Date of Award

8-2001

Level of Access Assigned by Author

Campus-Only Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Advisor

Rebecca J. Van Beneden

Second Committee Member

Bruce D. Sidell

Third Committee Member

Mary S. Tyler

Additional Committee Members

Robert Gundersen

Holly A. LaVoie

Abstract

A high prevalence of gonadal tumors has been documented in several populations of softshell clams (Mya arenaria) in eastern Maine. The etiology of these tumors is unknown. The hypothesis that dioxin contamination contributes to tumor formation is supported by several observations: 1) dioxin is a known carcinogen; 2) dioxin contamination of the local environment; 3) propensity for dioxin accumulation in the clam gonad. TCDD-induced changes in gene expression in laboratory-exposed clams included the up-regulation of an E3 ubiquitin-protein ligase structurally similar to human E6-AP. In human papillomavirus (HPV)-positive cells, E6-AP abnormally targets the tumor suppressor p53 for ubiquitin-mediated proteolytic degradation, which contributes to the formation of over 90% of human cervical cancers. A clam hect E3 was cloned and found to have significant amino acid identity and similar predicted secondary structure compared to human E6-AP, including the absolutely conserved cysteine residue required for thioester bond formation with ubiquitin. An ubiquitin-binding assay using wild-type clam E3 and a cysteine-to- alanine mutant (E3C-a) confirmed that the clam E3 functions as an ubiquitin- protein ligase. Based on structural and functional similarities, it is hypothesized that TCDD-induced expression of clam E3 targets p53 for degradation, leading to tumor formation.

In order to address this question, clam homologues for human p53 and p73 were cloned. M. arenaria p53 (Map53) and p73 (Map73) expression patterns were examined in two clam cancers: gonadal tumors and leukemia. Map73 was up- regulated in leukemic clam hemocytes, whereas Map53 levels were significantly lower in gonadal tumor-bearing females, suggesting roles for these proteins in different clam cancers. Further analysis of the gonadal cancers revealed a significant increase in E3 expression concomitant with a decrease in Map53. Binding studies also indicated that clam E3 physically associates with both Map53 and Map73, suggesting possible targeting of these proteins for degradation. However, E3-dependent degradation of Map53 and Map73 was not established using an in vitro mammalian system. Future investigation of E3- mediated degradation of Map53 and/or Map73 may require isolation of other clam-specific ubiquitination pathway components.

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