ABSTRACT
The process of dendritic cell (DC) maturation, critical for effective DC-based immunotherapy, also alters the proteasome such that peptides presented in the context of HLA class I are generated not by the constitutive proteasome, but by the immunoproteasome. Cytotoxic T lymphocytes (CTLs) induced by such DCs might not optimally recognize tumor cells normally expressing the constitutive proteasome. Using small interfering RNA (siRNA) transfection of DCs to inhibit expression of the 3 inducible immunoproteasome subunits in mature DCs, we found that such DCs expressed increased intracellular levels of constitutive proteasomes and presented an altered repertoire of tumor-antigenic peptides. When DCs generated from the monocytes of 3 patients with melanoma were transfected with immunoproteasome siRNA, induced to mature, and then trans-fected with RNA encoding defined melanoma antigens, these DCs were superior inducers of antigen-specific CTLs against autologous melanoma cells. This alteration of DC proteasome composition, which enhances the ability of mature antigen-loaded DCs to stimulate anti-tumor immune responses, may lead to more effective DC-based tumor immunotherapy.
Subject(s)
Antigens, Neoplasm/therapeutic use , Dendritic Cells/immunology , Melanoma/immunology , Proteasome Endopeptidase Complex/genetics , T-Lymphocytes, Cytotoxic/immunology , Antigen Presentation , Antigens, Neoplasm/genetics , Cell Line, Tumor , Dendritic Cells/metabolism , Humans , Immunotherapy/methods , Melanoma/therapy , RNA, Small Interfering/genetics , TransfectionABSTRACT
The activity of DNA topoisomerase I (Top1), an enzyme that regulates DNA topology, is impacted by DNA structure alterations and by the anticancer alkaloid camptothecin (CPT). Here, we evaluated the effect of the acetaldehyde-derived DNA adduct, N2-ethyl-2'-deoxyguanosine (N2-ethyl-dG), on human Top1 nicking and closing activities. Using purified recombinant Top1, we show that Top1 nicking-closing activity remains unaffected in N2-ethyl-dG adducted oligonucleotides. However, the N2-ethyl-dG adduct enhanced CPT-induced Top1-DNA cleavage complexes depending on the relative position of the N2-ethyl-dG adduct with respect to the Top1 cleavage site. The Top1-mediated DNA religation (closing) was selectively inhibited when the N2-ethyl-dG adduct was present immediately 3' from the Top1 site (position +1). In addition, when the N2-ethyl-dG adduct was located at the -5 position, CPT enhanced cleavage at an alternate Top1 cleavage site immediately adjacent to the adduct, which was then at position +1 relative to this new alternate Top1 site. Modeling studies suggest that the ethyl group on the N2-ethyl-dG adduct located at the 5' end of a Top1 site (position +1) sterically blocks the dissociation of CPT from the Top1-DNA complex, thereby inhibiting further the religation (closing) reaction.
Subject(s)
Camptothecin/pharmacology , DNA Adducts/chemistry , DNA Topoisomerases, Type I/metabolism , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/chemistry , Enzyme Inhibitors/pharmacology , DNA/chemistry , DNA/metabolism , Macromolecular Substances , Models, MolecularABSTRACT
7,8-Dihydro-8-oxoguanine (8-oxoG) is the most common form of oxidative DNA damage in human cells. Biochemical studies have shown that 8-oxoG decreases the DNA cleavage activity of human topoisomerase I, an enzyme vital to DNA metabolism and stability. We present the 3.1-A crystal structure of human topoisomerase I in noncovalent complex with a DNA oligonucleotide containing 8-oxoG at the +1 position in the scissile strand. We find that 8-oxoG reorganizes the active site of human topoisomerase I into an inactive conformation relative to the structures of topoisomerase I-DNA complexes elucidated previously. The catalytic Tyr-723-Phe rotates away from the DNA cleavage site and packs into the body of the molecule. A second active-site residue, Arg-590, becomes disordered and is not observed in the structure. The docked, inactive conformation of Tyr-723-Phe is reminiscent of the related tyrosine recombinase family of integrases and recombinases, suggesting a common regulatory mechanism. We propose that human topoisomerase I binds to DNA first in an inactive conformation and then rearranges its active site for catalysis. 8-OxoG appears to impact topoisomerase I by stabilizing the inactive, DNA-bound state.