NCI Program Project in Esophageal Carcinogenesis
Core D - Microbiology/Gene Expression Core
|Director:||Gary D. Wu, M.D.|
|Technical Director:||Sue Ann Keilbaugh, Ph.D.|
The Molecular Biology/Gene Expression Core Facility will provide two critical services to support the scientific goals of this Program Project, namely a digestive disease reagent bank and a gene expression facility, both of which will be focused towards esophageal cancer research:
Digestive disease reagent bank:
Each of the Program Project Leaders and their personnel have access to specific plasmids, expression constructs, reporter constructs, antibodies, RNA samples, and retroviral/adenoviral vectors that are relevant to the study of esophageal biology as it relates to carcinogenesis. As a result, these reagents have been organized and centralized. In addition, we will continue to store newly available reagents. One of the technicians is responsible for the acquisition, maintenance and distribution of these reagents. Nearly all the reagents provided by this bank are unique reagents that are not available commercially. For example, a common commercial source for reagents, ATCC, does not provide antibodies, RNA samples, or retroviral vectors. Furthermore, less than 10% of the cDNAs/expression vectors in the digestive disease reagent bank are available through ATCC. Tissue procurement, storage and RNA extraction will be centralized with the Morphology Core, and a database will be maintained.
Gene expression facility:
The ability to examine and quantify alterations in gene expression in fundamental to the understanding of molecular mechanisms that determine biological processes. High density gene arrays are now a technical reality. The cost of this technology, although previously prohibitive for most investigators, has very recently become affordable. This facility has 2 components: 1) arrays: both digestive organ custom arrays and Affymetrix commercial arrays; and 2) Real-time Quantitative PCR. The gene expression facility will be highly interactive, providing opportunities for the members of this Program Project to interact at multiple levels. Examples of these interactions include but are not limited to the following: 1) facilitate the analysis of wildtype and genetically engineered mice; 2) complement the analysis of gene expression performed by the Morphology Core through in situ hybridization; and 3) assist in the analysis of cell culture model systems.
Finally, the Molecular Biology/Gene Expression and Morphology Cores will work on a relational database that is web-based on behalf of the Project Leaders and their labs.
There are five databases that are currently available specifically for this Program Project:
1. Esophageal Tissue Database: To date, the Molecular Biology Core has collected over 400 specimens of the esophagus. Information about these tissue specimens can be obtained from this database using a searchable format. Choosing the “Query” button on the left panel will bring forward the search fields in this database. Search parameters include demographic information such as patient age, gender, race as well as tissue information (tissue type), tumor grade and TMN stage. Many of the tissue samples in this repository have been used to make tissue microarrays and have been stained for 17 different proteins listed on the lower half of this database. Semi-quantification of staining intensity has been graded by a pathologist and entered into the database. Clicking on an identifier for a specific tissue sample identified by this search will provide information on the status of the tissue sample.
2. Esophageal Cell Line Database:
a) Esophageal Transformed Cell Line Database: The Esophageal Cell Line Bank contains 19 transformed esophageal cell lines. In a searchable format, demographic and tumor characteristics such as differentiation, grade and/or stage can be obtained for each cell line. In addition, detailed status of 7 different oncogenes as well as cytokeratins is provided for each cell line in a searchable format. Searching this database will allow P01 Investigators to identify the appropriate transformed esophageal cell line to obtain from the Esophageal Cell Line Bank for their studies. Finally, a hyperlink is provided to the COSMIC database for nearly all of these cell lines.
b) Esophageal Nontransformed Cell Line Database: There are nontransformed, wild-type parental human and mouse esophageal cell lines maintained in the Esophageal Cell Line Bank (Human: EPC1, EPC2; Mouse MEK3N, MEK4N), all established by the P01 (previously, such cell lines did not exist). These cell lines are normal diploidy. The EPC1 and EPC2 cell lines in particular have also been immortalized with the introduction of hTERT to overcome replicative senescence. Subsequently, numerous genetic modifications have been introduced into these immortalized primarly esophageal cell lines, some of which have resulted in transformation based upon soft agar assays, nude mice xenotransplantation assays and organotypic culture assays.
3. Esophageal Organotypic Culture Database: There are three search fields in this database to identify the cell line used in the Organotypic Culture Experiment: 1) The identify of the parental cell line (EPC1, EPC2, MEK, MEK3N, MEK4N, T.Te, and TE); 2) The genetic modification introduced into the parental cell line (none, hTERT, AKT, IGFBP, p53, Ecaderin, Klf5, HIF1alpha, Notch, EGFR, Ras, p16, cyclin D1, cdx1, cdx2, c-myc, TGFR2, Rad); and 3) The immunoreagent (antibody) used to examine protein expression in each experiment (total of 39 different antibodies).
4. Esophageal Microarray Database: This database is currently under construction. It will serve as an archive for microarray data obtained from studies performed by P01 Investigators. Each experiment is identified by a specific name and a short experimental description. Upon the selection of a specific experiment, a full description of the experiment is provided as well as an opportunity to download a file containing experimental data.