CANCER PROTEOMICS PROJECT

20.CANCER PROTEOMICS PROJECT

    The Cancer Proteomics Project aims to clarify the molecular and cellular mechanisms of cancer promotion and progression using the combined power of the innovative techniques of transcriptome and proteome. A large-scale comparison of protein and gene expression profiling in various tissue culture models and animal models of cancer and in clinical samples is believed to lead to the identification of target molecules useful for the diagnosis and treatment of cancer.
Proteomic Analysis using two-dimensional differential gel electrophoresis (2D-DIGE)

    The proteomic alterations associated with cancer were examined using 2D-DIGE, in which thousands of proteins are labeled simultaneously with multifluorescence dyes and separated based on their isoelectric point and molecular weight. Our 2D-DIGE system has several advantages: 1. The internal control of Cy3-labeled proteins allows high reproducibility of 2D pattern and accurate quantification of the protein spots. 2. Using the specialized software for 2D-DIGE, the 2D profiles in multiple gels are compared rapidly and automatically. 3. High throughput analysis; 24 samples per day can be studied by 2D-DIGE. 4. The robotic device excises protein spots automatically with little operator intervention. 5. Results of 2D gel analysis and sequence information from mass spectrometry are integrated in a database for further study.
High-throughput protein identification by mass spectrometry

    Peptide finger printing and database search identify the spots of interests. The proteins in the gel are enzymatically digested and the masses of generated peptides are measured by mass spectrometry. The measured peptide masses are correlated with experimentally calculated peptide masses derived from proteins existing in sequence databases. Our original in-gel digestion protocol combined with three types of high performance mass spectrometry systems (Voyager DE Pro, Q-STAR and LCQ Deca) allows the high-throughput determination of protein sequences.
Establishment of intestinal epithelial cells capable of inducing stabilized beta-catenin

    Aberrant transactivation of a certain set of target genes by beta-catenin and TCF/LEF complexes has been identified in human colorectal cancer cell lines and implicated in early colorectal tumorigenesis. From an immortal undifferentiated rat intestinal untransformed epithelial cell line, IEC6, several clones capable of inducing a beta-catenin protein lacking the 89-amino acid phosphorylation site in its NH₂-terminus under the strict control of the tetracycline-regulatory system were established (IEC6-Tet OFF beta-catenin deltaN89). It is anticipated that these clones will allow precise pinpointing of the molecular and cellular changes that occur following the activation of the beta-catenin/TCF4 complex.
Gene expression profiling of beta-catenin mediated gene transactivation

    Using the IEC6-Tet OFF beta-catenin deltaN89 cells described above, a large-scale comparison of gene expression profiling was performed in order to define target genes of beta-catenin/TCF4-mediated gene transactivation. By hybridizing to oligonucleotide arrays (GeneChip, Affymetrix), more than 30,000 genes were examined for the differential expression before and after the induction of beta-catenin deltaN89. In addition to genes previously defined as target genes of beta-catenin and TCF/LEF complexes, such as cyclin D1 and axil (axin-2), several genes which were not described to be involved in a Wnt signaling pathway were identified.




20.CANCER PROTEOMICS PROJECT



	The Cancer Proteomics Project aims to clarify the molecular and cellular mechanisms of cancer promotion and progression using the combined power of the innovative techniques of transcriptome and proteome. A large-scale comparison of protein and gene expression profiling in various tissue culture models and animal models of cancer and in clinical samples is believed to lead to the identification of target molecules useful for the diagnosis and treatment of cancer. Proteomic Analysis using two-dimensional differential gel electrophoresis (2D-DIGE) The proteomic alterations associated with cancer were examined using 2D-DIGE, in which thousands of proteins are labeled simultaneously with multifluorescence dyes and separated based on their isoelectric point and molecular weight. Our 2D-DIGE system has several advantages: 1. The internal control of Cy3-labeled proteins allows high reproducibility of 2D pattern and accurate quantification of the protein spots. 2. Using the specialized software for 2D-DIGE, the 2D profiles in multiple gels are compared rapidly and automatically. 3. High throughput analysis; 24 samples per day can be studied by 2D-DIGE. 4. The robotic device excises protein spots automatically with little operator intervention. 5. Results of 2D gel analysis and sequence information from mass spectrometry are integrated in a database for further study. High-throughput protein identification by mass spectrometry Peptide finger printing and database search identify the spots of interests. The proteins in the gel are enzymatically digested and the masses of generated peptides are measured by mass spectrometry. The measured peptide masses are correlated with experimentally calculated peptide masses derived from proteins existing in sequence databases. Our original in-gel digestion protocol combined with three types of high performance mass spectrometry systems (Voyager DE Pro, Q-STAR and LCQ Deca) allows the high-throughput determination of protein sequences. Establishment of intestinal epithelial cells capable of inducing stabilized beta-catenin Aberrant transactivation of a certain set of target genes by beta-catenin and TCF/LEF complexes has been identified in human colorectal cancer cell lines and implicated in early colorectal tumorigenesis. From an immortal undifferentiated rat intestinal untransformed epithelial cell line, IEC6, several clones capable of inducing a beta-catenin protein lacking the 89-amino acid phosphorylation site in its NH₂-terminus under the strict control of the tetracycline-regulatory system were established (IEC6-Tet OFF beta-catenin deltaN89). It is anticipated that these clones will allow precise pinpointing of the molecular and cellular changes that occur following the activation of the beta-catenin/TCF4 complex. Gene expression profiling of beta-catenin mediated gene transactivation Using the IEC6-Tet OFF beta-catenin deltaN89 cells described above, a large-scale comparison of gene expression profiling was performed in order to define target genes of beta-catenin/TCF4-mediated gene transactivation. By hybridizing to oligonucleotide arrays (GeneChip, Affymetrix), more than 30,000 genes were examined for the differential expression before and after the induction of beta-catenin deltaN89. In addition to genes previously defined as target genes of beta-catenin and TCF/LEF complexes, such as cyclin D1 and axil (axin-2), several genes which were not described to be involved in a Wnt signaling pathway were identified.