KillerFLIP: A Novel Lytic Peptide Specifically Inducing Cancer Cell Death

One of the objectives in the development of effective cancer therapy is induction of tumor-selective cell death. Toward this end, we have identified a small peptide that, when introduced into cells via a TAT cell-delivery system, shows a remarkably potent cytoxicity in a variety of cancer cell lines and inhibits tumor growth in vivo, whereas sparing normal cells and tissues. This fusion peptide was named killer FLIP as its sequence was derived from the C-terminal domain of c-FLIP, an anti-apoptotic protein. Using structure activity analysis, we determined the minimal bioactive core of killerFLIP, namely killerFLIP-E. Structural analysis of cells using electron microscopy demonstrated that killerFLIP-E triggers cell death accompanied by rapid (within minutes) plasma membrane permeabilization. Studies of the structure of the active core of killer FLIP (-E) indicated that it possesses amphiphilic properties and self-assembles into micellar structures in aqueous solution. The biochemical properties of killerFLIP are comparable to those of cationic lytic peptides, which participate in defense against pathogens and have also demonstrated anticancer properties. We show that the pro-cell death effects of killer FLIP are independent of its sequence similarity with c-FLIP L as killer FLIP-induced cell death was largely apoptosis and necroptosis independent. A killer FLIP-E variant containing a scrambled c-FLIP L motif indeed induced similar cell death, suggesting the importance of the c-FLIP L residues but not of their sequence. Thus, we report the discovery of a promising synthetic peptide with novel anticancer activity in vitro and in vivo.

killerFLIP: a novel lytic peptide specifically inducing cancer cell death

One of the objectives in the development of effective cancer therapy is induction of tumor-selective cell death. Toward this end, we have identified a small peptide that, when introduced into cells via a TAT cell-delivery system, shows a remarkably potent cytoxicity in a variety of cancer cell lines and inhibits tumor growth in vivo, whereas sparing normal cells and tissues. This fusion peptide was named killerFLIP as its sequence was derived from the C-terminal domain of c-FLIP, an anti-apoptotic protein. Using structure activity analysis, we determined the minimal bioactive core of killerFLIP, namely killerFLIP-E. Structural analysis of cells using electron microscopy demonstrated that killerFLIP-E triggers cell death accompanied by rapid (within minutes) plasma membrane permeabilization. Studies of the structure of the active core of killerFLIP (-E) indicated that it possesses amphiphilic properties and self-assembles into micellar structures in aqueous solution. The biochemical properties of killerFLIP are comparable to those of cationic lytic peptides, which participate in defense against pathogens and have also demonstrated anticancer properties. We show that the pro-cell death effects of killerFLIP are independent of its sequence similarity with c-FLIPL as killerFLIP-induced cell death was largely apoptosis and necroptosis independent. A killerFLIP-E variant containing a scrambled c-FLIPL motif indeed induced similar cell death, suggesting the importance of the c-FLIPL residues but not of their sequence. Thus, we report the discovery of a promising synthetic peptide with novel anticancer activity in vitro and in vivo

Modulation of TRAIL resistance in colon carcinoma cells: Different contributions of DR4 and DR5

<p>Abstract</p> <p>Background</p> <p>rhTRAIL is a therapeutic agent, derived from the TRAIL cytokine, which induces apoptosis in cancer cells by activating the membrane death receptors 4 and 5 (DR4 and DR5). Here, we investigated each receptor's contribution to rhTRAIL sensitivity and rhTRAIL resistance. We assessed whether agonistic DR4 or DR5 antibodies could be used to circumvent rhTRAIL resistance, alone or in combination with various chemotherapies.</p> <p>Methods</p> <p>Our study was performed in an isogenic model comprised of the SW948 human colon carcinoma cell line and its rhTRAIL resistant sub-line SW948-TR. Effects of rhTRAIL and agonistic DR4/DR5 antibodies on cell viability were measured using MTT assays and identification of morphological changes characteristic of apoptosis, after acridine orange staining. Sensitivity to the different death receptor ligands was stimulated using pretreatment with the cytokine IFN-gamma and the proteasome inhibitor MG-132. To investigate the mechanisms underlying the changes in rhTRAIL sensitivity, alterations in expression levels of targets of interest were measured by Western blot analysis. Co-immunoprecipitation was used to determine the composition of the death-inducing signalling complex at the cell membrane.</p> <p>Results</p> <p>SW948 cells were sensitive to all three of the DR-targeting agents tested, although the agonistic DR5 antibody induced only weak caspase 8 cleavage and limited apoptosis. Surprisingly, agonistic DR4 and DR5 antibodies induced equivalent DISC formation and caspase 8 cleavage at the level of their individual receptors, suggesting impairment of further caspase 8 processing upon DR5 stimulation. SW948-TR cells were cross-resistant to all DR-targeting agents as a result of decreased caspase 8 expression levels. Caspase 8 protein expression was restored by MG-132 and IFN-gamma pretreatment, which also re-established sensitivity to rhTRAIL and agonistic DR4 antibody in SW948-TR. Surprisingly, MG-132 but not IFN-gamma could also increase DR5-mediated apoptosis in SW948-TR.</p> <p>Conclusions</p> <p>These results highlight a critical difference between DR4- and DR5-mediated apoptotic signaling modulation, with possible implications for future combinatorial regimens.</p

Rad51 and DNA-PKcs are involved in the generation of specific telomere aberrations induced by the quadruplex ligand 360A that impair mitotic cell progression and lead to cell death

Functional telomeres are protected from non-homologous end-joining (NHEJ) and homologous recombination (HR) DNA repair pathways. Replication is a critical period for telomeres because of the requirement for reconstitution of functional protected telomere conformations, a process that involves DNA repair proteins. Using knockdown of DNA-PKcs and Rad51 expression in three different cell lines, we demonstrate the respective involvement of NHEJ and HR in the formation of telomere aberrations induced by the G-quadruplex ligand 360A during or after replication. HR contributed to specific chromatid-type aberrations (telomere losses and doublets) affecting the lagging strand telomeres, whereas DNA-PKcs-dependent NHEJ was responsible for sister telomere fusions as a direct consequence of G-quadruplex formation and/or stabilization induced by 360A on parental telomere G strands. NHEJ and HR activation at telomeres altered mitotic progression in treated cells. In particular, NHEJ-mediated sister telomere fusions were associated with altered metaphase-anaphase transition and anaphase bridges and resulted in cell death during mitosis or early G1. Collectively, these data elucidate specific molecular and cellular mechanisms triggered by telomere targeting by the G-quadruplex ligand 360A, leading to cancer cell death

Ciliary Beating Recovery in Deficient Human Airway Epithelial Cells after Lentivirus Ex Vivo Gene Therapy

Primary Ciliary Dyskinesia is a heterogeneous genetic disease that is characterized by cilia dysfunction of the epithelial cells lining the respiratory tracts, resulting in recurrent respiratory tract infections. Despite lifelong physiological therapy and antibiotics, the lungs of affected patients are progressively destroyed, leading to respiratory insufficiency. Recessive mutations in Dynein Axonemal Intermediate chain type 1 (DNAI1) gene have been described in 10% of cases of Primary Ciliary Dyskinesia. Our goal was to restore normal ciliary beating in DNAI1–deficient human airway epithelial cells. A lentiviral vector based on Simian Immunodeficiency Virus pseudotyped with Vesicular Stomatitis Virus Glycoprotein was used to transduce cultured human airway epithelial cells with a cDNA of DNAI1 driven by the Elongation Factor 1 promoter. Transcription and translation of the transduced gene were tested by RT–PCR and western blot, respectively. Human airway epithelial cells that were DNAI1–deficient due to compound heterozygous mutations, and consequently had immotile cilia and no outer dynein arm, were transduced by the lentivirus. Cilia beating was recorded and electron microscopy of the cilia was performed. Transcription and translation of the transduced DNAI1 gene were detected in human cells treated with the lentivirus. In addition, immotile cilia recovered a normal beat and outer dynein arms reappeared. We demonstrated that it is possible to obtain a normalization of ciliary beat frequency of deficient human airway epithelial cells by using a lentivirus to transduce cells with the therapeutic gene. This preliminary step constitutes a conceptual proof that is indispensable in the perspective of Primary Ciliary Dyskinesia's in vivo gene therapy. This is the first time that recovery of cilia beating is demonstrated in this disease

Population specificity of the DNAI1 gene mutation spectrum in primary ciliary dyskinesia (PCD)

<p>Abstract</p> <p>Background</p> <p>Mutations in the <it>DNAI1 </it>gene, encoding a component of outer dynein arms of the ciliary apparatus, are the second most important genetic cause of primary ciliary dyskinesia (PCD), the genetically heterogeneous recessive disorder with the prevalence of ~1/20,000. The estimates of the <it>DNAI1 </it>involvement in PCD pathogenesis differ among the reported studies, ranging from 4% to 10%.</p> <p>Methods</p> <p>The coding sequence of <it>DNAI1 </it>was screened (SSCP analysis and direct sequencing) in a group of PCD patients (157 families, 185 affected individuals), the first ever studied large cohort of PCD patients of Slavic origin (mostly Polish); multiplex ligation-dependent probe amplification (MLPA) analysis was performed in a subset of ~80 families.</p> <p>Results</p> <p>Three previously reported mutations (IVS1+2-3insT, L513P and A538T) and two novel missense substitutions (C388Y and G515S) were identified in 12 families (i.e. ~8% of non-related Polish PCD patients). The structure of background SNP haplotypes indicated common origin of each of the two most frequent mutations, IVS1+2-3insT and A538T. MLPA analysis did not reveal any significant differences between patients and control samples. The Polish cohort was compared with all the previously studied PCD groups (a total of 487 families): IVS1+2-3insT remained the most prevalent pathogenetic change in <it>DNAI1 </it>(54% of the mutations identified worldwide), and the increased global prevalence of A538T (14%) was due to the contribution of the Polish cohort.</p> <p>Conclusions</p> <p>The worldwide involvement of <it>DNAI1 </it>mutations in PCD pathogenesis in families not preselected for ODA defects ranges from 7 to 10%; this global estimate as well as the mutation profile differs in specific populations. Analysis of the background SNP haplotypes suggests that the increased frequency of chromosomes carrying A538T mutations in Polish patients may reflects local (Polish or Slavic) founder effect. Results of the MLPA analysis indicate that no large exonic deletions are involved in PCD pathogenesis.</p

Mutations in Radial Spoke Head Genes and Ultrastructural Cilia Defects in East-European Cohort of Primary Ciliary Dyskinesia Patients

Primary ciliary dyskinesia (PCD) is a rare (1/20,000), multisystem disease with a complex phenotype caused by the impaired motility of cilia/flagella, usually related to ultrastructural defects of these organelles. Mutations in genes encoding radial spoke head (RSPH) proteins, elements of the ciliary ultrastructure, have been recently described. However, the relative involvement of RSPH genes in PCD pathogenesis remained unknown, due to a small number of PCD families examined for mutations in these genes. The purpose of this study was to estimate the involvement of RSPH4A and RSPH9 in PCD pathogenesis among East Europeans (West Slavs), and to shed more light on ultrastructural ciliary defects caused by mutations in these genes. The coding sequences of RSPH4A and RSPH9 were screened in PCD patients from 184 families, using single strand conformational polymorphism analysis and sequencing. Two previously described (Q109X; R490X) and two new RSPH4A mutations (W356X; IVS3_2–5del), in/around exons 1 and 3, were identified; no mutations were found in RSPH9. We estimate that mutations in RSPH4A, but not in RSPH9, are responsible for 2–3% of cases in the East European PCD population (4% in PCD families without situs inversus; 11% in families preselected for microtubular defects). Analysis of the SNP-haplotype background provided insight into the ancestry of repetitively found mutations (Q109X; R490X; IVS3_2–5del), but further studies involving other PCD cohorts are required to elucidate whether these mutations are specific for Slavic people or spread among other European populations. Ultrastructural defects associated with the mutations were analyzed in the transmission electron microscope images; almost half of the ciliary cross-sections examined in patients with RSPH4A mutations had the microtubule transposition phenotype (9+0 and 8+1 pattern). While microtubule transposition was a prevalent ultrastructural defect in cilia from patients with RSPH4A mutations, similar defects were also observed in PCD patients with mutations in other genes

MNS1 Is Essential for Spermiogenesis and Motile Ciliary Functions in Mice

During spermiogenesis, haploid round spermatids undergo dramatic cell differentiation and morphogenesis to give rise to mature spermatozoa for fertilization, including nuclear elongation, chromatin remodeling, acrosome formation, and development of flagella. The molecular mechanisms underlining these fundamental processes remain poorly understood. Here, we report that MNS1, a coiled-coil protein of unknown function, is essential for spermiogenesis. We find that MNS1 is expressed in the germ cells in the testes and localizes to sperm flagella in a detergent-resistant manner, indicating that it is an integral component of flagella. MNS1–deficient males are sterile, as they exhibit a sharp reduction in sperm production and the remnant sperm are immotile with abnormal short tails. In MNS1–deficient sperm flagella, the characteristic arrangement of “9+2” microtubules and outer dense fibers are completely disrupted. In addition, MNS1–deficient mice display situs inversus and hydrocephalus. MNS1–deficient tracheal motile cilia lack some outer dynein arms in the axoneme. Moreover, MNS1 monomers interact with each other and are able to form polymers in cultured somatic cells. These results demonstrate that MNS1 is essential for spermiogenesis, the assembly of sperm flagella, and motile ciliary functions

Inhibition of IGF-1R-dependent PI3K activation sensitizes colon cancer cells specifically to DR5-mediated apoptosis but not to rhTRAIL

Background: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) initiates apoptosis in tumor cells upon binding to its cognate agonistic receptors, death receptors 4 and 5 (DR4 and DR5). The activity of the insulin-like growth factor 1 (IGF-1) survival pathway is often increased in cancer, influencing both cell proliferation and apoptosis. We hypothesized that inhibiting the IGF-1 receptor (IGF-1R) using NVP-AEW541, a small molecular weight tyrosine kinase inhibitor of the IGF-1R, could increase death receptor (DR)-mediated apoptosis in colon cancer cells

Drosophila Dynein Intermediate Chain Gene, Dic61B, Is Required for Spermatogenesis

This study reports the identification and characterization of a novel gene, Dic61B, required for male fertility in Drosophila. Complementation mapping of a novel male sterile mutation, ms21, isolated in our lab revealed it to be allelic to CG7051 at 61B1 cytogenetic region, since two piggyBac insertion alleles, CG7051c05439 and CG7051f07138 failed to complement. CG7051 putatively encodes a Dynein intermediate chain. All three mutants, ms21, CG7051c05439 and CG7051f07138, exhibited absolute recessive male sterility with abnormally coiled sperm axonemes causing faulty sperm individualization as revealed by Phalloidin staining in Don Juan-GFP background. Sequencing of PCR amplicons uncovered two point mutations in ms21 allele and confirmed the piggyBac insertions in CG7051c05439 and CG7051f07138 alleles to be in 5′UTR and 4th exon of CG7051 respectively, excision of which reverted the male sterility. In situ hybridization to polytene chromosomes demonstrated CG7051 to be a single copy gene. RT-PCR of testis RNA revealed defective splicing of the CG7051 transcripts in mutants. Interestingly, expression of cytoplasmic dynein intermediate chain, α, β, γ tubulins and α-spectrin was normal in mutants while ultra structural studies revealed defects in the assembly of sperm axonemes. Bioinformatics further highlighted the homology of CG7051 to axonemal dynein intermediate chain of various organisms, including DNAI1 of humans, mutations in which lead to male sterility due to immotile sperms. Based on these observations we conclude that CG7051 encodes a novel axonemal dynein intermediate chain essential for male fertility in Drosophila and rename it as Dic61B. This is the first axonemal Dic gene of Drosophila to be characterized at molecular level and shown to be required for spermatogenesis