The objective of this research plan is to generate and characterize reagents (DNA probes and monoclonal antibodies) which will ultimately aid in the diagnosis, clinical management and/or therapy of pancreatic adenocarcinoma. This goal will be accomplished by identifying and characterizing oncogene activity previously detected in DNA of the human pancreatic adenocarcinoma cell line HPAF using the NIH 3T3 transformation assay. Monoclonal antibodies have previously been raised against the HPAF transfected NIH 3T3 cells which were selected for reactivity with HPAF cells and HPAF transfected NIH 3T3 cells and lack of reactivity with untransfected NIH 3T3 cells. These will be tested for potential immunodiagnostic uses against a variety of normal and malignant human tissue specimens. The biochemical characteristics of the antigens defined by these antibodies will be established and compared to other known cell products associated with oncogenes. We will also attempt to isolate genes coding for the antigens recognized by the antitransfectant antibodies by constructing and directly screening an HPAF cDNA expression library using the lambda gt 11 cloning and expression vector. If the monoclonal antibodies are unreactive with the cDNA expression library, then second generation monoclonal antibodies developed against denatured purified antigen will be used to screen the expression library. If this procedure was unsuccessful, then a transfectant cDNA library would be generated which could be positively selected against HPAF DNA and negatively selected against NIH 3T3 DNA to identify HPAF positive genes in the transfected cells. Genes selected in any of the above procedures would be used to select the intact gene from an HPAF genomic library, which would be used in transfection experiments to assess transforming capability. The sequence of these genes would be determined and used for comparison with other known sequences. Transfection studies will be repeated using DNA derived from fresh pancreatic tumor isolates to determine whether the observations made with the HPAF cell line are applicable to other pancreatic adenocarcinoma tumors.
| Hollingsworth, M A; Borowitz, M J; Everett, M L et al. (1990) A novel ribonucleoprotein complex defined by monoclonal antibodies to NIH 3T3 cells transfected with human pancreatic adenocarcinoma DNA. Cancer Res 50:6015-22 |
