Stimulated Motility in Tumor Cells

Clinical Sciences

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Abstract

We have been studying tumor cell motility as a component of the process of metastasis with a special emphasis on factors that could regulate this motility. To this end, we purified, cloned, and sequenced autotaxin, a tumor-secreted motility-stimulating glycoprotein from the conditioned medium of a human melanoma cell line. Utilizing reverse transciption followed by PCR, we have also isolated autotaxin from human breast carcinoma and teratocarcinoma cell lines. Northern blot analysis of multiple normal tissues indicated that this protein has the highest steady state levels in ovary, small intestine, brain and placenta. In addition to its motility-stimulating properties, autotaxin possesses type I phosphodiesterase, ATPase, nucleotide pyrophosphatase, inorganic pyrophosphatase, 5|-nucleotidase, autophosphorylation, and ATP binding activities. Recombinant autotaxin, purified to homogeneity, retains all of the above activities and stimulates motility at high picomolar to low nanomolar concentrations. Though the human melanoma cell line, A2058, has been used as the motility model in most of the purifications of autotaxin, we have found that certain breast and prostatic carcinoma cells, neuroblastoma cells, bovine aortic smooth muscle cells, as well as HUVEC|s (human umbilical vein cells) also respond to autotaxin as a chemoattractant. Studies utilizing site-directed mutagenesis of the phosphodiesterase active site demonstrated that an intact phosphodiesterase catalytic site is necessary for motility, but phosphorylation of the molecule does not appear to be a prerequisite. Since autotaxin has no known ATP binding site, we utilized a radiolabelled 8-azido-ATP to demonstrate ATP binding to autotaxin and to localize two regions on the molecule where the 8-azido-ATP crosslinks after exposure to light. The first region is the phosphodiesterase active site itself. The second region is 108 amino acids downstream, is unlike any reported ATP binding sites, and is sensitive to addition of salt. We have found that autotaxin possesses type I phosphodiesterase, ATPase, nucleotide pyrophosphatase, autophosphorylation, and ATP binding activities. Utilizing site- directed mutagenesis, we explored the relationship between the enzymatic properties of autotaxin and its capacity to stimulate motility. We found that ATX is the long sought enzyme that generates the bioactive phospholipids S1P and LPA from precursors.