sybr-green-i and Colorectal-Neoplasms

Mutation or epigenetic silencing of mismatch repair genes, such as MLH1 and MSH2, results in microsatellite instability (MSI) in the genome of a subset of colorectal carcinomas (CRCs). However, little is yet known of genes that directly contribute to tumor formation in such cancers. To characterize MSI-dependent changes in gene expression, we have now compared transcriptomes between fresh CRC specimens positive or negative for MSI (n=10 for each) with the use of high-density oligonucleotide microarrays harboring >44,000 probe sets. Correspondence analysis of the expression patterns of isolated MSI-associated genes revealed that the transcriptome of MSI+ CRCs is clearly distinct from that of MSI- CRCs. Such MSI-associated genes included that for AXIN2, an important component of the WNT signaling pathway. AXIN2 was silenced, apparently as a result of extensive methylation of its promoter region, specifically in MSI+ CRC specimens. Forced expression of AXIN2, either by treatment with 5'-azacytidine or by transfection with AXIN2 cDNA, resulted in rapid cell death in an MSI+ CRC cell line. These data indicate that epigenetic silencing of AXIN2 is specifically associated with carcinogenesis in MSI+ CRCs.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Aged, 80 and over; Axin Protein; Azacitidine; Benzothiazoles; Carrier Proteins; Cell Death; Cell Line, Tumor; Cell Proliferation; Cluster Analysis; Colorectal Neoplasms; CpG Islands; Cytoskeletal Proteins; Diamines; DNA Methylation; DNA Repair; DNA, Complementary; Epigenesis, Genetic; Female; Gene Silencing; Humans; Male; Microsatellite Repeats; Middle Aged; MutL Protein Homolog 1; MutS Homolog 2 Protein; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Organic Chemicals; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Time Factors; Transfection; Up-Regulation

The expression of carcinoembryonic antigen (CEA) mRNA was assessed in 102 lymph nodes (LNs) obtained from seven colorectal cancer patients by both the conventional non-quantitative RT-PCR and quantitative RT-PCR. The number of CEA-expressing cells was calculated compared with CEA-expressing MKN-45 cell line as a standard control. Using the quantitative RT-PCR, the relative number of CEA-expressing cells ranged between 1.3x103 and 5.7x106 in 16 histologically positive LNs and between 2.3x101 and 8.1x105 in 10 histologically negative and RT-PCR positive LNs. In both histologically and RT-PCR negative LNs, the relative cell number was <4.0x102. Our results demonstrated that quantifying the amount of metastasis might enhance the reliability of RT-PCR detection assay as a diagnostic tool for the detection of cancer micrometastases.

Topics: Adult; Aged; Aged, 80 and over; Benzothiazoles; Carcinoembryonic Antigen; Colorectal Neoplasms; Diamines; Female; Fluorescent Dyes; Humans; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Organic Chemicals; Quinolines; Reverse Transcriptase Polymerase Chain Reaction