== Representative growth curves show that Hsp53 expression significantly increases doubling time of H1299 cells (estimated at 108.1 hr). not likely the result of cytosolic sequestration since Map53, like Hsp53, localized almost exclusively to the nucleus. Functional similarity was observed in regulation by human MDM2, suggesting that the clam may have anmdm2homologue. Protein modeling identified an apparent MDM2 binding site in Map53, supporting the observation of a potential Map53/MDM2 interaction. Significant amino acid differences present in the Map53 tetramerization domain may potentially affect p53 protein/protein interactions. Taken together, these data suggest that the Map53 shares some functional similarity with human p53 as well as with other invertebrates, positioning the mollusk at a critical juncture in evolution of this gene family. Keywords:Mdm2, apoptosis, cell cycle arrest, bivalve, cancer, post-translational modification == 1. Introduction == The mammalian tumor suppressorp53is one of the most intensely investigated genes due to its dysregulation in the majority of human cancers (Vousden and Lane, 2007). P53 protein is a sequence-specific transcription factor best known for its role in maintaining genetic integrity following cellular stress, particularly in response to DNA damage. Under such conditions, p53 is stabilized by post-translational modifications (PTM), leading to nuclear accumulation and altered transcriptional activity. P53 binds to the p53-responsive elements of many target genes, thereby initiating cellular responses that include growth arrest and DNA repair, senescence or apoptosis. It remains unclear what actions or precursors trigger growth arrest versus apoptosis: tissue specificity, type and extent of DNA damage, post-translational modifications and other physiological conditions are hypothesized to play a role (Vousden, 2002;Sykes et al., 2006). Cells may remain viable following p53-initiated cell cycle arrest and DNA damage repair at the G1/S phase checkpoint (Venot et al., 1998). In this scenario, p53 acts as a transcription factor for p21(WAF1/CIP1), a cyclin-dependent kinase inhibitor capable of blocking cell cycle progression (El-Deiry et al., 1993). In the event DNA damage is too extensive for repair, cells may enter p53-mediated apoptosis, which involves activation of pro-apoptotic genes including members of the caspase family (Agarwal et al., 1998;Shengkan and Levine, 2001). This mechanism of action is crucial for resistance to neoplasia in humans. In the absence of cellular stress, p53 protein is maintained at low levels by the E3 protein-ubiquitin ligase, MDM2 (Alarcon-Vargas and Ronai, 2002;Oren, 2003). This effector protein modulates p53 levels through a negative feedback loop by both physically blocking the p53 transactivation domain and by targeting it for ubiquitin-mediated degradation (Moll and Petrenko, 2003). Constitutive expression ofmdm2in cell culture also appears to exhibit this feedback mechanism.In vivo, overexpression ofmdm2can inhibit the ability of p53 to arrest cells at the G1/S checkpoint in response to ionizing radiation.Mdm2overexpression has also been observed in human tumors that Daidzein lack inactivating mutations in p53 (Perry, 2004). A cDNA sequence with significant similarity to human p53 (Hsp53) in key functional domains was previously described from the soft-shell clam,Mya arenaria(Map53;Kelley et al., 2001). Conservation of functional domains, altered expression and sub-cellular localization of this purported p53 in neoplastic clam tissues (Kelley et al., 2001;Butler et al., 2004) suggest functional homology to mammalian p53. At least two types of neoplasia have been identified inM. arenaria: a germinoma (gonadal tumor;Barry and Yevich, 1975) and an unrelated proliferative hemocytic disease/disseminated neoplasia (reviewed inBarber, 2004). Previous Daidzein studies in this laboratory have shown a significant decrease in Map53 expression in neoplastic clam gonadal tissue compared to non-neoplastic tissue (Butler et al., 2004). In addition, clams exposed to the herbicide 2,4-dichlorophenoxyacetic acid exhibited a significant decrease in p53 protein expression concomitant with an altered tissue morphology in reproductive tissue (unpublished). An apparent dysregulation ofMap53also occurs Daidzein in the clam MYCC disseminated neoplasia, where neoplastic hemocytes exhibit aberrant cytosolic localization of p53 (Kelley et al., 2001;Walker et al., 2006,2008). This was recently confirmed byBttger et al. (2008). In light of the sequence similarities betweenHsp53andMap53, and the apparent dysregulation ofMap53under morphologically aberrant/pathologic conditions, we hypothesized that the two proteins function in a similar manner. This hypothesis was tested by transient transfection of a p53-null, human Daidzein non-small-cell-lung-carcinoma cell line (H1299) with plasmids expressing either human or clam p53. Function was assessed by monitoring the p53/Mdm2 feedback loop and expression of p53-mediated downstream markers of growth arrest and apoptosis. == 2. Materials.