The overall objectives of this renewal are to continue to define the molecular mechanisms controlling the phenotype-specific regulation of the polyamine catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT) in human lung cancer cells and the role of this process in the phenotype-specific cytotoxic response of these tumors to the antitumor polyamine analogues. We were the first to demonstrate a phenotype-specific response to the polyamine analogues that was associated with the superinduction of SSAT. Specifically, the non-small cell lung cancer (non-SCLC) phenotypes respond to analogue treatment with a much higher SSAT induction and are generally more sensitive to the polyamine analogues than are the SCLC phenotypes. Based in part on our results, phase II trials have been initiated with one of the highest SSAT inducing compounds, N1, N11-bis(ethyl)norspermine. During the course of the current granting period we have demonstrated that superinduction of polyamine catabolism and programmed cell death (PCD) are induced by the bis(alkyl)polyamines. H2O2production by polyamine catabolism plays a direct role in the early onset of PCD in non-SCLC lines. We have identified a cis-acting element in the human SSAT gene and a transcription factor (Nrf-2) that are involved in the phenotype-specific transcriptional regulation of SSAT. During our studies of SSAT and analogue response, we developed the first human polyamine transport deficient lines. This renewal will expand our studies into the role of SSAT induction and H2O2 production in tumor response to the analogues. We will use a conditional knockout strategy to ascertain if SSAT is required for cell survival, as our preliminary data suggest, or if it is necessary for rapid drug response. We will expand results demonstrating that nuclear/cytoplasmic transport is a major step in SSAT regulation. Finally, since there is a critical interplay between polyamine metabolism and polyamine transport that affects the accumulation and activity of the analogues, we will use the human polyamine transport deficient lines to clone components of the human polyamine transport system by a retroviral expression complementation technique. We project that understanding the regulation of SSAT in response to the polyamine analogues, in addition to the definition of proteins involved in their transport will aid in the more effective use of agents that target the polyamine metabolic pathway.
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