ABSTRACT
Topoisomerase 1 (TOP1) generates transient nicks in the DNA to relieve torsional stress encountered during the cellular processes of transcription, replication, and recombination. At the site of the nick there is a covalent linkage of TOP1 with DNA via a tyrosine residue. This reversible TOP1-cleavage complex intermediate can become trapped on DNA by TOP1 poisons such as camptothecin, or by collision with replication or transcription machinery, thereby causing protein-linked DNA single- or double-strand breaks and resulting in cell death. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a key enzyme involved in the repair of TOP1-associated DNA breaks via hydrolysis of 3'-phosphotyrosine bonds. Inhibition of TDP1 is therefore an attractive strategy for targeting cancer cells in conjunction with TOP1 poisons. Existing methods for monitoring the phosphodiesterase activity of TDP1 are generally gel based or of high cost. Here we report a novel, oligonucleotide-based fluorescence assay that is robust, sensitive, and suitable for high-throughput screening of both fragment and small compound libraries for the detection of TDP1 inhibitors. We further validated the assay using whole cell extracts, extending its potential application to determine of TDP1 activity in clinical samples from patients undergoing chemotherapy.
Subject(s)
Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/pharmacology , Oligonucleotides/metabolism , Phosphoric Diester Hydrolases/metabolism , Spectrometry, Fluorescence/methods , Base Sequence , Cost-Benefit Analysis , Drug Evaluation, Preclinical/economics , HEK293 Cells , High-Throughput Screening Assays , Humans , Kinetics , Mutation , Oligonucleotides/genetics , Phosphoric Diester Hydrolases/genetics , Spectrometry, Fluorescence/economicsABSTRACT
EMT-6 mammary carcinoma and B16 melanoma (B16M) cells are lethal and barely immunogenic in syngeneic BALB/c and C57BL/6 mice, respectively. We show that mice vaccinated with tumor cells pulsed with a MHC class I-restricted peptide develop a T cell response, not only to the peptide, but also to the unpulsed tumor. These mice display protective immunity against the unpulsed tumor, and their T cells adoptively transfer tumor-specific protection to immunodeficient SCID mice. Our data have implications for cancer vaccine strategies. Grafting a single well-defined foreign peptide on tumor cells might suffice to trigger anti-tumor immunity.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/immunology , Mammary Neoplasms, Animal/immunology , Melanoma, Experimental/immunology , Peptides/immunology , Adoptive Transfer , Animals , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Histocompatibility Antigens Class I/immunology , Mammary Neoplasms, Animal/therapy , Melanoma, Experimental/therapy , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, SCID , Peptides/metabolism , VaccinationABSTRACT
Aberrant regulation of phosphoinositide 3-kinase (PI3K) activity is implicated in various diseases such as cancer and diabetes. Thus, high-throughput screening (HTS) of small-molecule inhibitors for PI3 kinases is an appealing strategy for drug development. Despite the attractiveness of lipid kinases as drug targets, screening for inhibitors for PI3K activities has been hampered by limited assay formats adaptable for HTS. The authors describe a homogeneous, direct, and nonradioactive assay for highly sensitive detection of PI3Kalpha, beta, delta, and gamma activities, which is suitable for HTS. The assay is based on fluorescence superquenching of a conjugated polymer upon metal-ion-mediated association of phosphorylated and dye-labeled substrates. As a result of phosphorylation, quencher and polymer are brought into proximity, and fluorescent energy transfer occurs. This event can be monitored as either fluorescence quench of the polymer or as enhanced emission from the quencher. Ratiometric analysis of the wavelengths eliminates interferences from autofluorescing compounds, which are present in HTS libraries. The platform has been adapted for the 384-well microplate format and delivers Z factors of > 0.6 at substrate conversions as low as 7%. Using this assay platform, several unreported inhibitors and activators of PI3Ks were identified in an 84- compound screen.
Subject(s)
Drug Evaluation, Preclinical/methods , Phosphatidylinositol 3-Kinases/drug effects , Phosphoinositide-3 Kinase Inhibitors , Androstadienes/pharmacology , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Isoenzymes/antagonists & inhibitors , Isoenzymes/drug effects , Morpholines/pharmacology , Spectrometry, Fluorescence/methods , WortmanninABSTRACT
Herein we describe studies that indicate a cationic conjugated polyelectrolyte shows biocidal activity against gram-negative bacteria (Escherichia coli, E. coli, BL21, with plasmids for Azurin and ampicillin resistance) and gram-positive bacterial spores (Bacillus anthracis, Sterne, B. anthracis, Sterne). These studies were carried out with aqueous suspensions of the conjugated polyelectrolyte, with the polyelectrolyte in supported formats and with samples in which the conjugated polyelectrolyte was coated on the bacteria. The results are interesting in that the biocidal activity is light-induced and appears effective due to the ability of the conjugated polyelectrolyte to form a surface coating on both types of bacteria. The effects observed here should be general and suggest that a range of conjugated polyelectrolytes in different formulations may provide a useful new class of biocides for both dark and light-activated applications.
Subject(s)
Anti-Infective Agents/pharmacology , Electrolytes/chemistry , Spores, Bacterial/drug effects , Absorption , Bacillus anthracis/drug effects , Bacillus anthracis/metabolism , Cetylpyridinium/pharmacology , Escherichia coli/drug effects , Escherichia coli/metabolism , Fluorescent Dyes/pharmacology , Light , Methylene Blue/pharmacology , Microscopy, Fluorescence , Microscopy, Phase-Contrast , Models, Chemical , Quaternary Ammonium Compounds/pharmacology , Rose Bengal/pharmacologyABSTRACT
The rules that govern the engagement of antitumor immunity are not yet fully understood. Ags expressed by tumor cells are prone to induce T cell tolerance unless the innate immune system is activated. It is unclear to what extent tumors engage this second signal link by the innate immune system. Apoptotic and necrotic (tumor) cells are readily recognized and phagocytosed by the cells of the innate immune system. It is unknown how this affects the tumor's immunogenicity. Using a murine melanoma (B16m) and lymphoma (L5178Y-R) model, we studied the clonal sizes and cytokine signatures of the T cells induced by these tumors in syngeneic mice when injected as live, apoptotic, and necrotic cells. Both live tumors induced a type 2 CD4 cell response characterized by the prevalent production of IL-2, IL-4, and IL-5 over IFN-gamma. Live, apoptotic, and necrotic cells induced CD4 (but no CD8) T cells of comparable frequencies and cytokine profiles. Therefore, live tumors engaged the second signal link, and apoptotic or necrotic tumor cell death did not change the magnitude or quality of the antitumor response. A subclone of L5178Y-R, L5178Y-S cells, were found to induce a high-frequency type 1 response by CD4 and CD8 cells that conveyed immune protection. The data suggest that the immunogenicity of tumors, and their characteristics to induce type 1 or type 2, CD4 or CD8 cell immunity is not primarily governed by signals associated with apoptotic or necrotic cell death, but is an intrinsic feature of the tumor itself.
