Oncogene (2002) 21, 3253 ± 3257 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc Gene expression dierences between the microsatellite instability (MIN) and chromosomal instability (CIN) phenotypes in colorectal cancer revealed by high-density cDNA array hybridization Donncha S Dunican1,2, Peter McWilliam1, Orna Tighe1, Anne Parle-McDermott1,3 and David T Croke*,1 1Department of Biochemistry, Royal College of Surgeons in Ireland, 123, St Stephens' Green, Dublin 2, Ireland Two distinct pathways of tumorigenesis exist in sporadic loss of heterozygosity (LOH). The microsatellite colorectal cancer. The microsatellite instability pathway instability (MIN) phenotype is not as prevalent in (MIN), which is characterized by widespread micro- sporadic colorectal cancer, accounting for 15% of such satellite instability due to aberrant mismatch repair malignancies and has been ®rmly linked to a faulty machinery, accounts for 15% of all sporadic colorectal DNA mismatch repair system. Adenomatous polyposis cancers. The chromosomal instability (CIN) phenotype, coli (APC)orb-catenin mutations are the most which accounts for 85% of sporadic colorectal cancers, common initial molecular lesions in the CIN phenotype is characterized by gross chromosomal lesions but the (Kinzler and Vogelstein, 1996). A link between underlying mechanism remains unclear. We have microsatellite instability and mutant DNA repair genes addressed dierences in gene expression between the was discovered (Strand et al., 1993). Subsequently ®rm MIN and CIN colorectal cancer phenotypes in vitro by associations between DNA mismatch repair and colon the use of high density cDNA ®lters to compare gene cancer were demonstrated (Fishel et al., 1993; Bronner expression patterns between MIN and CIN colorectal et al., 1994). Unrepaired DNA mismatches in cancer cell-lines yielding a panel of 73 consistently transforming growth factor beta I receptor II dierentially expressed genes. Nine of these genes were (TGFbIRII) and insulin-like growth factor receptor II subjected to con®rmatory analysis by independent (IGFRII) (Parsons et al., 1995; Souza et al., 1996) are methods, of which six were con®rmed as being thought to be the driving force of tumorigenesis in dierentially expressed; PLK, RanBP2 and CCNA2 MIN tumours. p53 was initially a candidate gene in the were overexpressed in CIN lines while BTF3, H2AZ and molecular mechanism of CIN. However, a number of PTPD1 were overexpressed in MIN lines. These six cancers with mutant p53 have been shown to be diploid genes are involved in diverse processes, such as with no obvious chromosomal defects and to exhibit maintenance of chromatin architecture, DNA-damage MIN due to impaired mismatch repair (Eshleman et checkpoint and cell cycle regulation, which may al., 1998). Disruption of mitotic checkpoint assembly contribute to the CIN and MIN phenotypes. genes can lead to CIN because checkpoint defective Oncogene (2002) 21, 3253 ± 3257. DOI: 10.1038/sj/ cells can complete mitosis with inappropriately aligned onc/1205431 chromosomes (Paulovich et al., 1997). Recent data strongly support the hypothesis that carcinogen Keywords: colorectal cancer; microsatellite instability; exposure determines the type of instability in MIN chromosomal instability; gene expression and CIN cancers (Bardelli et al., 2001). Despite these ®ndings, the mechanism of CIN progression remains unclear. A number of studies have used cell lines derived The majority of colorectal tumours (85%) are sporadic from sporadic colon tumours as in vitro models of the in origin yet they exhibit close similarities to tumours MIN and CIN phenotypes (Cahill et al., 1998). We ONCOGENOMICS resulting from inherited colorectal cancer syndromes. report a global gene expression pro®ling approach used The chromosomal instability (CIN) phenotype, which to identify gene expression dierences within a panel of accounts for 85% of sporadic cases, exhibits gross MIN and CIN cell lines (selected on the basis of chromosomal abnormalities such as aneuploidy and Heinen et al., 1995). This utilized Gene Discovery Array (GDA) v1.3 ®lters (IncyteGenomics, USA) and SMART cDNA (Clontech, USA) to determine expres- *Correspondence: DT Croke; E-mail: [email protected] sion dierences between MIN (LS174T, SW48 and Current addresses: 2Department of Biomedical Sciences, Genes and HCT116) and CIN (Caco2, SW480 and HT29) Development Group, The University of Edinburgh, Hugh Robson sporadic colorectal cancer cell lines. The GDA ®lters 3 Building, Edinburgh EH8 9XD, UK; Department of Biochemistry, harbour over 18 000 cDNA species arrayed in Trinity College, Dublin 2, Ireland Received 2 October 2001; revised 19 February 2002; accepted 20 duplicate, of which approximately 4000 are known February 2002 genes and 14 000 are expressed sequence tags (ESTs). Gene expression profiles in colorectal cancer DS Duncan et al 3254 In order to rule out tissue-speci®c dierences, three previously to identify gene expression dierences pair-wise comparisons were performed between (in between HCT116 and Caco-2 (Zhang et al., 1997). each case) representative MIN and CIN colorectal The Unigene unique identi®er of each transcript was cancer cell-lines. Brie¯y, radiolabelled SMART total entered into the SAGE database and we were thus able cDNA preparations for each cell line pair were to determine if our results concurred with those hybridized in parallel to identical GDA v1.3 ®lters, obtained from the established technology of SAGE. expression patterns were visualized by phosphorima- The expression dierences observed by GDA ging (100 mM resolution) and imager ®les were analysed analysis were independently veri®ed by Northern blot using the Gene Discovery Array software (IncyteGe- and RT ± PCR for nine transcripts, selected on the nomics, USA). Individual expression pro®les were basis of current literature reports concerning the evaluated based upon three criteria: (a) consistency in transcripts and previous MIN/CIN ®ndings (see expression dierence across three pair-wise MIN-CIN above). The choice of veri®cation method was made comparisons; (b) an expression dierence of ®vefold or based on EST and SAGE tag abundances associated greater as recommended by IncyteGenomics; and (c) with a given transcript. These analyses con®rmed the only known genes were considered. A simple Perl script dierential expression of six of the nine genes: polo program (GeneMatch v1.0) was coded which allowed (Drosophila) like Kinase (PLK), ran binding protein 2 us to identify the most signi®cant expression dierences (RANBP2) and cyclin A2 (CCNA2) were overexpressed present in all three experiments. Approximately 70 in CIN; basic transcription factor 3 (BTF3), histone genes were found to be consistently overexpressed 2AZ (H2AZ) and protein tyrosine phosphatase D1 either in MIN or in CIN cell lines (Tables 1 and 2). As (PTPD1) were overexpressed in MIN. Dierential a control for our expression data we exploited the expression of H2AZ, BTF3, PTPD1 and RANBP2 public Serial Analysis of Gene Expression (SAGE) was evident using either method; expression dierences database (Lal et al., 1999). SAGE has been used ranged from 2 ± 3-fold (BTF3 and RANBP2) to greater Table 1 Genes exhibiting elevated expression in MIN cell lines Accession SAGE tags per SAGE tags per Gene transcript number Tentative gene function million in HCT116 million in Caco2 Hemoglobin, alpha 1 W04832 Oxygen transport ± ± Heat shock 27 kD protein 1 W69499 Activates apoptosis/casases ± ± Cell division cycle 2-like 1 N98592 Cell division 645 48 H2AZ HISTONE W45695 Nucleosome assembly 530 407 Spectrin, beta, non-erythrocytic 1 N70585 Cytoskeletal component of red blood cells ± ± Metallothionein-Ie H25252 Heavy metal transport 82 0 Transcription factor AP-2 N63770 Transcription factor/gene expression ± ± Isovaleryl Coenzyme A H37754 Metabolism 363 264 ETS-related transcription factor ELF-1 N74959 Transcription factor/gene expression ± ± Cathespin S precursor N53585 Cysteine protease; protein degradation/processing 64 72 Collagen, type V, alpha 1 W52829 Structural protein; bone architecture 0 32 BASIC TRANSCRIPTION AA203284 Transcription factor/gene expression 629 505 FACTOR 3 CTP synthetase H11731 Pyrimidine metabolism 49 32 Retinol-binding protein 3 H64488 Vit A regulation (in eye cells) ± ± Carbonic anhydrase I R46266 Hydration of CO2 ±± 40S ribosomal protein S19 AA046433 Monocyte chemotactic factor ± ± DNA primase R43686 DNA polymerase/DNA replication 0 48 Kinase insert domain receptor R35713 VEGF-A tyrosine kinase receptor ± ± Dual specificity phosphatase 5 R43931 Cell signalling 49 16 Integrin, alpha L T83460 T-cell adhesion to target cells ± ± HLA class I-histocompatability R74335 Cell signalling/cell communication 447 44 ADP-ribosylation factor 4 N52015 Purine metabolism 66 97 Microfibrallar protein R01211 Cytoskeletal organisation 0 16 Alu RNA transcript W31678 Unclassified ± ± Trans-1,2,dihydrobenzene-1,2-diol W63727 Aldehyde and ketone oxidation 0 32 dehydrogenase Ribonuclease, Rnase A family, R62231 RNA metabolism 545 0 1 pancreatic Elastin T79116 Cytoskeletal ± ± Prepro form of corticotropin R33777 Cell signalling/communication ± ± PROTEIN TYROSINE R23593 Gene/protein expression 32 0 PHOSPHATASE D1 A summary of the genes showing consistently elevated expression in the microsatellite instability (MIN) sporadic colorectal cancer cell lines LS174T, SW48 and HCT116 with respect to the chromosomal instability (CIN) cell lines Caco-2, SW480 and HT29. Tentative