Investigations of Mammalian Aminopropyltransferases

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DESCRIPTION(adapted from applicant's abstract): Polyamine content is increased during tumor promotion and in neoplastic growth. There is good evidence that the enzymes of the polyamine biosynthetic pathway are valid targets for the design of chemopreventive/chemotherapeutic agents and polyamine analogs such as N1,N11-bis(ethyl)norspermine (BE-3-3-3) are currently undergoing clinical trials as anti-tumor agents. The proposed experiments are aimed at several areas of polyamine physiology and analog action. These include the physiological role of the largest polyamine, spermine; and the importance and regulation of the enzyme spermidine/spermine-N1-acetyltransferase (SSAT), which is the rate limiting step in the conversion of the higher polyamines into putrescine. There are 3 specific aims: (A) To investigate the function(s) of spermine. All mammalian cells have the enzyme spermine synthase and produce spermine but there are no clear indications of any unique function of this polyamine. This will be investigated using cells lacking spermine synthase. Preliminary experiments indicate that these cells are considerably more sensitive to killing by BCNU and other agents that damage DNA. The role of spermine in protection of cells from DNA damage will therefore be the major initial focus for these studies. (B) To study the regulation and importance of SSAT. (B-1) SSAT is very highly inducible by polyamines and by some polyamine analogs such as BE-3-3-3. We have shown that these inducers stabilize the protein against intracellular degradation which occurs via the ubiquitin/proteasome system. Experiments will be carried out to elucidate the pathway of SSAT degradation and the mechanism by which polyamines prevent degradation. (B-2) The structure/function and substrate specificity of SSAT will be studied. These studies will include examination of mutant L156F which is present in CHO55.7Res cells described below. (B-3) The possibility that excess putrescine formation by SSAT enhances neoplastic growth will be tested using transgenic mice that we have generated that express SSAT in the skin from the K6 promoter. (C) To study the mechanism of the antiproliferative action of the polyamine analogs. Using a novel procedure to avoid transport mutants, we have isolated CHO cell lines resistant to BE-3-3-3 (CHO55.7Res). These cells do not express SSAT in response to the analog. Further experiments will test whether this is responsible for their resistance, investigate other potential differences between the control and CH055.7Res cells and use a similar but appropriately modified procedure to isolate human tumor cells resistant to BE-3-3-3. Cross-resistance to other polyamine analogs will be tested using the BE-3-3-3 resistant cells. We have already found a lack of cross-resistance of CHO55.7Res to another analog, CHEN-Spm, suggesting that this drug either acts at a different site. A similar protocol to that used to obtain the BE-3-3-3 resistant cells will be used to isolate cells resistant to CHE-Spm which will then be used to study its mechanism of action.