ADP-ribosylation of cyclophilin A by Pseudomonas aeruginosa exoenzyme S.

The virulence of the opportunistic pathogen Pseudomonas aeruginosa (Pa) is in part mediated by the type III secretion (TTS) of bacterial proteins into eukaryotic hosts. Exoenzyme S (ExoS) is a bifunctional Pa TTS effector protein, with GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) activities. Known cellular substrates of TTS-translocated ExoS (TTS-ExoS) ADPRT activity include proteins in the Ras superfamily and ERM family proteins.

DNA binding and antigene activity of a daunomycin-conjugated triplex-forming oligonucleotide targeting the P2 promoter of the human c-myc gene.

Triplex-forming oligonucleotides (TFO) that bind DNA in a sequence-specific manner might be used as selective repressors of gene expression and gene-targeted therapeutics. However, many factors, including instability of triple helical complexes in cells, limit the efficacy of this approach. In the present study, we tested whether covalent linkage of a TFO to daunomycin, which is a potent DNA-intercalating agent and anticancer drug, could increase stability of the triple helix and activity of the oligonucleotide in cells.

Inhibition of c-src transcription by mithramycin: structure-activity relationships of biosynthetically produced mithramycin analogues using the c-src promoter as target.

The aureolic acid antitumor antibiotic mithramycin (MTM) inhibits both cancer growth and bone resorption by cross-linking GC-rich DNA, thus blocking binding of Sp-family transcription factors to gene regulatory elements. Transcription of c-src, a gene implicated in many human cancers and required for osteoclast-dependent bone resorption, is regulated by the binding of Sp factors to specific elements in its promoter. Therefore, this gene represents an important anticancer target and a potential lead target through which MTM displays its so far uncharacterized action against osteoclastic bone resorption.

Selective inhibition of transcription of the Ets2 gene in prostate cancer cells by a triplex-forming oligonucleotide.

The transcription factor Ets2 has a role in cancer development and represents an attractive therapeutic target. In this study, we designed a triplex-forming oligonucleotide (TFO) directed to a homopurine:homopyrimidine sequence in the Ets2 promoter. Transcription factors of the Sp family bound to this sequence and mutation of the Sp1 site reduced Ets2 promoter activity.

Design of a novel triple helix-forming oligodeoxyribonucleotide directed to the major promoter of the c-myc gene.

Altered expression of c-myc is implicated in pathogenesis and progression of many human cancers. Triple helix-forming oligonucleotides (TFOs) directed to a polypurine/polypyrimidine sequence in a critical regulatory region near the c-myc P2 promoter have been shown to inhibit c-myc transcription in vitro and in cells. However, these guanine-rich TFOs had moderate binding affinity and required high concentrations for activity.

Antigene and antiproliferative effects of a c-myc-targeting phosphorothioate triple helix-forming oligonucleotide in human leukemia cells.

The c-myc gene is frequently deregulated and overexpressed in human cancers, and strategies designed to inhibit c-myc expression in cancer cells may have considerable therapeutic value. The purpose of the present work was to characterize the antigene and antiproliferative activity of a triple helix-forming oligonucleotide (TFO) targeted to a homopurine-homopyrimidine sequence in the P2 promoter of the c-myc gene. The TFO was synthesized with phosphorothioate (PS) internucleotide linkages to confer resistance to intra- and extracellular nucleases.

Inhibition of gene expression and cell proliferation by triple helix-forming oligonucleotides directed to the c-myc gene.

Triple helix-forming oligonucleotides (TFOs) bind with high affinity and specificity to homopurine-homopyrimidine sequences in DNA and have been shown to inhibit transcription of target genes in various experimental systems. In the present study, we evaluated the ability of 3'-amino-modified phosphodiester TFOs directed to four sites in the c-myc gene to inhibit gene expression and proliferation of human leukemia (CEM, KG-1, and HL-60) and lymphoma (Raji and ST486) cells. GT-rich TFOs were designed to target sequences located either upstream (myc1 and -2) or downstream (myc3 and -4) of the P2 promoter, which is the major c-myc promoter.

Differential sensitivity of human epithelial cells to Pseudomonas aeruginosa exoenzyme S.

Exoenzyme S (ExoS) is an ADP-ribosyltransferase produced and directly translocated into eukaryotic cells by the opportunistic pathogen Pseudomonas aeruginosa. Model systems that allow bacterial translocation of ExoS have found ExoS to have multiple effects on eukaryotic cell function, affecting DNA synthesis, actin cytoskeletal structure, and cell matrix adherence. To understand mechanisms underlying differences observed in cell sensitivities to ExoS, we examined the effects of bacterially translocated ExoS on multiple human epithelial cell lines.

ADP-ribosylation of oncogenic Ras proteins by pseudomonas aeruginosa exoenzyme S in vivo.

The exoenzyme S (ExoS)-producing Pseudomonas aeruginosa strain, 388, and corresponding ExoS knock-out strain, 388deltaexoS, were used in a bacterial and mammalian co-culture system as a model for the contact-dependent delivery of ExoS into host cells. Examination of DNA synthesis and Ras ADP-ribosylation in tumour cell lines expressing normal and mutant Ras revealed a decrease in DNA synthesis concomitant with ADP-ribosylation of Ras proteins after exposure to ExoS-producing bacteria, but not after exposure to non-ExoS-producing bacteria. Examination of normal H-Ras, K-Ras and N-Ras by two-dimensional electrophoresis after exposure to bacteria revealed differences in the degree of ADP-ribosylation by ExoS, with H-Ras being modified most extensively.

Interruption of multiple cellular processes in HT-29 epithelial cells by Pseudomonas aeruginosa exoenzyme S.

Exoenzyme S (ExoS), an ADP-ribosylating enzyme produced by the opportunistic pathogen Pseudomonas aeruginosa, is directly translocated into eukaryotic cells by bacterial contact. Within the cell, ExoS ADP-ribosylates the cell signaling protein Ras and causes inhibition of DNA synthesis and alterations in cytoskeletal structure. To further understand the interrelationship of the different cellular effects of ExoS, functional analyses were performed on HT-29 epithelial cells after exposure to ExoS-producing P.

Modification of Ras in eukaryotic cells by Pseudomonas aeruginosa exoenzyme S.

Genetic and functional data suggest that Pseudomonas aeruginosa exoenzyme S (ExoS), an ADP-ribosyltransferase, is translocated into eukaryotic cells by a bacterial type III secretory mechanism activated by contact between bacteria and host cells. Although purified ExoS is not toxic to eukaryotic cells, ExoS-producing bacteria cause reduced proliferation and viability, possibly mediated by bacterially translocated ExoS. To investigate the activity of translocated ExoS, we examined in vivo modification of Ras, a preferred in vitro substrate.

Effects of differential expression of the 49-kilodalton exoenzyme S by Pseudomonas aeruginosa on cultured eukaryotic cells.

Production of the ADP-ribosylating enzyme exoenzyme S (ExoS) by Pseudomonas aeruginosa has been associated with increased virulence. Previous studies, however, have been unable to confirm an effect of soluble ExoS in cell culture or animal model systems. To determine if bacteria must come in contact with target cells in order for an effect of ExoS to be observed, coculture systems were developed to compare the effects of ExoS- and non-ExoS-producing bacteria on eukaryotic cell function.

Enhancement of radiation dose to the nucleus by vesicular internalization of iodine-125-labeled A33 monoclonal antibody.

Unlabelled: In radioimmunotherapy, the emission characteristics of the radioisotope is critical in determining the radiation dose to the tumor compared to normal organs. If antibodies internalize and transport low-energy electron emitting isotopes close to the tumor cell nucleus, an improved therapeutic advantage is achieved.

Methods: Using fluorescent microscopy, we studied the subcellular distribution of an internalizing antibody, A33, which detects a restricted determinant on colon cancer cells.