Date of Award

1999

Degree Type

Thesis

Degree Name

Master of Science in Pharmaceutical Sciences

Specialization

Biomedical Sciences

Department

Biomedical Sciences

First Advisor

Bingfang Yan

Abstract

Basic helix-loop-helix (bHLH) proteins belong to a large family of transcription factors that are known to play important roles in cell proliferation, differentiation and oncogenesis. These proteins are structurally featured by a bHLH motif, which is responsible for protein dimerization and sequence-specific DNA binding (e.g., E-box). Recently we isolated a cDNA from a human liver library by a gene trapping method. Based on the Kozak rule, this cDNA encodes a protein with 415 amino acids, which is hereafter designated as CCAF. The objective of this thesis is to establish the molecular mass of this protein and to test the hypothesis that CCAF is a transcriptional modulator involving the regulation of cell cycle events.

To establish the molecular mass, CCAF was in vitro translated with TNT reticulocyte lysate and analyzed by autoradiography. Addition of the CCAF cDNA to the reaction mixture yielded a single product with a molecular weight of 52 kDa. This mass is consistent with the estimated weight and suggests that the Kozak favorable sequence indeed harbors the codon for translation initiation. In order to determine whether CCAF undergoes posttranslational modifications, immunochemical experiments were performed. An antibody was raised against a peptide derived from CCAF and subjected to affinity chromatography. This antibody detected a 52-kDa protein in the CCAF cDNA transfected cells but not in the control cells. These results further support the notion that the functional CCAF is a 52-kDa protein and undergoes little post-translational modifications.

To determine the expression of CCAF in different cell growing states, OLD cells derived from colon carcinomas were seeded at different densities. Likewise, this antibody detected the 52-kDa protein in the cells plated at all densities. However, proliferating cells expressed higher levels (-3-5 folds) than the quiescent cells. These studies were further extended to human colon carcinomas. Both Northern and Western blotting analyses detected abundant expression of CCAF in the carcinomas but not in the nearby normal tissues. These findings suggest that CCAF involves the regulation of cell cycle events and contributes to the oncogenic pathogenesis.

To determine the activity of CCAF in transcription regulation, transient cotransfection experiments were conducted with an E-box reporter. CCAF alone caused little change on the reporter enzyme activity. However, CCAF antagonized by 30% the transactivation activity conferred by E4 7, a bHLH protein that is known to transactivate E-box reporter. The antagonism on E47-mediated transactivation activity and the differential expression relating to cell growing and oncogenic states support the hypothesis that CCAF is a transcriptional modulator that involves the regulation of cell cycle events and plays a role in oncogenic pathology.

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