ABSTRACT
Controlled H-aggregation of single Pc-labeled oligonucleotides is utilized as a fluorescence quenching system to discern changes in enzyme activity for the discovery of inhibitors for Long Interspersed Element 1 endonuclease (L1-EN), which is involved in genome instability and implicated in many different diseases.
Subject(s)
Endodeoxyribonucleases/antagonists & inhibitors , Indoles/chemistry , Long Interspersed Nucleotide Elements , Spectroscopy, Near-Infrared/methods , Disease/genetics , Enzyme Activators/metabolism , Fluorescent Dyes/chemistry , Genomic Instability , Isoindoles , Oligonucleotides/chemistry , Oligonucleotides/geneticsABSTRACT
MLL leukemias are characterized cytogenetically by reciprocal translocations of the MLL gene at 11q23 and clinically by unfavorable outcomes. Evidence indicating that MLL leukemias are resistant to apoptosis encourages the identification of agents that induce cell death by other mechanisms. The AF4-mimetic peptide PFWT induces necrosis in the t(4;11) leukemia cell line, MV4-11. Treatment of MV4-11 cells with PFWT in combination with four chemotherapeutic compounds results in sequence-dependent synergy, induction of both apoptotic and necrotic cell death, and inhibition of MV4-11 clonogenicity. Therefore, PFWT holds promise as a therapy for MLL leukemias that augments the effects of several clinically available chemotherapeutic agents.
Subject(s)
Apoptosis/drug effects , DNA-Binding Proteins/chemistry , Gene Rearrangement , Leukemia/genetics , Leukemia/pathology , Myeloid-Lymphoid Leukemia Protein/genetics , Nuclear Proteins/chemistry , Peptide Fragments/pharmacology , Antimetabolites, Antineoplastic/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Blotting, Western , Cell Cycle/drug effects , Cell Proliferation/drug effects , Chromosomes, Human, Pair 11/genetics , Chromosomes, Human, Pair 4/genetics , Chromosomes, Human, Pair 9/genetics , Colony-Forming Units Assay , Cytarabine/pharmacology , Drug Synergism , Etoposide/pharmacology , Histone-Lysine N-Methyltransferase , Humans , Necrosis , Transcriptional Elongation Factors , Translocation, Genetic , Tumor Cells, CulturedABSTRACT
Despite ongoing success in the treatment of childhood acute lymphoblastic leukemia, patients harboring translocations involving the MLL gene at chromosome 11q23 remain resistant to treatment. To improve outcomes, novel therapeutics designed to target the unusual biology of these leukemias need to be developed. Previously, we identified an interaction between the two most common MLL fusion proteins, AF4 and AF9, and designed a synthetic peptide (PFWT) capable of disrupting this interaction. PFWT induced cell death in leukemia cells expressing MLL-AF4 with little effect on the colony forming potential of hematopoietic progenitor cells, suggesting the AF4-AF9 complex is an important pharmacological target for leukemia therapy and PFWT is a promising chemotherapeutic prototype. In these studies, we demonstrate that PFWT induces death by necrosis in MV4-11 cells. Cell death is characterized by rapid loss of plasma membrane integrity with maintenance of nuclear membrane integrity, and is independent of caspase activation, DNA fragmentation, and mitochondrial membrane depolarization. PFWT-mediated necrosis is inhibited by the serine protease inhibitor TLCK, suggesting this death pathway is regulated. Given the resistance of t(4;11) leukemias to conventional chemotherapeutic agents that induce apoptosis, further identification of the molecular events mediating this death process should uncover new avenues for therapeutic intervention.
Subject(s)
Apoptosis/drug effects , Leukemia/pathology , Myeloid-Lymphoid Leukemia Protein/metabolism , Nuclear Proteins/metabolism , Oncogene Proteins, Fusion/metabolism , Peptide Fragments/pharmacology , Blotting, Western , Cell Membrane/metabolism , Cell Nucleus/metabolism , Cells, Cultured , Colony-Forming Units Assay , Comet Assay , Flow Cytometry , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/ultrastructure , Humans , In Situ Nick-End Labeling , Leukemia/drug therapy , Leukemia/genetics , Membrane Potential, Mitochondrial , Necrosis , Nuclear Proteins/antagonists & inhibitors , Oncogene Proteins, Fusion/antagonists & inhibitors , Translocation, Genetic , Tumor Cells, CulturedABSTRACT
The non-receptor tyrosine kinase c-Src plays a central role in a variety of cell signaling pathways that regulate cell growth, differentiation, apoptosis, and other important cellular processes. An 85-amino acid N-terminal deletion construct of c-Src (DeltaN85 c-Src) has been structurally characterized and used extensively in biochemical and biophysical studies. In this report, we have established a relatively rapid, simplified purification of DeltaN85 c-Src from recombinant baculovirus-infected insect cells. Q-Sepharose anion-exchange and aminophenyl-ATP affinity chromatography were used to isolate 5mg of >98% pure DeltaN85 c-Src from 900 mg of total soluble protein. The specific activity of DeltaN85 c-Src (20 U mg(-1)) was found to be >or = 5-fold greater than previously reported values. A lag in the autophosphorylation kinetics of DeltaN85 c-Src was observed, and the reaction occurred with observed first-order rate constants k1=0.20+/-0.01 min(-1) and k2=0.38+/-0.01 min(-1) under the experimental conditions used. Steady-state kinetic analysis of peptide phosphorylation by DeltaN85 c-Src gave Km values of 99+/-23 microM and 190+/-30 microM for the peptide and ATP substrates, respectively, and a value of k(cat)=17+/-2s(-1). Overall, we present a dramatically improved purification strategy that represents a simplified, relatively rapid protocol for the isolation of milligram quantities of DeltaN85 c-Src required for rigorous structure-function and inhibition studies that rely on a pre-steady-state kinetic approach.
