Cellular multi-drug resistance (MDR), which often develops in cancer cells of patients subjected to anti-cancer treatment, remains a significant barrier to successful cancer therapy. One of the principal causes of cellular MDR development is an increased expression of ABC-transporter genes such as Abcb1 (mdr1) and Abcg2. Despite many years of intensive research, the natural biological role of mdr1 in the context of cancer has remained elusive.
While studying mechanisms of muscle regeneration we have identified the expression of mdr1 in muscle precursor cells (MPC) and its up-regulation by muscle growth factors. Further, we have shown that MPCs that express these transporters, namely, the muscle SP cells, have some characteristic in common with stem cells (Israeli et al, J. Cell Physiol 2004, 201:409-19; Benchaouir et al Exp Cell Res 2004, 294:254-68). Indeed expression of these transporters in stem cells has been described, initially in the hematopoietic system, and latter in many other tissues. Expression of drug transporters in stem cells could protect these precious cells against exhaustion. However, the mdr1ab(-/-) x Abcg2(-/-) triple-KO mouse has normal tissue viability. We therefore hypothesized that the expression of these transporters in stem cells is dispensable for normal tissue maintenance (homeostatic maintenance) but will be needed during the activation of a regeneration “genetic program” after extensive tissue damage.
To test this hypothesis in the context of muscle regeneration we created the mdr1ab(-/-) x dystrophin(-/-) triple KO mouse. This mouse that undergoes chronic muscle regeneration, has reduced muscle regeneration capacity compared to control dystrophin(-/-) mouse, and therefore in support of our hypothesis (Israeli et al, Exp Cell Res 2007, 313:2438-50).
Based on this data we proposed that the activation of mdr1 and Abcg2 expression in cells in tumor tissues that were subjected to anti-cancer treatment is part of the activation, in transformed just as in a normal tissue, of a regeneration genetic program (Israeli et al, 2005, J Theor Biol. 7;232, 41-5).
Expression in cancer stem cells of mdr1, Abcg2 and of the SP phenotype is in agreement with this hypothesis. Better understanding the role of these transporters following cancer therapy may help designing new strategies to overcome MDR in cancer.
A Series Of Antibacterial Proteins Made From Inactive Cyt-Like ORF Of Bacillus Thuringiensis Subsp. Israelensis Using The Microgene Polymerization Reaction ITSKO M1,2, ZARITSKY A1 1 Department of Life Sciences, Ben-Gurion University of the Negev, POB 653, Be'er-Sheva 84105, Israel, 2 Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
Background: Insecticidal crystal proteins of Bacillus thuringiensis belong to two unrelated toxin families: receptors-specific Cry against insects and Cyt that lyse a broad range of cells, bacteria included, via direct binding to phospholipids. A new cyt-like gene (cyt1Ca) encoding a 60 kDa Open Reading Frame, has recently been discovered in B. thuringiensis subsp. israelensis. Neither bactericidal/larvicidal activity of cyt1Ca expressed in Escherichia coli nor hemolytic effect of His-tagged purified Cyt1Ca has been observed.
Results: In an attempt to endow inactive Cyt1Ca with Cyt1Aa-like antibacterial activity, two amino acids were replaced by QuickChange mutagenesis, E117V and N125A, so as to raise the hydrophobicity of the corresponding region, considered being the membrane-active motif. Serendipitously, the primers used for QuickChange mutagenesis displayed the intrinsic ability to expand into multiple head-to-tail tandem-repeats in the so-called Microgene Polymerization Reaction (MPR). The clones thus obtained include varying lengths of multiple repeats of the amino acid sequence VIEVLKSLLGIALA, corresponding to head-to-tail polymerization of the primer, translated in frame with Cyt1Ca. These versions of Cyt1Ca caused instant arrest in biomass growth and decreased viability upon expression in E. coli. Multiple insertions into the polypeptide of the non-mutated motif VIEELKSLLGINLA were also lethal. To expose toxicity of the latter motif in the original Cyt1Ca, cyt1Ca was appropriately truncated.
Conclusions: 1) The toxicity of the above motif is ascribed to its amphiphilic nature; toxicity was not displayed in the original Cyt1Ca because of possible motif sequestration by other parts of the protein due to its inherent folding, which may provide a safety mechanism to protect the host bacterium. 2) Combination of MPR with QuickChange can thus be exploited to design and synthesize polypeptides with antibacterial motifs, multiplied within the frame of a given protein.
Supported by an Eshkol Scholarship (to MI) and BSF Grant (to AZ).
The Role Of Lipid Rafts In Host-Pathogen Interactions:
Involvement Of Lactosylceramide-Enriched Lipid Rafts In Innate Immunity And Ycobacterial Infection- IWABUCHI K1,2, NAKAYAMA H1, IWAHARA C1, TAKAMORI K1, OGAWA H1 1 Inst. Env. Gender Specific Med. Juntendo Univ. Grad. Sch. Med., 2 Infect. control Nurs., Juntendo Univ. Grad. Sch. of Health Care and Nurs.
The innate immune system is the first line of defense against invading microorganisms, including bacteria, fungi, and viruses. Phagocytes, such as neutrophils and macrophages, play important roles in the innate immune system by recognizing pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) expressed on the cell surface, and then engulfing and eliminating pathogens. It has been suggested that membrane microdomains/lipid rafts of phagocytes are involved in these innate immune responses. Proteomic analyses of lipid rafts from plasma membranes of phagocytes have provided new insight into lipid raft-mediated processes occurring during phagocytosis. Several types of pathogens, however, have developed mechanisms to attach to and enter host cells using lipid rafts. Even after being engulfed by phagocytes, a particular group of pathogens, including Mycobacteria, Afipia felis, and Shigella, can avoid degradation by escaping from the vacuolar compartment or preventing phagosome maturation, utilizing the lipid rafts of host cells. Although the molecular mechanisms underlying these phenomena remain largely unknown, detailed understanding of these lipid raft-associated host-pathogen interactions will open new avenues for the design of effective therapeutic agents for infectious diseases.
Lactosylceramide (LacCer, CDw17), a neutral glycosphingolipid, forms glycosphingolipid-enriched microdomains, which are coupled with the Lyn protein, a member of the Src family of kinases on the plasma membrane of human neutrophils. LacCer-enriched lipid rafts act as PRRs and mediate superoxide generation, migration, and phagocytosis through the binding of ligand to LacCer. In this talk, we will discuss the membrane lipid raft-associated immune functions of phagocytes, focusing on the molecular mechanisms of LacCer-enriched lipid raft-mediated phagocytosis. We also introduce evidence indicating that LacCer-enriched lipid rafts may be involved in escape mechanisms by which Mycobacteria escape from killing by human neutrophils.
