Mass culture and characterization of tumor cells from a naturally occurring invertebrate cancer model: applications for human and animal disease and environmental health.

On the northeastern coast of the United States and Canada, Mya arenaria, the soft shell clam, develops a diffuse, hemopoetic tumor (a fatal leukemia-like cancer) resulting from inactivation of p53-like family member proteins.These malignant cells provide a model for an unrelated set of human cancer cells that are also characterized by mortalin-based cytoplasmic sequestration of wild-type p53 tumor suppressor protein (mortalin is the mitochondrial Hsp70 protein). Here we describe methods for mass culture and long-term storage of tumor cells from this cancer. These are the first successful efforts at maintaining malignant cells from any marine invertebrate in vitro. Following passage (subculture), these cultures undergo transition from primary cultures to non-immortalized cell lines that continue to proliferate and do not re-differentiate the normal hemocyte phenotype. We also characterize normal clam hemocytes and the pathology of cancerous clam hemocytes in vitro and in vivo using light and electron microscopy, cyto- and immunocytochemistry, molecular biology, and a phagocytosis assay. Our protocols provide biomedical and environmental researchers with ready access to this naturally occurring cancer model. We discuss the clam cancer model regarding (a) human health and disease; (b) animal health, disease, and aquaculture; (c) environmental health monitoring; and (d) future research directions.

Genotoxic stress-induced expression of p53 and apoptosis in leukemic clam hemocytes with cytoplasmically sequestered p53.

In nature, the soft shell clam, Mya arenaria, develops a fatal blood cancer in which a highly conserved homologue for wild-type human p53 protein is rendered nonfunctional by cytoplasmic sequestration. In untreated leukemic clam hemocytes, p53 is complexed throughout the cytoplasm with overexpressed variants for both clam homologues (full-length variant, 1,200-fold and truncated variant, 620-fold above normal clam hemocytes) of human mortalin, an Hsp70 family protein. In vitro treatment with etoposide only and in vivo treatment with either etoposide or mitoxantrone induces DNA damage, elevates expression (600-fold) and promotes nuclear translocation of p53, and results in apoptosis of leukemic clam hemocytes. Pretreatment with wheat germ agglutinin followed by etoposide treatment induces DNA damage and elevates p53 expression (893-fold) but does not overcome cytoplasmic sequestration of p53 or induce apoptosis. We show that leukemic clam hemocytes have an intact p53 pathway, and that maintenance of this tumor phenotype requires nuclear absence of p53, resulting from its localization in the cytoplasm of leukemic clam hemocytes. The effects of these topoisomerase II poisons may result as mortalin-based cytoplasmic tethering is overwhelmed by de novo expression of p53 protein after DNA damage induced by genotoxic stress. Soft shell clam leukemia provides excellent in vivo and in vitro models for developing genotoxic and nongenotoxic cancer therapies for reactivating p53 transcription in human and other animal cancers displaying mortalin-based cytoplasmic sequestration of the p53 tumor suppressor, such as colorectal cancers and primary and secondary glioblastomas, though not apparently leukemias or lymphomas.