A very sensitive coupled luminescent assay for cytotoxicity and complement-mediated lysis

Corey, Michael J.; Kinders, Robert J.; Brown, Lisha G.; Vessella, Robert L.

doi:10.1016/s0022-1759(97)00098-7

Cited by 47 publications

(31 citation statements)

References 14 publications

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“…Based on these results and on our previous studies (14), we propose that lung cancer cells may develop a protective mechanism against complement attack by expressing and binding factor H to their cell membranes. Several studies have also suggested the importance of factor H in the protection of other tumor cells against complement activation (12,13,25,37). For the first time, we demonstrate the importance of factor H expression for the protection of cancer cells in an in vivo model.…”

Section: Discussionsupporting

confidence: 69%

Down-Regulation of Human Complement Factor H Sensitizes Non-Small Cell Lung Cancer Cells to Complement Attack and Reduces In Vivo Tumor Growth

Ajona¹,

Hsu²,

Corrales³

et al. 2007

The Journal of Immunology

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Malignant cells are often resistant to complement activation through the enhanced expression of complement inhibitors. In this work, we examined the protective role of factor H, CD46, CD55, and CD59 in two non-small cell lung cancer cell lines, H1264 and A549, upon activation of the classical pathway of complement. Complement was activated with polyclonal Abs raised against each cell line. After blocking factor H activity with a neutralizing Ab, C3 deposition and C5a release were more efficient. Besides, a combined inhibition of factor H and CD59 significantly increased complement-mediated lysis. CD46 and CD55 did not show any effect in the control of complement activation. Factor H expression was knockdown on A549 cells using small interfering RNA. In vivo growth of factor H-deficient cells in athymic mice was significantly reduced. C3 immunocytochemistry on explanted xenografts showed an enhanced activation of complement in these cells. Besides, when mice were depleted of complement with cobra venom factor, growth was recovered, providing further evidence that complement was important in the reduction of in vivo growth. In conclusion, we show that expression of the complement inhibitor factor H by lung cancer cells can prevent complement activation and improve tumor development in vivo. This may have important consequences in the efficiency of complement-mediated immunotherapies.

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Section: Discussionsupporting

confidence: 69%

Down-Regulation of Human Complement Factor H Sensitizes Non-Small Cell Lung Cancer Cells to Complement Attack and Reduces In Vivo Tumor Growth

Ajona¹,

Hsu²,

Corrales³

et al. 2007

The Journal of Immunology

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“…The presence of X52.1 (22 nM) enhanced complement-mediated lysis of this cell line by 73%. Subsequently we developed an ultra-sensitive luminescent assay for cell death and membrane damage, which we used to observe an 11-fold enhancement of complement-mediated killing of puromycin-treated Raji cells by X52.1 versus the complement-only background (26). Untreated Raji cells were much less sensitive to complement-mediated lysis than puromycintreated Raji, but X52.1 still enhanced the rate of lysis by approximately 12-fold (Table I).…”

Section: Resultsmentioning

confidence: 99%

Mechanistic Studies of the Effects of Anti-factor H Antibodies on Complement-mediated Lysis

Corey¹,

Kinders²,

Poduje³

et al. 2000

Journal of Biological Chemistry

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We have recently reported that complement factor H, a negative regulator of complement-mediated cytotoxicity, is produced and secreted by most bladder cancers. This observation was exploited in the development of the BTA stat™ and BTA TRAK™ diagnostic assays, both of which make use of two factor H-specific monoclonal antibodies in sandwich format. Here we show that both antibodies exert interesting effects on the biochemistry of complement activation in in vitro systems. Antibody X13.2 competes with C3b for association with factor H and strongly inhibits factor H/factor I-mediated cleavage of C3b, thereby evidently inactivating a negative regulator of complement; yet, the antibody strongly inhibits complement-mediated lysis as well. Conversely, antibody X52.1, which does not compete with C3b and has no effect on solution-phase cleavage of C3b, is capable of enhancing complement-mediated lysis of various cell types, including cancer cells, by over 10-fold. Our observations indicate that it is possible to deconvolute the biochemical roles of factor H in complement by means of appropriate inhibitors, a finding with potentially valuable implications for both basic research and cancer therapy.

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“…In SK-MEL-93-2, a human melanoma cell line, factor H is the dominant factor regulating the inactivation of cell-bound C3b and is involved in the control of the classical pathway of complement (42). An antifactor H antibody also enhances complement-mediated killing of Raji cells, a cell line obtained from a Burkitt's lymphoma (43). Members of the SIBLING family can protect murine erythroleukemia and human myeloma and breast cancer cells from complement attack, likely by sequestration of factor H to the cell surface (44,45).…”

Section: Discussionmentioning

confidence: 99%

Expression of Complement Factor H by Lung Cancer Cells

et al. 2004

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The complement system is important in immunosurveillance against tumors. However, malignant cells are usually resistant to complementmediated lysis. In this study, we examine the expression of factor H, an inhibitor of complement activation, and factor H-like protein 1 (FHL-1), its alternatively spliced form, in lung cancer. We also evaluate the potential effect of factor H/FHL-1 in the protection of lung cancer cells against the activation of the complement cascade. By Northern blot analysis we demonstrate a high expression of factor H and FHL-1 in most non-small cell lung cancer cell lines, although neuroendocrine pulmonary tumors (small cell lung carcinoma and carcinoid cell lines) had undetectable levels. Western blot analysis of conditioned medium showed the active secretion of factor H and FHL-1 by cells that were positive by Northern blot. Expression of factor H/FHL-1 mRNA was also shown in a series of non-small cell lung cancer biopsies by in situ hybridization. Interestingly, many cultured lung cancer cells were able to bind fluorescence-labeled factor H to their surfaces. Deposition of C3 fragments from normal human serum on H1264, a lung adenocarcinoma cell line, was more efficient when factor H/FHL-1 activity was blocked by specific antibodies. Blocking factor H/FHL-1 activity also enhanced the release of anaphylatoxin C5a and moderately increased the susceptibility of these cells to complement-mediated cytotoxicity. In summary, we demonstrate the expression of factor H and FHL-1 by some lung cancer cells and analyze the contribution of these proteins to the protection against complement activation.

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A very sensitive coupled luminescent assay for cytotoxicity and complement-mediated lysis

Cited by 47 publications

References 14 publications

Down-Regulation of Human Complement Factor H Sensitizes Non-Small Cell Lung Cancer Cells to Complement Attack and Reduces In Vivo Tumor Growth

Down-Regulation of Human Complement Factor H Sensitizes Non-Small Cell Lung Cancer Cells to Complement Attack and Reduces In Vivo Tumor Growth

Mechanistic Studies of the Effects of Anti-factor H Antibodies on Complement-mediated Lysis

Expression of Complement Factor H by Lung Cancer Cells

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