The eventual goal of this work is the development of a pharmacological method to elevate levels of a known, tumor-suppressing protein, antizyme, and thereby reduce cancer promotion and tumor formation in humans. The polyamines, spermidine and spermine, and their diamine precursor, putrescine, are aliphatic cations essential for mammalian cell growth and viability. Cancer cells require elevated levels of the polyamines, and inhibitors that prevent the requisite polyamine synthesis or cellular uptake can block the promotional stage of tumor formation. Polyamine homeostasis in normal cells is largely dependent upon the activity of a regulatory protein, antizyme, which is synthesized in response to elevations in either spermidine or spermine. In turn, this small protein mediates both rapid degradation of the initial enzyme in polyamine biosynthesis, ornithine decarboxylase, and down regulation of the polyamine uptake system. By engineering oncogene-transfected cells to over express antizyme it is possible to inhibit both their transformed phenotype and their tumor-forming ability in animals.
The specific aims of the proposed study are structured to test the hypothesis that polyamine analogs can be used to gratuitously stimulate antizyme production, and thereby block the promotional phase of tumor formation. (1) Putrescine mimics that specifically stimulate transcription of the antizyme gene will be identified and characterized. (2) Polyamine analogs and oligoamines that specifically promote translation of the antizyme message in intact mammalian cells will be identified. (3) The potential of selected antizyme-inducing analogs to increase and sustain antizyme levels in mammalian cells will be evaluated. (4) The ability of selected analogs to reverse a transformed cell phenotype will be assessed using H-ras-transfected NIH 3T3 cells. Antizyme induction studies will use rat liver (HTC) and hamster (CHO) cells, and will involve detection of antizyme mRNA by northern-blot analysis, antizyme activity by ODC enzyme inhibition assays, and antizyme protein by immunodetection on western-blots. These studies will provide an essential foundation for further exploration of the chemical induction of antizyme as a means of chemoprevention in animals.
| Mitchell, John L A; Simkus, Carrie L; Thane, Thynn K et al. (2004) Antizyme induction mediates feedback limitation of the incorporation of specific polyamine analogues in tissue culture. Biochem J 384:271-9 |
| Mitchell, John L A; Leyser, Aviva; Holtorff, Michelle S et al. (2002) Antizyme induction by polyamine analogues as a factor of cell growth inhibition. Biochem J 366:663-71 |
