NOVEL APPROACHES TO MODULATE ORNITHINE DECARBOXYLASE ACTIVITY AND TO DETERMINE A ROLE FOR ORNITHINE DECARBOXYLASE IN CELL TRANSFORMATION

Open Access
Author:
Ackermann, Joseph Michael
Graduate Program:
Pharmacology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
December 21, 2004
Committee Members:
  • Anthony Edward Pegg, Committee Chair
  • Melvin Lee Billingsley, Committee Member
  • Mark Kester, Committee Member
  • Lisa M Shantz, Committee Member
  • Charles D Smith, Committee Member
  • Kent Eugene Vrana, Committee Member
Keywords:
  • polyamines
  • ornithine decarboxylase
  • gene silencing
  • DNazyme
  • ribozyme
  • antisense oligodeoxynucleotides
  • difluoromethylornithine
  • DFMO
  • resonance energy transfer
  • RET
  • FRET
  • catalytic nuleic acids
Abstract:
Ornithine decarboxylase (ODC) is known to have an important role in cell transformation, but that role is not well understood. The scientific and clinical value of reducing the activity of ODC, a key enzyme in the biosynthesis of polyamines, is well appreciated. Polyamines are necessary components for cell growth and manipulation of polyamine homeostasis may be an effective strategy for the treatment of a number of disorders, including neoplastic diseases. Indeed, -difluoromethylornithine (DFMO), a specific enzyme-activated irreversible inhibitor of ODC is currently in clinical trials as a potential chemopreventative/therapeutic agent and is clinically used for other disorders. However, the effectiveness of DFMO is limited by the drug's pharmacokinetic profile and the unique regulation of ODC. Targeting ODC mRNA may be an effective method to reduce ODC activity due to the enzyme's extensive regulation at the translational and protein levels. The half-life of ODC is among the shortest of any known protein. Due to the rapid turnover of ODC protein, cells are reliant on translation of new protein to maintain or elevate ODC activity. A number of reports have indicated that little to no change in ODC mRNA content is often detected upon substantial increases or decreases in ODC protein content and activity. Reversible inhibitors of ODC protein have a disadvantage because they can stabilize ODC from degradation. The initial inhibition of ODC leads to increased ODC transcription and active ODC can accumulate in the cells. Conversely, the efficacy of irreversible inhibitors may be limited by the short half-life of ODC. Therefore targeting ODC mRNA, rather than protein, may be a better strategy. An approach to develop an effective 10-23 DNAzyme and hammerhead ribozyme against ODC is described in these studies. DNAzymes and ribozymes bind to a cognate RNA substrate via Watson-Crick base pairing hybridization arms and subsequently catalyze cleavage of a phosphodiester bond of the RNA. The major difference between the two is the base composition; DNAzymes are composed of DNA, ribozymes of RNA. DNAzymes capable of cleaving the target ODC RNA were identified in vitro and further characterized by the effect each had on ODC protein and activity levels using in vitro translated ODC RNA. ODC protein levels and activity correlated well with the RNA cleavage activity of the DNAzyme. A DNAzymes that exhibited good activity was optimized for use in cell culture studies. The length of the DNAzyme hybridization arms was altered and kinetics were performed to identify the most catalytically efficient configuration. One DNAzyme, DZ IV, with equal length arms of nine nucleotides proved to be the most catalytically efficient. In HEK 293 cells, DZ IV was able to reduce the amount of translated ODC protein resulting in ~80% reduction in ODC activity—a statistically significant enhancement over the antisense effect of a catalytically inactive DNAzyme. The effectiveness of DZ IV was evaluated an in vivo model with clinical significance. DFMO is currently undergoing clinical trials for chemoprevention in skin cancer and topical application of DFMO is FDA-approved for the treatment of unwanted facial hair. ODC activity is induced in the skin in response to tumor promoters, including 12-O-tetradecanoylphorbol-13-acetate (TPA). Topical DZ IV reduced TPA-induced ODC activity by ~50 % over vehicle control in the dermis of mice. The result is more impressive considering that TPA treatment induces ODC activity ~1000-fold over basal levels. Hammerhead ribozymes targeted against ODC were designed to sites identified by a ribozyme site selection screen. Two of the identified sites cleaved ODC mRNA within 5 nucleotides from the DZ IV cleavage site indicating that this is an accessible site within the mRNA. Four of the ribozymes that most effectively cleaved in vitro transcribed RNA were cloned into a novel expression vector. The ribozymes were evaluated in cell culture, however an accurate assessment could not be made due to non-specific silencing from the expression vector. The ribozymes may be active but must be evaluated with a different expression system. ODC is known to have an important role in cell transformation, but that role is not well understood. A better understanding of the role of ODC in transformation may identify effectors downstream of ODC that are better drug targets. NIH/3T3 cells transfected with an activated Ras mutant (3T3+HRas(61L) cells) display a transformed phenotype, but the transformed phenotype can be reversed by co-expression of a dominant-negative ODC (3T3+HRas(61L)+ODC-dn cells). Microarray analysis was performed on the 3T3, 3T3+HRas (61L) and 3T3+HRas(61L)+ODC-dn cells and genes potentially downstream of ODC that are essential for Ras transformation in 3T3 cells were identified. In conclusion, these results indicate that targeting ODC at the mRNA level is an effective way to modulate ODC activity. An effective DNAzyme has been demonstrated to be active in vitro and in vivo and can be used as a tool to study the function and translational regulation of ODC and the potential of DNAzymes for therapeutic uses. Identification of effectors downstream of ODC that cooperate in transformation may provide better chemopreventative/therapeutic drug targets than ODC.