrant chromosome was present. This unusual pattern Discussion of changes with a low number of structural chromosomal aberrations has been observed in further murine The TGFa/p53 þ /À transgenic mouse has been shown to tumor cell lines (Weaver et al., 1999; Wu et al., 2002). be an informative tumor model for pancreatic adeno- Whereas in human pancreatic adenocarcinoma cell lines carcinoma (Wagner et al., 2001). During analysis of the various structural chromosomal aberrations are present secondary genetic changes, which are induced during (Curtis et al., 1998), in murine tumors nondisjunction tumor progression, it was surprising to observe that this seems to be the preferred type of chromosomal genetically modified system canalyses a relative strict aberration. In spite of this, the pattern of genetic cascade of consecutive genetic changes (Schreiner et al., changes, as determined by CGH, is very similar when submitted). A common finding is amplification of human and mouse cell lines are compared.

Oncogene c-Myc and Egfr amplification in the pancreatic cell line TD2 B Schreiner et al 6807 small to get detected by conventional cytogenetic analysis and also, a HSR chromosome may be difficult to recognize as such in an aberrant mouse karyotype. Upon continuous growth, we observed that the presence of c-Myc amplification in the form of DMs or HSR is variable. At low passage, only DMs were observed. At higher passage, metaphases with an HSR chromosome dominated, but no combination of DMs and HSR chromosome in one and the same metaphase was detected (Figure 3). The HSR integration did not take place on the original chromosome 15, but on another chromosome. This is the usual finding for this type of gene amplification (Wahl, 1989; Schwab, 1999). In most metaphases, the HSR is integrated on chromosome 6. But as is evident from Figure 4, the integration on chromosome 6 is variable. Further evidence for variation between the presence of c-Myc amplification as DMs or HSR provides hybridization of respective metaphases with the WCP 6 probe. Also on the extrachromosomal DMs the probe detects material from chromosome 6. The most simple interpretation of this unusual observation is that these DMs in their former history did integrate on MMU 6, but later switched over to the extrachromosomal DM state again. This interpretation is in contrast to the commonly held view of the emergence of DMs and HSRs (Wahl, 1989). This is seen as a one-way process from extrachromosomal episome formation, via DM development to stable integration as HSR into another chromosome. But, obviously, the c-Myc locus is subject to enhanced mobility in the cell line described here. The length of the c-Myc amplification unit has been determined up to now only in human tumor cell lines (Maurer et al., 1987). FISH with BAC clones mapped to chromosome 15 indicates a length of the c-Myc amplification unit of 1.65 Mb. This amplification unit includes the Pvt1 locus (Figure 5). Therefore, the unit is Figure 7 (a–e) Gene expression tested by Northern blot analysis considerably larger than the human c-Myc amplification of c-Myc (a), Egfr (c), c-Rel (d) and b-actin (b, e) as control and units in the cell lines HL-60 and COLO320, which are NIH3T3 cells as reference. (f) Analysis of the methylation status of 120–250 kb long (Maurer et al., 1987). the p16Ink4a-promoter region. Primers for the methylated and The organization of the amplification unit on proxi- unmethylated sequences were used for cell line TD2, normal mal chromosome 11 is quite different. Here, a large unit pancreatic cells (NP) and normal DNA which was not treated with bisulfite (NPnt); (N) negative control. (g) EMSA of TD2 cells extending at least from Egfr at 9 cM (16.8 Mb) to Stk10 showing NF kB/Rel activity (lane 1). By supershift assay using at 16.0 cM (35.6 Mb) is amplified. The amplified unit is antibodies against RelA and NFkB1 binding specificity was integrated in tandem and in inverted order on a marker confirmed chromosome. This marker chromosome is made up of chromosome 11 in its proximal part and of chromosome It is shown here that only the combined analysis by 5 in its distal part. Therefore, Egfr amplification takes cytogenetic and molecular techniques is sufficient to place on the endogenous chromosome, on which Egfr detect all relevant tumor-specific chromosomal changes. maps. The large amplification unit is visible even by The classical cytogenetic banding analysis and also the conventional cytogenetic techniques by a periodic ladder- elegant SKY analysis did not reveal c-Myc amplifica- like banding pattern on an easily identifiable large tion. This became evident only by CGH analysis and marker chromosome (Figure 6). This marker chromo- here again, only due to the fact that amplification was some is present in one copy and was observed in each really high. Bei real-time PCR c-Myc amplification up to metaphase every time during passaging the cell line. 15–16 times was observed. On a nearly triploid By real-time PCR, a threefold amplification of the chromosomal background – as is given for cell line Egfr locus was observed (Figure 2). On the triploid TD2 – about 45–48 extra copies of c-Myc are present. chromosomal background of cell line TD2, this indi- By FISH analysis with c-Myc-specific genomic probes, cates the presence of five to six extra copies of Egfr, the c-Myc amplification became visible in the form of c-Rel and Stk10, though on the single marker chromo- DMs, or as an HSR (Figure 3). The DMs are far too some only three units get detectable at first sight

Oncogene c-Myc and Egfr amplification in the pancreatic cell line TD2 B Schreiner et al 6808 (Figure 1). On enforced analysis of elongated metaphase Cytogenetic analysis chromosomes, the green signals of the c-Rel probe Chromosomes were prepared at passage no. 23, 30, 103 and and the red signals of the Stk10 probe were sometimes 119. GTG banding was done by standard techniques. WCPs observed as double signals. This would be in line with were used according to the manufacturer’s protocol (Cambio an inverted order of the amplification units on the Ltd, Cambridge, UK). Bacterial artificial chromosomes marker chromosome, as is shown in the schematic (BACs) of MMU 11 were chosen from the Baylor College of drawing (Figure 6c). An inverted order is due to an Medicine database: ‘Mouse Chromosome 11 Bac mapping’ amplification mechanism, which differs from that just (www.mousegenome.bcm.tmc.edu) and further gene-specific described for the c-Myc amplification. These differences BACs from the Ensembl Mouse Genome Server (www.ensem- are: (1) the amplified sequences remain on the original bl.org/Mus_musculus/) version 3.1.1. BACs from MMU 15D2-3 were elected according to the map provided by the Egfr-encoding chromosome 11; (2) the amplification Ensembl Mouse Genome Server, contig 15006. All BACs are unit is extremely large, encompassing more than a from library RPCI-23 or RPCI-24 and purchased at the cytogenetic band with at least 35.6 Mb; and (3) the BACPAC resource center (www.chori.org/bacpac). For FISH, degree of amplification itself is limited; only five standard techniques were applied. extra copies of the Egfr unit are present. These are characteristics of an amplification model according to Spectral karyotyping (SKY) the breakage/fusion/bridge model, as extensively out- The chromosome preparations were hybridized with the mouse lined by Ma et al. (1993). According to this model, SKY probe mixture (SkyPaintt, Applied Spectral Imaging amplification occurs by an intrachromosomal mechan- Ltd, Israel), according to the manufacturer’s instructions. The ism and leads to an inverted repeat organization of SKY probe mixture was applied to the preparations and the amplification unit. This inverted organization causes hybridized for 48 h in a humidified chamber. Washing and the described ladder-like appearance of the FISH signals detection was applied using standard techniques. For image on the marker chromosome (Toledo et al., 1992; acquisition, the SpectraCube system SD200 including a triple- Hellman et al., 2002). It is obvious that both of these band-pass filter was connected to a fluorescence microscope different amplification mechanisms – intrachromosomal (Axiophot, Zeiss, Germany), assisted by Spectral Imaging by a breakage/fusion/bridge cycle for the Egfr region Software, version 2.5. The final analysis of the metaphases was and extrachromosomal via DMs for c-Myc – are performed with the help of the Sky View program, version 1.6.1 (Applied Spectral Imaging Ltd, Israel). realized in the same cell line. This is only possible in a cell line with enhanced genetic instability. In cell line Comparative genomic hybridization (CGH) TD2, this is thought to be due to the loss of p53 activity at an early stage during tumor induction (Paulson et al., DNA from the cell line TD2 (passage 25) and normal female 1998). mouse tissue as reference DNA were used. CGH was It was remarkable to observe such a large amplifica- performed as described (Kallioniemi et al., 1992), with slight tion unit around the Egfr locus, which raises the modifications (Schreiner et al., in preparation). The DNA from cell line TD2 was labeled with biotin-16-dUTP, the suspicion that besides Egfr there might be another normal reference DNA with digoxigenin-11-dUTP, using gene on proximal chromosome 11, whose activity is standard nick translation. beneficial for tumor growth. One attractive candidate gene would be c-Rel. In pancreatic tumor tissue, NF-kB Real-time PCR has been shown to be active (Wang et al., 1999). One of the several different dimerization partners of DNA amplification was quantified using real-time PCR NF-kB is c-Rel (Gilmore, 1999). To prove if c-Rel analysis (TaqMan, ABI PRISM 7700 Sequence Detection System, PE Applied Biosystems, Norwalk, CT, USA). PCR or other members of this transcription factor family reaction, denaturation at 951C for 2 min followed by 40 cycles bind to the NF-kB-affinity sequence, EMSAs were of 951C for 15 s and 601C for 1 min (Sybr Green PCR Core performed on nuclear extracts of TD2 cells. These Reagents, PE Applied Biosystems) were performed using assays show a constitutive activity of NF-kB; however, genomic DNA extracted from TD2 cells as well as normal the complex seems to consist of RelA/NF-kB1 dimers DNA as reference. The relative quantitation of the target (Figure 7g). DNA level was calculated with the comparative CT method The cell line established here from murine pancreatic according to the manufacturer’s protocol. adenocarcinomas bears all genetic changes of the original tumor. The TD2 cell line has been shown to be also Electromobility shift assay (EMSA) a potent inducer of pancreatic adenocarcinomas in Nuclear extracts were prepared from TD2 cells according to vivo (Greten et al., 2002). In future, this cell line will standard techniques. Protein concentration was determined by prove a valuable in vitro model for tumor therapeutic Bradford assay (Bio-Rad Laboratories, Munich). The oligo- studies. nucleotide used for binding reaction contained the high- affinity kB sequence of mouse as present in the k-light-chain enhancer. The oligonucleotide sequence, EMSA and supershift Materials and methods assays are described elsewhere (Steinle et al., 1999).

Establishment of the cell line TD2 Northern blot analysis The tumor cell line TD2 is derived from a pancreatic tumor of Total cellular RNA was isolated from TD2 cells at cell culture a male TGFa/p53 þ /À transgenic mouse (Greten et al., 2002). passage 86 and 136, as well as from NIH3T3 cells with the

Oncogene c-Myc and Egfr amplification in the pancreatic cell line TD2 B Schreiner et al 6809 Qiagen RNeasy purification kit. About 25 mg RNA was used GGG TAT TGA ATT TTC GC 30) and p16-M2 (50CAC GTC for Northern blot analysis, which was conducted according to ATA CAC ACG ACC CTA AAC CG 30) (Bardeesy et al., standard protocol. Probes for c-Myc, Egfr and c-Rel were 2002). To amplify the unmethylated allele, a nested PCR generated by PCR. The b-actin probe was purchased from reaction was necessary. First, the PCR reaction was performed Clontech, CA, USA. All probes were labeled with the with primers FM6 (50TTT TTA GAG GAA GGA AGG AGG rediprime II random prime labeling system (Amersham GAT TT 30) and p16-UB (50ACC CAC ACA TCA ACA CAA Biosciences, UK) in the presence of [a-32P]dCTP. Autoradio- CCC TAA 30) (Patel et al., 2000). In the second PCR assay, the graphs were obtained by exposing the membrane for 8–24 h at nested primers Un1-p16 (50GTA ATT GGG TGG GTA TTG À701C. The signals were detected by Fujifilm FLA-300 and AAT TTT TGT G 30) and Un2-p16 (50CAC ACA TCA TAC BAS Reader (V 2.26, raytest, Straubenhardt, Germany) and ACA CAA CCC TCC ACC/T A 30) were used. quantified by Aida Image Analyzer (V 2.31).

ink4a R16 methylation assay Acknowledgements Total genomic DNA (700 ng) was restricted with EcoRI at We thank Antje Kollak, Alexandra Kilian and Beate Knobl 371C for at least 6 h. The bisulﬁte treatment was performed as for excellent technical assistance. This work was supported by described by Hajkova et al. (2002). The methylated sequence grants from the Deutsche Forschungsgemeinschaft SFB 518 was ampliﬁed with the primers p16-M1 (50CGA TTG GGC (Teilprojekt B6) and IZKF Ulm (Teilprojekt C4) to RMS.

References

Bardeesy N, Morgan J, Sinha M, Signoretti S, Srivastava S, Maurer BJ, Lai E, Hamkalo BA, Hood L and Attardi G. Loda M, Merlino G and DePinho RA. (2002). Mol.Cell. (1987). Nature, 327, 434–437. Biol., 22, 635–643. Patel AC, Anna CH, Foley JF, Stockton PS, Tyson FL, Bardeesy N, Sharpless NE, DePinho RA and Merlino G. Barrett JC and Devereux TR. (2000). Carcinogenesis, 21, (2001). Semin. Cancer Biol., 11, 201–218. 1691–1700. Caldas C, Hahn SA, da Costa LT, Redston MS, Schutte M, Paulson TG, Almasan A, Brody LL and Wahl GM. (1998). Seymour AB, Weinstein CL, Hruban RH, Yeo CJ and Kern Mol. Cell. Biol., 18, 3089–3100. SE. (1994). Nat. Genet., 8, 27–32. Sandgren EP, Luetteke NC, Palmiter RD, Brinster RL and Curtis LJ, Yong L, Gerbault-Sereau M, Kuick R, Dutrillaux Lee DC. (1990). Cell, 61, 1121–1135. A-M, Goubin G, Fawcett J, Cram S, Dutrillaux B, Hanash S Sandgren EP, Quaife CJ, Paulovich AG, Palmiter RD and and Muleris M. (1998). Genomics, 53, 42–55. Brinster RL. (1991). Proc. Natl. Acad. Sci. USA, 88, 93–97. Gilmore TD. (1999). Oncogene, 18, 6842–6844. Schwab M. (1999). Semin. Cancer Biol., 9, 319–325. Greten FR, Weber CK, Greten TF, Schneider G, Wagner M, Steinle AU, Weidenbach H, Wagner M, Adler G and Schmid Adler G and Schmid RM. (2002). Gastroenterology, 123, RM. (1999). Gastroenterology, 116, 420–430. 2052–2063. Toledo F, Le Roscouet D, Buttin G and Debatisse M. (1992). Hajkova P, EI Maarri O, Engemann S, Oswald J, Olek A and EMBO J., 11, 2665–2673. Walter J. (2002). Methods Mol. Biol., 200, 143–154. Wagner M, Greten FR, Weber CK, Koschnick S, Mattfeldt T, Hellman A, Zlotorynski E, Scherer SW, Cheung J, Vincent JB, Deppert W, Kern H, Adler G and Schmid RM. (2001). Smith DI, Trakhtenbrot L and Kerem B. (2002). Cancer Genes Dev., 15, 286–293. Cell, 1, 89–97. Wahl GM. (1989). Cancer Res., 49, 1333–1340. Hruban RH, Goggins M, Parsons J and Kern SE. (2000). Clin. Wang W, Abbruzzese JL, Evans DB, Larry L, Cleary KR and Cancer Res., 6, 2969–2972. Chiao PJ. (1999). Clin. Cancer Res., 5, 119–127. Kallioniemi A, Kallioniemi OP, Sudar D, Rutovitz D, Weaver ZA, McCormack SJ, Liyanage M, du MS, Coleman Gray JW, Waldman F and Pinkel D. (1992). Science, 258, A, Schrock E, Dickson RB and Ried T. (1999). Genes 818–821. Chromosom. Cancer, 25, 251–260. Ma C, Martin S, Trask B and Hamlin JL. (1993). Genes Dev., Wu Y, Renard CA, Apiou F, Huerre M, Tiollais P, Dutrillaux 7, 605–620. B and Buendia MA. (2002). Oncogene, 21, 1518–1526.

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