identification of methylation-associated gene expression in neuroendocrine pancreatic tumor cells

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Original Paper

Pancreatology 2007;7:352–359 DOI: 10.1159/000107270

Identification of Methylation-Associated Gene Expression in Neuroendocrine Pancreatic Tumor Cells

Nils Habbe

a, b Tillmann Bert

a Babette Simon

a

Departments of a Gastroenterology and Endocrinology, and b

Surgery, Philipps-University Marburg, Marburg , Germany

estingly, 5-aza-2 � -deoxycytidine treatment led to re-expres-sion of cofillin whereas matriptase expression levels were sig-nificantly lower. Both genes have been associated with metastastic spread and tissue invasion. The other differen-tially expressed genes play an unknown role in the course of neuroendocrine tumorigenesis. Conclusion: DNA methyla-tion appears to be an important molecular mechanism in the process of neuroendocrine pancreatic tumorigenesis and metastatic spread. The definition of DNA methylation pat-terns associated with neuroendocrine pancreatic tumors might open up the potential for a new sensitive diagnostic tool and might serve as a new antitumor target.

Copyright © 2007 S. Karger AG, Basel and IAP

Introduction

Development of tumors of the neuroendocrine gastro-enteropancreatic (GEP-NET) system is a rare and indo-lent event, with an average age-adjusted annual incidence of 2/100,000 [1] . Carcinomas of the neuroendocrine pan-creas, due to the anatomical location that makes early detection very difficult, can lead to severe morbidity and a rapid deterioration of the patient’s health. These types of neoplasm may secrete neuropeptides with clinical manifestations. Most of these tumors have a poor prog-nosis, and even using modern methods of histopatholo-gy, a reliable assessment of the patient’s prognosis cannot be made [2] .

Key Words

Carcinoid � Methylation � Neuroendocrine tumor � Epigenetics � Pancreas

Abstract

Background: CpG islands methylation is the main epige-netic modification found in human tumors leading to tran-scriptional silencing of certain tumor suppressor genes. Reacquisition of p16/CDKN2A tumor suppressor gene ex-pression by 5-aza-2 � -deoxycytidine results in concurrent growth inhibition of neuroendocrine pancreatic tumor cells. However, the growth suppressive effects of 5-aza-2 � -deoxy-cytidine is unlikely to be solely attributable to the restored p16/CDKN2A function, but rather a consequence of re-ex-pression of additional genes silenced by de novo methyla-tion. In an effort to validate DNA methylation as an important mechanism in neuroendocrine tumorigenesis and metastat-ic spread, we attempted to isolate methylation-specific tran-scripts in neuroendocrine pancreatic tumor cells. Methods: Differentially expressed methylation-associated genes were identified by cDNA-representational difference analysis (cDNA-RDA). Differential expression was confirmed by semi-quantitative RT-PCR using insert specific primers. Results: We identified 48 differently expressed gene fragments and methylation-associated expression was confirmed by semi-quantitative RT-PCR. 52,3% (25 of 48) showed elevated ex-pression levels after 5-aza-2 � -deoxycytidine treatment, whereas 47.7% revealed lower expression levels. 7 fragments showed homology to genes with unknown function. Inter-

Received: September 7, 2006 Accepted after revision: February 21, 2007 Published online: August 15, 2007

Dr. med. Nils Habbe Department of Surgery, Philipps-University Marburg Baldingerstrasse DE–35043 Marburg (Germany) Tel. +49 642 1286 2582, Fax +49 642 1286 8995, E-Mail [email protected]

© 2007 S. Karger AG, Basel and IAP1424–3903/07/0074–0352$23.50/0

Accessible online at:www.karger.com/pan

Methylation-Associated Gene Expression Pancreatology 2007;7:352–359 353

Epigenetics is defined as the inheritance of cellular information independent of the DNA nucleotide se-quence, in contrast to genetics, which is concerned with the information given from the nucleotide sequence [3] . Modulation of gene expression by alterations in DNA methylation pattern is often observed during malignant development in cancers [4–6] . Two major mechanisms have been identified so far: first, global hypomethylation of wide areas along the genome, predominantly in re-petitive DNA and endoparasitic sequences causes ampli-fication of proto-oncogenes, karyotypic instability, chro-mosomal recombinations and oncogene activation [7–9] . Second, hypermethylation of promotor regions contain-ing CpG islands leads to transcriptional silencing of tu-mor suppressor genes, whereas methylation within a gene can cause mutational events [10] . The methylation-mediated transcriptional silencing is caused by a variety of events including methylation of cytosines and estab-lishment of heterochromatin with modification of the histone tails, especially via methylation of either histone 3 lysine 9 (methyl-K9-H3) or lysine 27 (methyl-K27-H3) [11–15] .

Many approaches have been taken to use methylation patterns as biomarkers for early detection of cancer, clas-sification of tumors, response to drug treatment or as markers predictive of outcome [13, 15] .

In previous works, we could prove that restoring the expression of the p16/CDKN2A tumor suppressor gene by 5-aza-2 � deoxycytidine led to growth inhibition in the neuroendocrine pancreatic tumor cell line QGP-1 [16] . Assuming that the growth-suppressive effects of 5-aza-2 � -deoxycytidine are unlikely to be solely attributable to the restored p16/CDKN2A function but rather a conse-quence of reexpression of additional genes silenced by de novo methylation, the next step was to determine the dif-ferences in gene expression that might explain the growth suppressive effects between wild-type QGP-1 cells and cells treated with 5-aza-2 � -deoxycytidine to identify nov-el genes being transcriptionally silenced by methylation, employing cDNA-representational difference analysis (RDA) combined with RT-PCR. cDNA-RDA is a sensitive and efficient PCR-based method combining substractive hybridization and selective enrichment of genes while providing an efficient depletion of ubiquitous genes from both cDNA populations and cloning the fragments that are most differentially expressed [17, 18] .

Furthermore, we identified genes which are silenced after treatment with 5-aza-2 � -deoxycytidine. The poten-tial mechanisms underlying this lower expression will be further discussed.

Materials and Methods

Cell Lines The cell line derived from neuroendocrine pancreatic carci-

noma, QGP-1, was grown in RPMI 1640 (Gibco-BRL) containing 10% fetal bovine serum supplemented with penicillin 100 U/ml and streptomycin 100 �g/ml at 37 ° C.

5-aza-2 � -Deoxycytidine-Treatment Cells were grown in media containing 1 �M 5-aza-2�-deoxy-

cytidine for 72 h and harvested by trypsinization. Total RNA was isolated using RNeasy Mini Kit (QIAgen) according to the proto-col provided by the manufacturer.

cDNA-RDA RNA Preparation . Total RNA (200 � g) of each treated and un-

treated cells was used for mRNA isolation by poly(A) + selection using magnetic beads (PolyATract System IV, Promega). Double-stranded cDNA synthesis was performed with 5 � g of the result-ing mRNA (cDNA Synthesis System, GIBCO-BRL). The cDNA strands were digested by DpnII (New England Biolabs, Beverly, Mass., USA) and extracted with phenol-chloroform and precipi-tated in ethanol. cDNA fragments were ligated to R-BGL-24-oligomers in the presence of R-BGL-12-oligomers (Metabion). Li-gated cDNA fragments were diluted and amplified for 21 cycles with R-BGL-24 as primer at an annealing temperature of 72 ° C. The resulting amplicons were extracted with phenol-chloroform and digested by DpnII to remove the R-BGL-24 adapters. These steps were performed in parallel for treated and untreated QGP-1 samples.

Tester Preparation. Amplicons prepared from untreated and treated RNA were each used as tester in independent reactions. For tester preparation, 15 � g of amplicon was separated and visu-alized on a 1.5% agarose gel (Sigma). The size range of 100–1,000 bp was excised and extracted from the gel slices using the QIA Ex kit (Qiagen, Valencia, Calif., USA). 2 � g of the gel purified tester was ligated to new oligomers, J-BGL-24. As a new step, PCR was performed using the new ligated tester as template with J-BGL-24 as primer. This step ensures that ligation has worked before sub-stractive hybridization takes place in order to control quality and economize material.

Substractive Hybridization. In the first round of substractive hybridization, 40 � g of driver and 0.4 � g of tester were mixed, extracted with phenol-chloroform and precipitated with ethanol. The resulting pellet was washed twice in 70% ethanol, dried and dissolved in 4 � l of hybridization buffer (30 m M EPPS, pH8.0, 3 m M Na 2 EDTA). Mineral oil was used to overlay this droplet which then was denatured at 98 ° C for 5 min in a thermal cycler. After this, the reaction tube was immediately cooled down to 67 ° C and 1 � l 5 M NaCl was added. The tester-driver mixture was incubated at 67 ° C for 20 h.

Amplification of Difference Products . The hybridization mix-ture was diluted in TE (10 m M Tris-Cl, 1 m M EDTA, pH 8.0) in the presence of 40 � g of yeast tRNA to a final volume of 400 � l. The diluted mixture was subjected to 10 cycles of PCR amplifica-tion using J-BGL-24 as a primer. The resulting PCR product was extracted with phenol-chloroform, precipitated with ethanol in the presence of glycogen and treated with mung bean nuclease (New England Biolabs) for 35 min at 30 ° C to remove single-stranded DNA consisting primarily of unhybridized driver se-

Habbe /Bert /Simon

Pancreatology 2007;7:352–359354

quences. The mung bean nuclease reaction mixture was dena-tured at 95 ° C for 5 min and chilled on ice. The first difference product (DP1) was generated from the mung bean nuclease mix-ture by 19 cycles of PCR amplification using J-BGL-24 as a prim-er. In the next new round of substraction, the difference product of the previous substraction was used as the tester in combination with a fresh aliquot of the initial amplicon from the opposite source as driver (40 � g). The second difference product (DP2) was generated with a tester/driver ratio of 1: 800. DP2 were again di-gested by DpnII, separated and visualized on a 2% agarose gel (Sigma), showing a size range from 100 to 500 bp, excised in 3 fractions and extracted from the gel slices (QIA EX kit, Qiagen). After being excised, the DP2s were cloned into the Bam HI site of the pBluescript SK II+ vector (Stratagene, La Jolla, Calif., USA), transformed into Escherichia coli DH10b and plated on LB-ampi-cillin agar. The clones were picked and submitted into a liquid LB-ampicillin medium. Plasmid preparation was performed us-ing the NucleoSpin Plasmid Kit (Macherey-Nagel).

Sequencing The plasmid fragments containing the sequence of the dif-

ferentially expressed genes were sequenced using the BigDye-Terminator Cycle Sequencing Kit (Abi Prism, Perkin Elmer) and the AbiPrism Sequencer 310. T3 and T7 primer were used rep-resenting sequences in the vicinity of the multiple cloning site. All sequences were transferred into a Microsoft Word document format. Sequence homology and chromosomal localizationwere obtained from BLAST algorithm . Only sequences longer than 80 bp with more than 95% homology to known genes were accepted for annotation. Functional prediction was performed by using the information at Unigene, LocusLink and Medline databases.

RT-PCR Verification of the cDNA-RDA Results Gene-specific primers were designed for all gene fragments

cloned from the second difference products except for fragments being to small to be enriched by PCR. PCR reaction was per-formed twice to verify different expression. A PCR mastermix, containing 1 � l of cDNA template, 1 � l of each gene-specific for-ward and reverse primer (10 pmol, primer sequences are available on request to the corresponding author), 2.5 � l 10 ! PCR buffer (Qiagen), 2.5 � l of dNTP mix (2 m M of each dATP, dCTP, dUTP and dGTP), 5 � l Q-solution (Qiagen) and 0.1 � l (1 U) Taq-poly-merase (Qiagen) and sterile water to 25 � l total volume per reac-tion. The Robocycler Gradient 40 � was used for PCR. The PCR protocol consisted of an initial denaturation step at 95 ° C for 5 min followed by 1 min at 95 ° C, 1 min at the primer specific an-nealing temperature and 1 min at 72 ° C. 20 cycles were performed with a final step at 72 ° C for 10 min. The resulting PCR fragments were separated by electrophoresis on 2% agarose gel stained with ethidium bromide.

Results

In this present study, we applied cDNA-RDA screen-ing and RT-PCR verification to identify genes that might be important in the carcinogenesis of neuroendocrine tu-

mors. The major aim was to isolate methylation-depen-dent differentially expressed genes, whose function could be restored by 5-aza-2 � -deoxycytidine-treatment.

Identification of Methylation-Dependent Expressed cDNA Fragments in OGP-1 Cells To identify and isolate differentially expressed genes

after treatment with 5-aza-2 � -deoxycytidine in the neu-roendocrine pancreatic tumor cell line QGP-1, cDNA representational difference analysis was performed with two PCR-coupled rounds of substractive hybridization and selective amplification. Whereas the representations show a smear of products ranging from 100 to 500 bp, DP2 has visible bands from 100 to 450 bp in size. After excision of each 3 bands from the second difference prod-uct ( fig. 1 ), plasmid preparation was performed and the isolated clones were subdivided into two classes. Treat-ment-mediated re-expressed clones were named D-clones, silenced clones were named U-clones. In order to identify the expressed genes and to map them to a chro-mosomal location, we analyzed the sequences using the NCBI BLAST program. Thereby, we identified 49 D-clones and 18 U-clones with more than 95% homology to known genes.

To verify the differential expression of the identified genes, gene-specific primer sequences were designed for all gene fragments isolated by RDA except for those too small for RT-PCR verification. All genes were considered as differentially expressed if RT-PCR revealed the same result twice in different reactions. Furthermore, we did not analyze inter- or intra-assay variance of DNA meth-ylation depending on cell culture conditions, as all condi-

DP2 (+)M

501 bp �

331 bp �

242 bp �

Fig. 1. Difference product 2 showing distinct bands of treatment mediated upregulated genes after two rounds of subtraction and selective amplification. M = Marker; DP2(+) = difference product 2 of upregulated genes.


tions were the same for the treated or untreated OGP-1 cells and all steps were equally performed on both groups of the QGP-1 cells. The genes were subdivided into groups corresponding to the hallmarks of cancer as described by Hanahan and Weinberg [19] in 2000 and summarized in tables 1–4 .

Demethylating Treatment of QGP-1 Cells Leads to Differential Expression of Genes Considered in Cellular Proliferation-, Signal Transduction-, Ribosomal Biosynthesis- and Protein Homeostasis-Associated Genes After 5-aza-2 � -deoxycytidine-treatment, we identified

23 genes associated with proliferation control mecha-nisms, signal transduction and the genes encoding for ribosomal proteins and protein homeostasis as summa-rized in table 1 . Expression levels of these were increased in 11 gene fragments (48%), whereas 12 genes (52%) re-vealed lower expression levels.

Demethylation in QGP-1 Cells Affects Differential Expression of Apoptosis-Associated Genes We identified 7 genes associated to another hallmark

of cancer, namely the evasion of apoptosis. RT-PCR-based verification showed a stronger expression of cytokeratin 18, the encoding gene of the p53 effector protein (PERP) and death-associated protein (DAXX) 6 after 5-aza-2 � -deoxycytidine treatment. In contrast, 3 genes of this group revealed a significantly lower expression or were transcriptionally silenced. After 5-aza-2 � -deoxycytidine treatment, transaldolase 1 and inositol 1,3,4-triphosphate 5/6 kinase were still expressed on a lower level, whereas the transcription of galectin 9 was completely silenced. One isolated fragment, encoding for a small subunit of calpain, could not be verified as differentially expressed.

Differential, Methylation-Dependent Gene Expression of Metastasis and Immunological Escape-Associated Genes in QGP-1 Cells Performing cDNA-RDA, we isolated and verified the

differential expression of four genes associated with tis-

Table 1. Genes differentially expressed after 5-aza-2�-deoxycytidine treatment known to be involved in cellular proliferation, signal transduction, ribosomal biosynthesis and protein homeostasis in QGP-1 cells

Accession Name REG Gene map locus

Upregulated genesNM_021727 fatty acid desaturase 3 (FADS3) d 11q12–q13.1NM_021098 T-type calcium channel alpha 1H subunit (CACNA1H) d 16p13.3NM_001961 eukaryotic translation elongation factor 2 (EEF2) d 19pter–q12NM_005918 malate dehydrogenase 2 (MDH2) d 7p12.3–q11.2NM_004990 methionine-t-RNA-synthetase (MARS) d 12q13.2NM_006826 tyrosine 3-monooxygenase activation protein theta polypeptide (YWHAQ) d 2p25.1NM_002696 polymerase (RNA) II polypeptide G d 11q13.1NM_006319 CDP-diacylglycerol-inositol 3 phosphatidyltransferase d 16p21.1NM_006184 nucleobindin 1 (NUB1) d 19q13.2–q13.4NM_000967 ribosomal protein L3 (RPL3) d 22q13NM_000445 plectin 1 (PLEC1) d 8q24

Downregulated genesNM_012179 F-box only protein 7 (FBXO7) f 22q12–q13NM_153280 ubiquitin-activating enzyme E1 (UBE1) f Xp11.23NM_006196 poly (rC) binding protein 1 (PCBP1) f 2p13–p12BC006182 calmodulin 3 f 19q13.2–q13.3NM_002046 glyceraldehyde-3-phosphate-phosphatase dehydrogenase (GAPDH) f 12p13.31NM_005619 reticulon 2 (RTN2) f 19q13.32NM_001002 ribosomal protein large P0 (RPLP0) f 12q24.2NM_024923 nucleoporin 210 (NUP210) f 3p25.1NM_004145 myosin-IXb (MYOB9) f 19p13.1NM_021034 interferon-induced transmembrane protein 3 (IFITM3, 1-8U) f 11p15.5NM_007273 repressor of estrogen receptor activity (REA) f 12p13NM_002795 proteasome subunit, beta type 3 (PSMB3) f 17q12

Habbe /Bert /Simon


Table 2. Genes differentially expressed after 5-aza-2�-deoxycytidine treatment in QGP-1 cells concerning ap-optosis


Upregulated genesNM_022121 p53 effector protein (PERP) d 6q24NM_000224 cytokeratin 18 (KRT18) d 12q13NM_001350 death-associated protein 6 (DAXX) d 6p21.3

Downregulated genesNM_006755 transaldolase 1 (TA1) f 11p15.5–p15.4NM_014216 inositol 1,3,4-triphosphate 5/6 kinase (ITPK1) f 14q31NM_001749 calpain, small subunit 1 f 19q13.13NM_009587 galectin 9 (GAL9) f 17q11.1

Table 3. Genes differentially expressed after 5-aza-2�-deoxycytidine treatment known to be involved in metas-tasis and immunological escape associated genes in QGP-1 cells


Upregulated genesNM_014770 PIKE-A d 12q13.2NM_015937,2 phosphatidyl inositol glycan class T (CGI-06) d 20q12–q13.2NM_005146 squamous cell carcinoma antigen (SART1) d 11q13.1NM_005507 cofilin 1 d 11q13NM_020314 esophageal cancer-associated protein (ECAP) d 16p13.11NM_002116 MHC class I antigen d 6p21.3NM_000942 cyclophilin B d 15q21–q22

Downregulated genesNM_033453 inosine triphosphatase f 20pNM_021978 matriptase (ST14) f 11q24–q25NM_006986 melanoma antigen D1 (MAGE D1) f Xp11.23NM_002276 cytokeratin 19 (KRT 19) f 17q21.2

Table 4. Genes differentially expressed after 5-aza-2�-deoxycytidine treatment with unknown function


Upregulated genesNM_016016 CGI-69 d 17q12NM_004140 lethal giant larvae homolog 1 (LLGL1) d 17p12–p11.2NM_014297 YF13H12 d 19q13.32

Downregulated genesAF289557 pp5382 f 11q13NM_017623 cyclin M3 f 2p12–p11.2NM_032118 FLJ12953 f 2p13.1AF 465980 YAN 0650 f mitochondrium


sue invasion and metastasis. Two genes, cofilin and the phosphatidylinositol 3-kinase enhancer-A ( PIKE-A ) were transcriptionally silenced in wild-type QGP-1 cells and transcription could be restored after treatment with 5-aza-2 � -deoxycytidine. Interestingly, RT-PCR confirma-tion revealed a lower expression of matriptase and cyto-keratin 19 after treatment with 5-aza-2 � -deoxycytidine as shown in figure 2 .

Another mechanism supporting tumor growth is im-munological escape ability. We identified 9 genes show-ing 5-aza-2 � -deoxycytidine-treatment mediated differen-tial gene expression. Five of these gene fragments, MHC class I antigen (HLA-A), cyclophillin B, esophageal can-cer-associated protein, phosphatidylinositol glycan class T and nucleobindin, revealed significantly higher expres-sion levels after treatment, whereas the expression of the squamous cell carcinoma antigen (SART 1), which was transcriptionally silenced before, could be restored by 5-aza-2 � -deoxycytidine. In addition, we were able to iden-tify lower expression levels in the melanoma antigen D1 (MAGE D1), FK 506 binding protein 2 and inosine tri-phosphatase.

Identification of Methylation-Dependent Expressed Novel Genes with Unknown Function in QGP-1 Cells Interestingly, our analysis revealed 7 fragments en-

coding for genes whose function was not identified thus far and were differentially expressed after 5-aza-2 � -de-oxycytidine treatment. Transcription could be enforced

in 3 genes, CGI-69 , LGLL1 and YF13H12 , suggesting a methylation-mediated transcriptional silencing in QGP-1 cells. In contrast, the 4 gene fragments, named pp5382 , cyclin M3 , FLJ12953 and YAN 0650 , showed decreased ex-pression levels after demethylating treatment. The func-tion of these genes remains to be elucidated.

Discussion

We identified 48 genes which are differently expressed in QGP-1 cells after 5-aza-2 � -deoxycytidine using cDNA-RDA. Treatment of cell lines with 5-aza-2 � -deoxycytidine is an established method to investigate methylation-de-pendent gene expression and is still used by several groups, and different gene expression levels are contrib-uted to the demethylating treatment [17] . The RDA was developed to isolate differences in complex genomes, i.e. genetic lesions in tumors resulting from DNA deletion, rearrangement or insertions. Hubank and Schatz [18] employed the RDA technique on the cDNA level to deter-mine differentially expressed mRNA species in cell pop-ulations. cDNA-RDA combined with RT-PCR verifica-tion is a simple but powerful method that allows the iden-tification of different expression levels in an unbiased manner.

Increased gene expression levels could be verified by RT-PCR in 25 gene fragments (52.3%), whereas 23 (47.7%) fragments revealed lower expression levels after demeth-ylating treatment. One isolated fragment revealed no dif-ferential expression. Of these 48 gene fragments, 41 were already known whereas the specific functions of each of the remaining 7 genes are still to be elucidated. Some of the isolated genes revealed multiple functions which led to some difficulties in classifying these genes.

Differential, methylation-dependent gene expression is a common event in tumorigenesis. Remarkably, we were able to show that demethylation led to significantly lower expression in nearly 50% of all fragments isolated in this study. The underlying mechanism of this finding remains unclear, and there are several possibilities which might explain the lower expression after 5-aza-2 � -deoxy-cytidine-treatment. For example, treatment of QGP-1 cells with 5-aza-2 � -deoxycytidine could relieve methyla-tion of a repressor element or promoter methylation of a repressor of the gene that is then silenced. Even genes with increased expression after 5-aza-2 � -deoxycytidine treatment could lead to silencing of genes by feedback mechanisms. Every one of the 41 known gene fragments shown in the tables has been described in tumorigenesis

+DAC

Cofilin

PIKE-A

MT-SP 14

187 bp

140 bp

129 bp

–QGP

–NC

–PC

–M

Fig. 2. Examples of gene fragments revealing differential expres-sion after treatment with 5-Aza-CdR. Molecular weight marker (M), gene fragment containing plasmids serve as positive control (PC), water as negative control (NC), 5-aza-2 � -deoxycytidine (DAC). Lanes 4 and 5 show wild-type QGP-1 and 5-aza-2 � -deoxy-cytidine-treated QGP-1 cDNA, respectively. Cofilin and PIKE-A reveal higher expression levels after treatment, matriptase expres-sion is significantly lower.

Habbe /Bert /Simon


so far, but none has been reported to play a pivotal role in neuroendocrine carcinogenesis.

Within the large set of genes implied in cellular prolif-eration and protein trafficking identified by the cDNA-RDA we were especially interested in genes leading to-wards metastatic potential. Out of the isolated gene frag-ments concerning metastasis and tissue invasion, cofilin and PIKE-A are most interesting, as we were able to re-store the expression of both genes suggesting a methyla-tion-dependent transcriptional silencing. Cofilin is a key enzyme for the depolymerization of actin filaments thus inducing lamellipodia cell movement. Although lower expression levels of cofilin were found in a HCC-cell line known for a high metastatic ability, inhibition of cofilin expression reduces the invasion of cancer cells by reduc-ing the stability and assembly of invadopodia [20, 21] . Furthermore, overexpression of cofilin enhances the mo-tility of glioblastoma tumor cells in a concentration-de-pendent fashion, which might lead to their invasiveness [22] . Therefore, restoring the expression of cofillin by 5-aza-2 � -deoxycytidine treatment might affect or even con-tribute to metastatic spreading of neuroendocrine pan-creatic tumors. In contrast to our results, cofilin expres-sion was lower after 5-aza-2 � -deoxycytidine treatment in PaCa44 cells [23] .

The other gene, the phosphatidylinositol 3-kinase en-hancer-A (PIKE-A) codes for a recently discovered pro-tein, which possesses GTPase activity and is co-amplified with CDK4 in a variety of human cancers. Overexpres-sion of PIKE-A in glioblastoma cells leads to an increased cell invasion [24] . PIKE-A is not expressed in wild-type QGP-1 cells, but expression could be restored by 5-Aza-CdR treatment. The effect of overexpression of PIKE-A after 5-aza-2 � -deoxycytidine treatment still needs to be examined in neuroendocrine pancreatic tumor cells, as PIKE-A expression might lead to different effects in dif-ferent cell types. Taken together, we demonstrated that transcriptionally silenced genes leading towards tissue invasion or apoptosis inhibition are recovered by 5-aza-2 � -deoxycytidine treatment, implying a potential risk of demethylating agents, but the functional effects of this reexpression need to be carefully elucidated.

A novel finding is the lower expression of matriptase after 5-aza-2 � -deoxycytidine treatment in QGP-1 cells. Matriptase is a serine protease with extracellular matrix degrading ability. Antisense-transfection-mediated inhi-bition of matriptase in ovarian carcinoma cells leads to a reduced invasion into the extracellular matrix and small-er tumors in mouse model experiments [25] . List et al. [26] recently demonstrated that the modest increase of

matriptase expression is sufficient for the introduction of spontaneous squamous cell carcinomas in transgenic mice, possibly through activation of the PI3K-Akt path-way. Furthermore, matriptase is an efficient activator of hepatocyte growth factor, which stimulates proliferation, migration and cell survival whose deregulation plays a pivotal role in cancer onset and progression [27] . In QGP-1 cells, 5-aza-2 � -deoxycytidine treatment led towards a complete transcriptional silencing of matriptase repre-senting a powerful inhibitor of this gene, suggesting an antimetastatic function.

Interestingly, 7 genes were isolated whose function re-mains unclear. There is one report concerning YF13H12 describing an overexpression of this gene in 52% of 46 gastric carcinoma samples compared to normal tissue, suggesting a role in carcinogenesis [28] . Further studies have to elucidate the function in neuroendocrine tumor-igenesis.

As 5-azacytidin has been approved by the FDA for the treatment of myelodysplastic syndrome, demethylating agents have become an important weapon against cancer [29] . Furthermore, several demethylating drugs, such as zebularine and procaine, that are less toxic and can be administered orally, are exciting new options for antican-cer chemotherapy. Therefore, it is necessary to investigate every biological and epigenetic effect of these drugs. This study reveals that demethylating drugs lead to differen-tial expression of a large set of genes. These genes can be associated to every hallmark of cancer, showing adverse effects on cancer control mechanisms in some cases. In conclusion, we identified 48 genes differentially expressed in 5-aza-2 � -deoxycytidine-treated QGP-1 cells using RDA and RT-PCR. These clones will be useful to identify epigenetic alterations in neuroendocrine pancreatic tu-mors and may also be useful markers for early detection and prediction of prognosis in such tumor entities.

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