Protection of human breast cancer cells from complement-mediated lysis by expression of heterologous CD59 (original) (raw)
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European Journal of Immunology, 1992
Human umbilical vein endothelial cells (HUVEC) were found by Western blot analysis to express three membrane-bound C regulatory proteins, decayaccelerating factor (DAF), membrane cofactor protein (MCP) and CD59. DAF was detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 70-kDa molecule under nonreducing conditions in 2 % deoxycholate extracts of HUVEC, MCP as a 63-kDa protein and CD59 as a 20-kDa molecule. Northern blot analysis revealed the presence of two species of mRNA expressed in HUVEC,which hybridized to a cDNA probe specific for DAF, with sizes of about 2.0 kb and 2.7 kb. MCP mRNA was detected at 4.2 kb and a CD59 cDNA probe hybridized with three mRNA species with sizes of about 800, 1400 and 2000 bp. DAF and CD59 were released from the surface of HUVEC by phosphatidylinositol-phospholipase C, demonstrating that both are attached to the cell membrane by means of a glycolipid anchor.The relative contribution of DAF, MCP and CD59 in regulating the sensitivity to lysis of HUVEC by autologous complement was determined by incubation of sensitized endothelial cells with F(ab'). fragment5 of polyclonal antibodies raised against these proteins. The susceptibility of sensitized cells to lysis by homologous complement was markedly increased in the presence of F(ab')z anti-CD59 and to a lesser, but significant, extent in the presence ofF(ab')z anti-DAF. F(ab')z anti-MCP did not significantly alter the susceptibility of HUVEC to complement-mediated lysis.
European Journal of Immunology, 2005
Expression of the complement-regulatory proteins (CRP) CD46, CD55 and CD59 represents a strategy used by tumor cells to evade complement-dependent cell cytoxicity stimulated by monoclonal antibodies. We have isolated two single-chain variable fragments (scFv) to CD55 and CD59 from a human phage-display library and from these scFv we have produced two miniantibodies (MB), MB-55 (against CD55) and MB-59 (against CD59), containing the human hinge-CH2-CH3 domains of IgG1. The specificity of the two MB for the corresponding CRP was assessed by ELISA using purified CD46, CD55 and CD59. MB-55 and MB-59 neutralized the inhibitory activity of CD55 and CD59, respectively, restoring the complement-mediated lysis of sheep and guinea pig erythrocytes that was otherwise inhibited by the two CRP. FACS analysis revealed binding of MB-55 and MB-59 to the lymphoma cell line Karpas 422. The two MB induced a two-fold increase in the complement-dependent killing of these cells stimulated by Rituximab, a chimeric anti-CD20 monoclonal antibody. Transfection of HEK293T cells with vectors encoding MB-55 or MB-59 markedly reduced the expression of CD55 and CD59. We conclude that the human antibodies MB-55 and MB-59 may represent a therapeutic tool to increase the complement-dependent killing activity of Rituximab in the treatment of non-Hodgkin's lymphoma.
Breast Cancer Research and Treatment, 1994
GB24 is a mouse monoclonal antibody raised against a common trophoblast-lymphocyte cross-reactive antigen. GB24 detects the membrane cofactor protein (MCR CD46), a member of the complement regulatory protein family, which serves as a cofactor for factor 1 mediated cleavage of C3b. This study investigated the reactivity of GB24 on 38 breast carcinomas and 34 normal/benign breast tissues by immunochemistry as well as the reactivity of F2B7-2, an antibody specific to the decay accelerating factor (DAE CD55) of the complement. GB24 staining was present on both normal tissue and benign lesions, but very strong diffuse reactivity was observed on carcinomas. This reactivity increased with the tumor grade. By contrast, malignant tumor cells lacked DAF expression. F2B7-2 antibody reacted weakly with benign epithelial cells. Results were studied by computer assisted image analysis to accurately define the mean optical densities. The densitometric analysis of MCP positive carcinomas showed a high intensity of the staining. Expression of MCP and DAF on MCF-7 cell lines was analyzed by flow cytometry. MCF-7 cell lines were strongly stained by mAb GB24 only. These data suggest that selectively enhanced expression of the antigen recognized by GB24 is associated with malignant breast disorders. This high expression, which may reflect a protective mechanism by which tumor cells survive complement activation, may prove useful as a marker of malignant transformation.
The Role of the Complement Inhibitor CD59 on Breast Cancer Cells
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Cellular & molecular immunology, 2011
Many monoclonal antibodies (mAbs) have been extensively used in the clinic, such as rituximab to treat lymphoma. However, resistance and non-responsiveness to mAb treatment have been challenging for this line of therapy. Complement is one of the main mediators of antibody-based cancer therapy via the complement-dependent cytolysis (CDC) effect. CD59 plays a critical role in resistance to mAbs through the CDC effect. In this paper, we attempted to investigate whether the novel CD59 inhibitor, recombinant ILYd4, was effective in enhancing the rituximab-mediated CDC effect on rituximab-sensitive RL-7 lymphoma cells and rituximab-induced resistant RR51.2 cells. Meanwhile, the CDC effects, which were mediated by rituximab and anti-CD24 mAb, on the refractory multiple myeloma (MM) cell line ARH-77 and the solid tumor osteosarcoma cell line Saos-2, were respectively investigated. We found that rILYd4 rendered the refractory cells sensitive to the mAb-mediated CDC effect and that rILYd4 exh...
Decay-accelerating factor protects human tumor cells from complement-mediated cytotoxicity in vitro
Journal of Clinical Investigation, 1988
The disialoganglioside GD2 is expressed on a wide spectrum of human tumor typesf including neuroblastomas and melanomas. Upon binding of 3F8, a murine monoclonal antibody (MAb) specific for GD2, neuroblastomas and some melanomas are sensitive to killing by human complement, whereas some melanomas are not. To investigate the mechanism underlying these differences in complement mediated cytotoxicity, complementinsensitive melanoma cell lines were compared with respect to expression of the decay-accelerating factor (DAF), a membrane regulatory protein that protects blood cells from autologous complement attack. While DAF was undetectable among neuroblastomas, it was present in complement-insensitive melanomas. When the function of DAF was blocked by anti-DAF MAb, C3 uptake and complement-mediated lysis of the insensitive melanoma lines were markedly enhanced. F(ab')2 fragments were as effective in enhancing lysis as intact anti-DAF MAb. The DAF-negative and DAF-positive melanoma cell lines were comparably resistant to passive lysis by cobra venom factor-treated serum. The data suggest that in some tumors, DAF activity accounts for their resistance to complement-mediated killing. The ability to render these cells complement-sensitive by blocking DAF function may have implications for immunotherapy.
Sublytic complement attack protects tumor cells from lytic doses of antibody and complement
European Journal of Immunology, 1992
Sublytic complement attack protects tumor cells from lytic doses of antibody and complement* Sublytic doses of the membrane attack complex (MAC) of complement are known to exert multiple stimulatory effects on metabolically active cells. Results presented herewith demonstrate that pretreatment of the human leukemic cells K562 and HL-60 with sublytic doses of antibody and normal human serum protects them from lytic complement concentrations, a phenomenon proposed to be called "complement-induced protection". C7-and C8-deficient human sera are ineffective in inducing resistance unless they are reconstituted with purified human C7 and C8, respectively. The complement-induced protection is inhibitable by actinomycin D and cycloheximide indicating that the increased complement resistance depends on RNA and protein synthesis triggered by the sublytic complement doses. Free extracellular Ca2+ is also required to achieve maximal protection, indicating a role for Ca2+ ions in the cell stimulatory events which culminate in increased complement resistance. Quantitative analysis of bound complement components indicated that similar amounts of C3 and C9 molecules are deposited on "protected" and control cells during complement activation.The "protected" K562 and RL-60 cells regain sensitivity to lytic MAC doses after about 8 or 3 h, respectively, of culture in growth medium, in the absence or presence of actinomycin D and cycloheximide. The "induced protection" is not species restricted and protection from human complement can be induced in K562 cells by treatment with sublytic doses of antibody and rabbit or guinea pig sera.
Persistent complement activation on tumor cells in breast cancer
The American journal of pathology, 1992
The neoantigens of the C5b-9 complement complex, IgG, C3, C4, S-protein/vitronectin, fibronectin, and macrophages were localized on 17 samples of breast cancer and on 6 samples of benign breast tumors using polyclonal or monoclonal antibodies and the streptavidin-biotin-peroxidase technique. All the tissue samples with carcinoma in each the TNM stages presented C5b-9 deposits on the membranes of tumor cells, thin granules on cell remnants, and diffuse deposits in the necrotic areas. When chemotherapy and radiation therapy preceded surgery, C5b-9 deposits were more intense and extended. The C5b-9 deposits were absent in all the samples with benign lesions. S-protein/vitronectin was present as fibrillar deposits in the connective tissue matrix and as diffuse deposits around the tumor cells, less intense and extended than fibronectin. IgG, C3, and C4 deposits were present only in carcinoma samples. The presence of C5b-9 deposits is indicative of complement activation and its subsequent...
Clinical and Experimental Immunology, 2003
Nucleated cells employ several strategies to evade killing by homologous complement. We studied complement resistance in the human carcinoma cell lines (CA) T47D (mammary), SKOV3 (ovarian), and PC-3 (prostate) with emphasis on the following mechanisms of defense: 1. Expression and shedding of the membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59; 2. Resistance based on protein phosphorylation; 3. Cell surface expression of sialic acid residues; 4. Desensitization to complement upon exposure to sublytic complement doses. Anti-mCRP antibody blocking experiments demonstrated that CD59 is the main mCRP protecting these CA from complement. Soluble CD59 was also found in supernates of PC-3 > SKOV3 > T47D cells. Second, inhibitors of PKC, PKA and MEK sensitized the CA to lysis, thus implicating these protein kinases in CA complement resistance. Third, removal of sialic acid residues with neuraminidase also sensitized CA to lysis. Finally, exposure of CA to sublytic doses of complement conferred on them enhanced resistance to lytic complement doses in a PKC-dependent process. Combined treatment of CA with anti-CD59 antibodies, PD98059 (a MEK inhibitor) and neuraminidase produced a large enhancement in CA sensitivity to complement. Our results show that CD59 and sialic acid residues present on the cell surface, and intracellular processes involving protein phosphorylation act additively to secure CA resistance to complement-mediated lysis. Therefore, the effectiveness of antibody-and complement-based cancer immunotherapy will markedly improve by suppression of the various complement resistance mechanisms.