No abstract

The thymidylate synthase inhibitor 5-fluorouracil (5-FU) continues to play a pivotal role in the treatment of cancer. A downstream event of thymidylate synthase inhibition involves the induction of a self-defeating base excision repair process. With the depletion of TTP pools, there is also an increase in dUMP. Metabolism of dUMP to the triphosphate dUTP results in elevated pools of this atypical precursor for DNA synthesis. Under these conditions, there is a destructive cycle of dUMP incorporation into DNA, removal of uracil by the base excision repair enzyme uracil-DNA glycosylase (UDG), and reincorporation of dUMP during the synthesis phase of DNA repair. The end point is DNA strand breaks and loss of DNA integrity, which contributes to cell death. Evidence presented here indicates that both the nuclear and the mitochondrial isoforms of UDG are modulated by FdUrd (and 5-FU) treatment in certain cell lines but not in others. Modulation occurs at the transcriptional and post-translational levels. Under normal conditions, nUDG protein appears in G 1 and is degraded during the S to G 2 phase transition. The present study provides evidence that, in certain cell lines, FdUrd mediates an atypical turnover of nUDG. Additional data indicate that, for cell lines that do not down-regulate nUDG, small interfering RNA-mediated knockdown of nUDG significantly increases resistance to the cytotoxic effects of FdUrd.Results from these studies show that nUDG is an additional determinant in FdUrd-mediated cytotoxicity and bolster the notion that the self-defeating base excision repair pathway, instigated by elevated dUTP (FdUTP) pools, contributes to the cytotoxic consequences of 5-FU chemotherapy. (Cancer Res 2006; 66(17): 8829-37)

The C-terminal region of peripherin/rds contains three predicted α-helical domains. One of these domains, corresponding to amino acids 311-322, form an amphiphilic α-helix previously shown to promote membrane fusion. The present studies were conducted to determine how the additional α-helical regions of the peripherin/rds C-terminus affect complex formation with rom-1, glycosylation, intracellular localization and membrane fusion properties. Bovine peripherin/rds and rom-1 were epitope tagged with an amino-terminal FLAG-tag or amino-terminal hemagglutinin (HA)-tag, respectively, and cloned into the pCI-neo expression vector for transient transfection into COS cells. Similarly, four C-terminal peripherin/rds truncation mutants (Δ1, Δ2, Δ3 and Δ4), corresponding to deletions of −19, −29, −39 and −59 amino acids were designed to disrupt the α-helical domains. Immunofluorescence microscopy and enzymatic digestions demonstrated that full-length peripherin/rds and the four C-terminal deletion mutants were localized to intracellular membranes and were all Endo-H sensitive. Western blotting and immunoprecipitation studies showed that the FLAG-tagged bovine peripherin/rds (full-length) was expressed as a 76 kDa dimer, which associates with HA-tagged rom-1 to form a higher order complex. The deletion mutants were also able to associate with rom-1. However, when analyzed using non-denaturing tricine electrophoresis, full-length peripherin/rds and the Δ1, Δ2 and Δ3 mutants formed homo-oligomeric complexes, while the Δ4 mutant appeared to form only homodimers suggesting a region upstream of amino acid 300 may be involved in C-terminal interactions. Membrane fusion was then evaluated using fluorescence resonance energy transfer (RET) techniques. Intracellular COS cell membranes containing full-length peripherin/rds fused with rod outer segment plasma membrane vesicles. This fusion was inhibited with the addition of a synthetic peptide (PP-5) corresponding to the fusion domain of peripherin/rds. In contrast, fusion was negligible with any of the C-terminal truncation mutants. Collectively, these results suggest that in addition to the fusion domain, other regions of the peripherin/rds C-terminus are required for fusion. Most interesting is the observation that the last 19 amino acids, a region downstream of the fusion peptide that is deleted in the Δ1 mutant, appear to be necessary for fusion. This region corresponds to the epitope for anti-peripherin/ rds monoclonal antibody 2B6, which is shown to partially inhibit peripherin/rds mediated membrane fusion.

We have previously demonstrated that human cells contain multiple forms of uracil-DNA glycosylase (Caradonna, S. J., Ladner, R., Hansbury, M., Kosciuk, M., Lynch, F., and Muller, S. J. (1996) Exp. Cell Res. 222, 345-359). One of these is an M r 29,000 processed form of the highly conserved uracil-DNA glycosylase (UDG1) located in the mitochondria. The others are located in the nucleus and migrate as a group of at least three distinct bands within the 35,000 -37,000 molecular weight range. In this report, we perform a detailed characterization of the M r 35,000 -37,000 purified proteins. To accomplish this, uracil-DNA glycosylases were affinity purified from HeLa cell nuclear extracts. The proteins were separated by SDS-PAGE, and their identities were verified by renaturation and activity assays. The three protein bands were individually digested with cyanogen bromide, and the resulting peptide fragments were analyzed by direct amino acid sequencing. Peptide sequence, derived from each band, was identical and corresponded to a recently identified isoform of UDG1. This isoform (UDG1A) has a unique 44-amino acid N-terminal region and a C-terminal region that is identical to UDG1. To begin to study the signals required for nuclear targeting, the N-terminal regions of UDG1 and UDG1A were isolated and cloned into pEGFP-N2 to generate fusions with a redshifted variant of green fluorescent protein (GFP). When these constructs were transfected into NIH3T3 cells, UDG1/pEGFP was targeted to the mitochondria, and UDG1A/pEGFP was targeted to the nucleus. Further studies, using deletion mutants, demonstrate that the nuclear localization signal resides within the first 20 amino acids of UDG1A. To investigate the possibility that the heterogeneity observed on SDS-PAGE results from post-translational modification(s), the UDG/ pEGFP fusion constructs were transfected into NIH3T3 cells, and the cells were metabolically labeled with [ 32 P]orthophosphate. Results from these experiments show that UDG1A is a phosphoprotein. Subsequent phosphoamino acid analysis revealed that UDG1A is phosphorylated on both serine and threonine residues. As a final characterization, RNase protection assays were performed to examine expression of each of these isoforms. These studies demonstrate that UDG1A is expressed in a wide variety of cell types and that message levels are elevated in transformed cells.