GTMB 7 - Gene Therapy & Molecular Biology

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Li et al: MET amplification in liver tumorset al, 1995). Mutation of the p53 gene was detected inapproximately 36% of advanced HCC (Murakami et al,1991) and was also implicated in tumor progression(Teramoto et al, 1994). Overexpression was reported forseveral oncogenes such as N-ras, c-myc and fos(Arbuthnot et al, 1991). However, oncogene amplificationappears to be rarely the underlying mechanism of cancerdevelopment in these cases. Amplifications associatedwith HCC have been found on 11q13 (Nishida et al,1994), involving both INT2 and cyclin D1. Nishida andcolleagues (1994) showed that the cyclin D1 gene wasamplified 3 to 16 fold in about 11% of HCC samplesanalyzed, with a concomitant 6 to 10 fold overexpression.Based on this finding they suggested that amplificationand overexpression of the cyclin D1 gene might beresponsible for rapid growth of a subset of HCC.The rapid emergence of microarray technology hasallowed new approaches to tumor analysis. The mostcommon application of this technology has focused on theuse of cDNA arrays for the large-scale analysis of geneexpression to monitor tumor progression (Sgroi et al,1999) or for cancer typing (Anbazhagan et al, 1999).Oligonucleotide arrays have enabled rapid re-sequencingfor genotyping or point mutation analysis, such as p53mutation detection (Hacia, 1999). Applying ComparativeGenomic Hybridization (CGH) to microarrays of largegenomic clones has also been successful, allowing thedetection of gross chromosomal abnormalities that resultin copy number changes for a given sequence, such asgene amplifications or deletions (e.g. LOH), (Solinas-Toldo et al, 1997; Pinkel et al, 1998; Muller, 2001). Suchsub-chromosomal aneuploidies are known to befundamental causes of cancer and many other humandiseases, often leading to the over- or under-expression ofgenes.II. Materials and methodsWe have developed a CGH-based microarray system(Genosensor System) and a microarray to specifically detectabnormalities of 52 genomic loci that have been associated withformation of various human solid tumors (Müller et al, 2002).The arrays consist of 3 repeats each of 52 P1, PAC or BAC cloneDNAs that are arrayed on a chromium-coated glass surface. Forhybridization to this array, genomic DNA samples were extractedfrom human liver tumors or from histopathologically non-tumorliver sections from the same patient. After purification (GentraKits, Gentra Systems, Inc., Minneapolis, MN), the genomic DNAsamples were then labeled by nick translation (NicktranslationKit, Vysis, Inc., Downers Grove, IL) in the presence ofSpectrum-Green dUTP (green fluorophore). Genomic DNA froma normal human male donor was chemically labeled with a redfluorophore (Vysis, Inc., Downers Grove, IL), and served as areference. The test probe (green) and reference probe (red) werethen mixed with unlabeled human cot-1 DNA and co-hybridizedto the microarrays. After removal of un-hybridized probes, thearray was imaged by a multi-color CCD based image analysissystem, and fluorescence intensities were determined for eachtarget spot. Under the assumption that the hybridization kineticsfor a given sequence are equal for the test and reference DNA,the signal intensity is proportional to the copy number of thatsequence in the hybridization mixture. The test/referenceintensity ratio for each target genomic locus (average of 3 spots)was normalized by dividing with the average ratio of all"normal" targets, resulting in an estimate for the copy numberchange of that specific sequence compared to the rest of thegenome.III. ResultsAs shown in Figure 1, the DNA extracted from thetumor tissue of HCC patient #21 was found to have anaverage normalized ratio of 4.2 ± 1 by Genosensoranalysis for the MET target locus (average of 5experiments), and 6.4 ± 0.8 by Southern analysis (3experiments; see below). Since the reference sample usedhere was from a normal human male and has 2 copies ofthe MET sequence, this ratio suggests that there are onaverage between 8.4 to 12.8 copies per cell (4.2 or 6.4 x 2)of the MET gene in the HCC tumor sample. Thisamplification is considered a significant finding, as it isthe first time to be reported in this type of cancer.Since microarray or Southern analysis yields anestimate for the copy number of a sequence averaged overall cells from which the DNA was extracted, the METamplification level was confirmed by fluorescent in situhybridization (FISH). The tumor tissue from the sameHCC patient was formalin-fixed, paraffin-embedded, andsectioned. FISH was performed with SpectrumGreenlabeled DNA from a BAC clone containing the MET gene.SpectrumOrange labeled CEP 7 DNA (containingchromosome 7-specific centromere DNA sequences;Vysis) was co-hybridized as a control. The signal for both,the MET gene and chromosome 7 were counted under afluorescent microscope after counterstaining with DAPI.As expected, the majority (60%) of the cells contained 2copies of chromosome 7 per nucleus, while approximately40 % of cells have an average of 25 copies of MET(Figure 2). Since the remaining 60% of cells have only 2copies of the MET gene, the DNA extracted from thistumor section should have 11 copies of the MET gene,which is in good agreement with the microarray andSouthern data.For further confirmation and comparisons additionalSouthern blot analyses were carried out with EcoR1-digested DNA from 32 tumor samples including 6 HCC,16 colorectal liver metastasis (CLM), 3cholangiocarcinomas, 2 adenomas, 2 fibrolamellar (HCCvariant), and 3 unclassified liver tumors. Normal humangenomic DNA (control) and DNA from the non-tumorliver tissue of HCC patient #21 were also included in theSouthern blot analyses. A 360bp DNA fragment (1) wasamplified by polymerase chain reaction (PCR) in thepresence of the following pair of primers, primer H1: 5'-TCTTGATTACCTGCATTTGC-3' and primer H2: 5'-TGGGGCAAGAAGGCCTCTCT-3' from a BAC clonecontaining the entire MET gene. The 360bp MET probewas labeled by PCR in the presence of 32 P-labelled dCTPand hybridized to the Southern blot. A probe generatedfrom a genomic clone on 11q13 was re-hybridized to thesame Southern blot for normalization, after the MET probewas stripped from the blot.100
Gene Therapy and Molecular Biology Vol 7, page 101Figure 1: Genosensor and Southern analysis of HCC samples. Genomic DNA (8 µg for DNA from tumor tissue and 8 µg for DNA fromnormal tissue) was digested with Eco R1, run on agarose gels and blotted. Southern hybridization was performed with a P32 labeled 360bp MET probe as described in the text. A composite image (red, green and blue) of a Gneosensor oncogene array after hybridizationwith a mixture of sample 21T DNA (green) and normal refernce DNA (red) is shown after counterstaining with DAPI.The level of MET gene amplification was determinedusing a PhosphoImager (Molecular Dynamics). Some ofthe results are shown in Figure 1. Among the 6 HCCsamples analysed, 2 MET gene amplifications wereobserved (6.4 and 2.5 fold after normalization). MET geneamplifications were also observed in thecholangiocarcinoma and CLM samples. Two of the threecholangiocarcinoma patients had MET gene amplificationsin their tumour specimens at a level of 6.5 fold and 1.6fold, respectively. Of the 16 patients with CLM, three hadMET gene amplifications of 2.3, 2.1 and 1.8 fold,respectively. Of specific interest is the finding that both,the tumor as well as non-tumor tissues from the sameHCC patient (No. 21) showed a similar level of METamplification (6.4 fold and 6.1 fold, respectively),suggesting that MET amplification may precede malignanthistopathological changes. This patient developed HCC inthe background of a cirrhotic liver complicating hepatitisC infection. Liver cirrhosis provides a pre-malignant fieldchange for HCC development.of the MET oncogene in hepatocellular carcinomasstrongly suggests a role here as well.This finding in combination with multiple otherreports of cancer associated gene amplificationsunderscores the need for a rapid, quantitative detectionmethod for such genetic changes. The microarray basedmethod described here is consistent (within a factor of 2)with other established methods (FISH, Southern blotting),and therefore suitable for the screening of geneamplifications. Since this method is non-radioactive,simpler, faster, and more economical than either FISH orSouthern, especially when the mutated genetic locus is notknown, it lends itself to applications in clinicaldiagnostics.IV. DiscussionHepatocyte growth factor (HGF) plays an importantrole in the growth, progression and angiogenesis ofvarious tumors and is known to specifically promotehepatocyte proliferation and liver regeneration. Inaddition, it may also be involved in tumor invasion andprogression (Tamatani et al, 1999). Overexpression andamplification of the HGF receptor (MET gene) have beenimplicated in progression of colorectal cancer (Di Renzoet al, 1995), by a mechanism where the elevated level ofthe MET gene product confers a selective growthadvantage to tumor cells (Di Renzo et al, 1991). In thecontext of this information, our finding of amplificationsFigure 2: FISH on interphase nuclei of patient #21. FISH wasperformed on formalin-fixed, de-parafinized tumor tissuesections. A BAC clone containing the MET gene was labeledwith SpectrumGreen by nick translation and used as a probe. ASpectrumOrange labeled chromosome 7-specific centromereprobe (CEP7; Vysis Inc.) was co-hybridized as reference.101
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