Cellular and biochemical characterization of the polyamine transport system in various tumor cell lines.

摘要

Polyamine transport is an active process associated with all cell types and known to be critically involved in the regulation and maintenance of intracellular polyamine pools. This thesis is comprised of four related sections focusing on different aspects of polyamine transport which have not previously been studied. In the first section, polyamine transport activity was shown to display a biphasic increase in synchronized human lung carcinoma A549 cells during cell cycle progression. Transport-deficient A549-4 cells were found to be much more sensitive to polyamine inhibitors than parental cells. In the second section, certain human prostate tumor cell lines were found to be unique among mammalian cell lines in these inability to regulate transport in response to polyamine inhibitor or analog treatment. In the third section, a new and potent polyamine-conjugated N1-azidosalicylamido-spermine was used in combination with photoaffinity labeling to identify certain polyamine binding proteins (pbps) on the surface of murine leukemia L1210 cells. Following labeling, partial chromatographic purification and partial amino acid sequencing analysis, one of their proteins, pbp118, was determined to be dipeptidyl peptidase IV (DPPIV, also known as CD26). To our knowledge, pbp118/DPPIV/CD26 is the first polyamine binding protein to be identified on the surface of mammalian cells and the relationship to polyamine transport is hypothesized. In the final section, photoaffinity labeling was further developed for application to solid-tumor A549 cells using N1-ASA-Spm and four pbps were found. These pbps demonstrated specificity for polyamine binding and at least certain of them appear to be specifically involved in polyamine transport.;Overall, it is concluded that polyamine transport is an important cellular process from both biologic and therapeutic perspectives. Leads deriving from the present studies have obvious therapeutic potential and warrant further exploration. Although photoaffinity labeling provided meaningful information regarding cell surface pbps, we conclude that definitive findings relating pbps to transport will require combining this approach with others such as genetic manipulations.