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Characterization of a putative truncated Calcium-sensing receptor form human ovarian tumors


EMSL Project ID
2106

Abstract

Rationale: We have determined that the wild-type calcium-sensing receptor [CaR] plays an important role in regulating the proliferative behavior of normal ovarian surface epithelial cells, the cell type responsible for ovarian cancer. In a study of 55 ovarian tumor samples from patients at OHSU, we have observed a highly immunoreactive band of 65 kDa which is specifically recognized by our anti-CaR antibody [antibody binding is blocked in the presence of excess competitive petide]. Statisical analysis indicates a positive correlation between increasing 65 kDa expression and tumor stage and grade at a probability of 0.0017. This 65 kDa protein could represent a truncated version of the CaR resulting from alternate splicing, frame-shift mutation, or abnormal proteolytic processing; it is also formally possible that it is a distinct protein expressing an epitope recognized by our antibody, generated against a synthetic peptide. Objective: To characterize the 65 kDa protein recognized by anti-CaR antibodies and over-expressed in stage III/IV ovarian cancer. Approach: After partial biochemical purification of the full length and truncated CaR, the appropriate fractions are size-fractionated by SDS-PAGE and Coomassie stained bands corresponding to the immunoreactive proteins at 120 kDa and 65 kDa are excised and subjected to in-gel tyrptic digestion. The resulting peptide fragments have been subjected to MALDI-TOF analysis, and several peptides with masses corresponding to predicted fragments of the published human CaR have been identified. MS-MS analysis is requested to establish whether or not these peptide fragments are from the human CaR, and to confirm the identification of the CaR. Fragments of most interest at present are 859.442, 1356.65, and 1938.059 Da.

Project Details

Project type
Exploratory Research
Start Date
2000-11-29
End Date
2002-09-24
Status
Closed

Team

Principal Investigator

Karin Rodland
Institution
Pacific Northwest National Laboratory

Team Members

David Springer
Institution
Pacific Northwest National Laboratory