BOETTGER, Stefanie A.; LOW , Ben E.; HAMILTON-HOUDEK, Kimberley; WALKER, Charles W.; The University of New Hampshire; The University of New Hampshire; The University of New Hampshire; The University of New Hampshire: Cytoplasmic anchoring of p53 family member proteins by heat shock protein family member mortalin II in leukemic clam hemocytes.

Mechanisms causing cytoplasmic sequestration andinactivation of the tumor suppressor p53 are currently investigated only in vertebrate models, which are expensive to maintain and highly regulated. We use leukemic hemocytes from the softshell clam, Mya arenaria, both in vivo and in vitro, to investigate cytoplasmic anchoring of p53 family member proteins by mortalin (mot-2). Clam mot-2 displays >70% sequence homology with human mot-2. Mot-2 also displays co-localization with cytoplasmically sequestered clam p53 and p63/73 proteins.MKT-077 (a rhodocyanin dye binding to mortalin at the p53 binding site) were administered in vitro and leukemic clam hemocytes were examined for the over-expression of p53 and p63/73, cellular localization of p53, p63/73 and mot-2 and cell viability prior to and following drug exposure. Real time PCR (ABI Gene 5700) showed that p53 and p63/73 had increased expression levels at 14 and 24h following drug exposure. Western blots and immunocytochemistry indicated translocation of both p53 and p63/73 proteins from the cytoplasm into the nucleus followed by an increase in apoptotic cells and a subsequent decrease in viable cells/ml hemolymph. Following drug treatment, mot-2 distribution in the cytoplasm switched from perinuclear to pan-cytoplasmic. Changes in mot-2 distribution indicate that etoposide treatment may result in dissociation of mortalin and p53 and p63/73 followed by movement of these both transcription factors into the nucleus and apoptosis of tumor cells. (Supported by NCI CA88883 and Hatch to CWW)