INVESTIGATIONS OF POLYAMINE FUNCTION USING TRANSGENIC MOUSE MODELS WITH SPERMIDINE SYNTHASE AND S-ADENOSYLMETHIONINE DECARBOXYLASE OVEREXPRESSION
Open Access
- Author:
- Shi, Chenxu
- Graduate Program:
- Physiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 13, 2011
- Committee Members:
- David J Feith, Ph D, Dissertation Advisor/Co-Advisor
David J Feith, Committee Chair/Co-Chair
Anthony Edward Pegg, Committee Member
Lisa M Shantz, Committee Member
Edward Joseph Gunther, Committee Member
Scot R Kimball, Committee Member
Barbara Ann Miller, Committee Member - Keywords:
- Polyamines
S-adenosylmethionine decarboxylase
Spermidine synthase
Transgenic mice
Nonmelanoma skin cancer - Abstract:
- Polyamines, including putrescine, spermidine, and spermine, are ubiquitous polycationic compounds that are essential for cell growth and differentiation. To better understand cellular function of polyamines, two transgenic mouse models with either widespread constitutive overexpression of spermidine synthase (SpdS) or regulated expression of S-adenosylmethionine decarboxylase (AdoMetDC) in the skin were created and characterized. SpdS is a polyamine biosynthetic enzyme, and it transfers an aminopropyl group from decarboxylated S-adenosylmethionine (dcAdoMet) to putrescine to produce spermidine. We generated a transgenic mouse line that overexpresses human spermidine synthase from a composite cytomegalovirus- immediate early gene enhancer/chicken β-actin promoter (CAG) in order to determine the impact of elevated SpdS activity on murine development and physiology, and to enable simultaneous overexpression of SpdS and other polyamine biosynthetic enzymes by combining transgenic models. CAG-SpdS mice were viable and fertile and exhibited a reduced tissue spermine:spermidine ratio that correlated with the level of SpdS activity. We also demonstrated that the combined overexpression of both SpdS and spermine synthase (SpmS) in CAG-SpdS/CAG-SpmS bitransgenic mice did not impair murine viability or lead to overt developmental abnormalities but instead normalizes the elevated tissue spermine:spermidine ratios of CAG-SpmS mice. Finally, upon breeding of CAG-SpdS mice to MHC (α-myosin heavy chain)-AdoMetDC mice with a >100-fold increase in cardiac AdoMetDC activity, CAG-SpdS/MHC-AdoMetDC bitransgenic animals were produced at the expected frequency and exhibited cardiac polyamine levels comparable to MHC-AdoMetDC littermates. Taken together these results indicate that SpdS levels are unlikely to exert significant regulatory effects on cellular polyamine content and function. Previous studies suggest that the increased levels of polyamines, especially putrescine, play a critical role in skin tumor development. AdoMetDC is a rate-limiting enzyme in the polyamine biosynthetic pathway. It catalyzes the decarboxylation of S-adenosylmethionine (AdoMet) to generate dcAdoMet that serves as the aminopropyl donor for the production of spermidine and spermine from the precursor putrescine. We utilized the Tet-off system to generate transgenic mice with regulated expression of AdoMetDC in skin basal keratinocytes directed by the Keratin 5 promoter to enhance our understanding of the role played by AdoMetDC in controlling polyamine levels and of polyamine function in epithelial carcinogenesis. We found that untreated transgenic mice displayed a 7 to 8-fold increase in epidermal and dermal AdoMetDC activity at 7 weeks of age, and a corresponding increase in the enzymatic product dcAdoMet levels. These mice also exhibited a thin fur phenotype that could be eliminated by doxycycline treatment. A skin chemical carcinogenesis experiment on these mice demonstrated that AdoMetDC overexpression resulted in a 46% reduction in tumor multiplicity compared to controls after 25 weeks of tumor promotion. A further study of tumor growth upon transgene silencing by doxycyline suggested that the tumor suppressive effect was the result of blocking the growth of initiated cells at a microscopic, dormant but still viable state in the skin. These findings will help with future development of polyamine-targeted therapies to prevent and treat cancer.