Zvi Fishelson

Cancer resistance to complement-dependent cytotoxicity (CDC): Problem-oriented research and development

The number of anti-cancer antibodies in therapy and in clinical trials is increasing gradually while their curative efficacy remains rather limited due to the resistance of tumor cells to complement-dependent cytotoxicity (CDC). An updated review of the various defense mechanisms complement is confronting when tackling a tumor cell is presented. The mechanisms discussed are: membrane and secreted complement regulatory proteins, heat shock proteins, extracellular proteases and protein kinases, cell surface sialylation and intracellular survival anti-lytic signals. Projected treatment strategies are depicted for each of the complement resistance mechanisms. It is conceivable that the therapeutic capacity of anti-cancer antibodies will be amplified once combined with a reagent that sensitizes the cancer cells to CDC.

Mortalin inhibitors sensitize K562 leukemia cells to complement-dependent cytotoxicity

Mortalin, the mitochondrial hsp70, is a vital constitutively expressed heat shock protein. Its elevated expression has been correlated with malignant transformation and poor cancer prognosis. Cancer cells exhibit increased resistance to complement‐dependent cytotoxicity, partly due to their capacity to eliminate the complement membrane attack complex (MAC) from their cell surface. As we have previously reported, mortalin and the complement membrane attack complexes are released in membrane vesicles from complement attacked cells. As shown here, knock down of mortalin with specific siRNA reduces MAC elimination and enhances cell sensitivity to MAC‐induced cell death. Similar results were obtained with MKT‐077, a cationic rhodacyanine dye that inhibits mortalin. Treatment of human erythroleukemia K562 and colorectal carcinoma HCT116 cells with MKT‐077 sensitizes them to cell death mediated by MAC but not by streptolysin O. Pre‐treatment of cells with MKT‐077 also reduces the extent of MAC‐mortalin vesiculation following a sublytic complement attack. In the presence of MKT‐077, the direct binding of mortalin to complement C9, the major MAC component, is inhibited. The tumor suppressor protein p53 is a known mortalin client protein. The effect of MKT‐077 on complement‐mediated lysis of HCT116 p53+/+ and p53−/− cells was found to be independent on the presence of p53. Our results also demonstrate that recombinant human mortain inhibits complement‐mediated hemolysis of rabbit erythrocytes as well as zinc‐induced C9 polymerization. We conclude that mortalin supports cancer cell resistance to complement‐dependent cytotoxicity and propose consideration of mortalin as a novel target for cancer adjuvant immunotherapy.

Complement Nomenclature 2014

The first update since 1981 of the nomenclature used in the field of complement has been completed by the Complement Nomenclature Committee established under the auspices of the International Complement Society (ICS) and by the boards of the ICS and the European Complement Network (ECN). Recommended names of complement pathways, proteins, protein complexes, protein fragments and receptors are listed. Authors are urged to use these names in their published and presented works.

Programmed Necrotic Cell Death Induced by Complement Involves a Bid-Dependent Pathway

The membrane attack complex (MAC) of the complement system induces a necrotic-type cell death. Earlier findings suggested that Bcl-2 protects cells from MAC-induced necrosis. Here we examined the involvement of Bid, a proapoptotic protein, in MAC-induced cytotoxicity. Bid knockout (Bid−/−) mouse embryonic fibroblasts (MEF) and primary fibroblasts were damaged by complement but to a significantly lower extent than wild-type (WT) fibroblasts. Bid silencing with small interfering RNA duplexes led to elevated resistance of mouse fibroblasts, human K562, and Jurkat cells to lysis by complement. Bid−/− MEF were also resistant to toxic doses of streptolysin O, melittin, and A23187. Analysis of complement protein deposition on fibroblasts demonstrated that less complement C3 and C9 bound to Bid−/− than to WT cells, even though expression of the membrane complement inhibitors Crry and CD59 was relatively reduced on Bid−/− cells. Bid was rapidly cleaved in WT MEF subjected to lytic doses of MAC. Pretreatment of the cells with the pan-caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone reduced Bid cleavage and cell lysis. These results indicate that complement MAC activates two cell death pathways, one involving caspases and Bid and one that is Bid-independent.

Caveolin-1 and Dynamin-2 Are Essential for Removal of the Complement C5b-9 Complex via Endocytosis * □

Background: Cells resist complement-dependent cytotoxicity by elimination of the membranolytic C5b-9 complex from their surface. Results: Modulations of caveolin-1 and dynamin-2 expression and activity affect C5b-9 endocytosis and cell death. Conclusion: Elimintion of C5b-9 and complement resistance depend on caveolae formation, dynamin activity and lipid rafts. Significance: Targeting of key factors in the C5b-9 elimination pathway may enable us to regulate C5b-9 homeostasis and cell resistance to complement-dependent cytotoxicity. The complement system, an important element of both innate and adaptive immunity, is executing complement-dependent cytotoxicity (CDC) with its C5b-9 protein complex that is assembled on cell surfaces and transmits to the cell death signals. In turn, cells, and in particular cancer cells, protect themselves from CDC in various ways. Thus, cells actively remove the C5b-9 complexes from their plasma membrane by endocytosis. Inhibition of clathrin by transfection with shRNA or of EPS-15 with a dominant negative plasmid had no effect on C5b-9 endocytosis and on cell death. In contrast, inhibition of caveolin-1 (Cav-1) by transfection with an shRNA or a dominant negative plasmid sensitized cells to CDC and inhibited C5b-9 endocytosis. Similarly, both inhibition of dynamin-2 by transfection with a dominant negative plasmid or by treatment with Dynasore reduced C5b-9 endocytosis and enhanced CDC. C5b-9 endocytosis was also disrupted by pretreatment of the cells with methyl-β-cyclodextrin or Filipin III, hence implicating membrane cholesterol in the process. Analyses by confocal microscopy demonstrated co-localization of Cav-1-EGFP with C5b-9 at the plasma membrane, in early endosomes, at the endocytic recycling compartment and in secreted vesicles. Further investigation of the process of C5b-9 removal by exo-vesiculation demonstrated that inhibition of Cav-1 and cholesterol depletion abrogated C5b-9 exo-vesiculation, whereas, over-expression of Cav-1 increased C5b-9 exo-vesiculation. Our results show that Cav-1 and dynamin-2 (but not clathrin) support cell resistance to CDC, probably by facilitating purging of the C5b-9 complexes by endocytosis and exo-vesiculation.

Involvement of the c-jun N-terminal kinases JNK1 and JNK2 in complement-mediated cell death

Cell death and survival signals activated by the complement membrane attack complex C5b-9 play important roles in complement-associated diseases and in antibody-based cancer therapy. Here, we investigated the involvement of the JNK mitogen-activated protein kinase in C5b-9-induced cell lysis. Necrotic-type cell death regulation by JNK1 and JNK2 was selectively studied in mouse fibroblasts and human K562, HeLa and 293T cells. C5b-9 induced higher JNK activation than C5b-8. Pretreatment with a JNK inhibitor reduced cell sensitivity to complement-mediated lysis. KO cells deficient in either JNK1 or JNK2 were less sensitive to lysis than WT cells. This correlated with lower C3 and C5b-9 deposition on KO cells. Furthermore, silencing of JNK1 or JNK2 expression by RNA interference decreased cell lysis by complement. Reconstitution of JNK2 into JNK2-/- cells and over expression of JNK2 in WT cells increased C3 and C5b-9 deposition as well as cell sensitivity to complement-mediated lysis. Pretreatment of cells with the phosphotyrosine phosphatase inhibitor phenylarsine oxide increased JNK activation and JNK-dependent complement-mediated necrotic death of WT and JNK2-/- KO cells but not of JNK1-/- KO cells. The JNK inhibitor and PAO had no effect on complement-mediated lysis in cells lacking Bid, suggesting involvement of Bid in the JNK lytic pathway. Our results demonstrate that complement C5b-9 induce a JNK/Bid-dependent and JNK-independent necrotic cell death. Both JNK1 and JNK2 have cytotoxic potential, however JNK2 is the primary signal transducer.

Mortalin/Grp75 Binds to Complement C9 and Plays a Role in Resistance to Complement-Dependent Cytotoxicity

Background: Mortalin was shown to contribute to removal of the complement membranolytic C5b-9 complex from the target cell surface. Results: Modulations of mortalin expression and activity affect deposition of C5b-9 and cell death. Conclusion: Mortalin, through its ATPase domain, regulates the C5b-9 deposition and confers resistance to complement-dependent cytotoxicity. Significance: Mortalin is a potential therapeutic target in autoimmune diseases and in cancer immunotherapy. Mortalin/GRP75, the mitochondrial heat shock protein 70, plays a role in cell protection from complement-dependent cytotoxicity (CDC). As shown here, interference with mortalin synthesis enhances sensitivity of K562 erythroleukemia cells to CDC, whereas overexpression of mortalin leads to their resistance to CDC. Quantification of the binding of the C5b-9 membrane attack complex to cells during complement activation shows an inverse correlation between C5b-9 deposition and the level of mortalin in the cell. Following transfection, mortalin-enhanced GFP (EGFP) is located primarily in mitochondria, whereas mortalinΔ51-EGFP lacking the mitochondrial targeting sequence is distributed throughout the cytoplasm. Overexpressed cytosolic mortalinΔ51-EGFP has a reduced protective capacity against CDC relative to mitochondrial mortalin-EGFP. Mortalin was previously shown by us to bind to components of the C5b-9 complex. Two functional domains of mortalin, the N-terminal ATPase domain and the C-terminal substrate-binding domain, were purified after expression in bacteria. Similar to intact mortalin, the ATPase domain, but not the substrate-binding domain, was found to bind to complement proteins C8 and C9 and to inhibit zinc-induced polymerization of C9. Binding of mortalin to complement C9 and C8 occurs through an ionic interaction that is nucleotide-sensitive. We suggest that to express its full protective effect from CDC, mortalin must first reach the mitochondria. In addition, mortalin can potentially target the C8 and C9 complement components through its ATPase domain and inhibit C5b-9 assembly and stability.

A Role for the NF-κB Pathway in Cell Protection from Complement-Dependent Cytotoxicity

Nucleated cells are equipped with several mechanisms that support their resistance to complement-dependent cytotoxicity (CDC). The role of the NF-κB pathway in cell protection from CDC was examined. Elevated sensitivity to CDC was demonstrated in cells lacking the p65 subunit of NF-κB or the IκB kinases IKKα or IKKβ, and in cells treated with p65 small interfering RNA. Pretreatment with the IKK inhibitor PS-1145 also enhanced CDC of wild-type cells (WT) but not of p65−/− cells. Furthermore, reconstitution of p65 into p65−/− cells and overexpression of p65 in WT cells lowered their sensitivity to CDC. The postulated effect of p65 on the JNK-mediated death-signaling pathway activated by complement was examined. p65 small interfering RNA enhanced CDC in WT cells but not in cells lacking JNK. JNK phosphorylation induced by complement was more pronounced in p65−/− cells than in WT cells. The results indicate that the NF-κB pathway mediates cell resistance to CDC, possibly by suppressing JNK-dependent programmed necrotic cell death.

A novel mutation in the C3 gene and recurrent invasive pneumococcal infection: a clue for vaccine development.

BACKGROUNDnWe identified a 4 year-old boy born to a consanguineous marriage with C3 deficiency after three episodes of invasive pneumococcal disease. The efficacy of anti-pneumococcal vaccination in C3 deficient patients is not clear.nnnOBJECTIVESnOur objective was to identify the genetic defect resulting in his C3 deficiency and measure his ability to mount an adaptive immune response.nnnMETHODSnFibroblast cell lines were generated from the patient and parents. DNA was isolated and the C3 gene sequenced. Quantitation of C3 expression was performed by immunoprecipitation of (35)S-methionine labeled protein. Isotype specific anti-pneumococcal antibodies present in the patients sera was quantitated after administration of Prevnar-7 and Pneumovax vaccines.nnnRESULTSnPneumococcal types 14, 10B and 29 were identified from the blood on three separate occasions over a period of 20 months. C3 levels in the blood was <10, 71, and 66 for the patient, mother and father, respectively (90-180mg/dl, normal). Sequencing revealed a homozygous deletion of one nucleotide located in exon 31 (delA in position 3997 of cDNA) which resulted in a transcriptional stop signal thirteen codons later. The parents were heterozygous for the mutation. No detectable C3 was noted by immunoprecipitation. The patient mounted adequate antibody responses to the protein-conjugated Prevnar and tetanus vaccines but not to the polysaccharide antigen based Pneumovax vaccine. Major immunoglobulin class levels were normal.nnnCONCLUSIONnC3 deficiency results in the selective impairment to mount a response against polysaccharide-based antigens. Protein-conjugated vaccines are likely to be efficacious in immunizing against encapsulated organisms in these patients.

‘Rejected’ vs. ‘rejecting’ transcriptomes in allogeneic challenged colonial urochordates

In botryllid ascidians, allogeneic contacts between histoincompatible colonies lead to inflammatory rejection responses, which eventually separate the interacting colonies. In order to elucidate the molecular background of allogeneic rejection in the colonial ascidian Botryllus schlosseri, we performed microarray assays verified by qPCR, and employed bioinformatic analyses of the results, revealing disparate transcription profiles of the rejecting partners. While only minor expression changes were documented during rejection when both interacting genotypes were pooled together, analyses performed on each genotype separately portrayed disparate transcriptome responses. Allogeneic interacting genotypes that developed the morphological markers of rejection (points of rejection; PORs), termed rejected genotypes, showed transcription inhibition of key functional gene groups, including protein biosynthesis, cell structure and motility and stress response genes. In contrast, the allogeneic partners that did not show PORs, termed rejecting genotypes, showed minor expression changes that were different from those of the rejected genotypes. This data demonstrates that the observed morphological changes in the rejected genotypes are not due to active transcriptional response to the immune challenge but reflect transcription inhibition of response elements. Based on the morphological and molecular outcomes we suggest that the rejected colony activates an injurious self-destructive mechanism in order to disconnect itself from its histoincompatible neighboring colony.