Date of Award

12-2005

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Marine Biology

Advisor

Rebecca J. Van Beneden

Second Committee Member

J. Malcolm Shick

Third Committee Member

Paul D. Rawson

Abstract

Gonadal tumors in the soft-shell clam, Mya arenaria, have been observed in certain eastern Maine populations. The etiology of the tumors is unknown, but environmental factors including cell cycle disrupting contaminants are likely involved. The studies reported here focus on the role of the tumor suppressor protein p53, which functions as a transcription factor in cell cycle regulation. In response to DNA damage, p53 protein is stabilized and can activate pathways leading to either cell cycle arrest or apoptosis, thereby preventing the perpetuation of damaged DNA. If mutated, p53 loses its protective function, potentially leading to tumor formation. Mya arenaria p53 (map53) has been identified through sequence similarity to known vertebrate and invertebrate p53s. Earlier histological and gene expression studies linked an undifferentiated cell state in clam gonadal tissue with altered Map53 protein levels. These undifferentiated cells were observed in clams exposed in the laboratory to 2,4-dichlorophenoxyacetic acid (2,4-D) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and resemble cells seen in gonadal tissue of field-collected tumor-bearing clams. The Map53 sequence shows homology to Homo sapiens p53 (Hsp53) in key functional domains. These observations regarding Map53 led to the hypothesis that Map53 and Hsp 53 function in a similar manner. To assess this possible functional similarity, constructs were made using Map53 or wild-type Hsp53 cDNA under direction of a constitutive promoter, and transfected into the mammalian p53-null cell line H1299. The expression of p53, p21WAFI/CIP1 retinoblastoma (Rb), activated caspase-3, and actin was determined by western blot analysis. p53-mediated GI phase growth arrest was evaluated by flow cytometry (cell cycle analysis), p21WAFl/CIPl and Rb expression. Apoptosis was measured using activated caspase-3 expression and flow cytometry as indicators. HI299 cells transfected with mp53 entered a transient state of growth arrest as determined by both protein expression and flow cytometry data. HI299 cells did not show signs of either apoptosis (activated caspase-3 expression) or growth arrest (a halt in cell cycle progression) as a consequence of Map53 transfection. Rather, those cells transfected with Map53 acted in a manner similar to the controls. Results suggest that Map53 functions in a different manner than Hsp53 within the HI299 cell system.

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