Co-expression of the complement regulatory proteins human DAF and CD59 with an IRES-mediated dicistronic mammalian vector enhances their cell protective effects

Xu, Li; Shao, Huanjie; Wu, Wen-Lan; Cao, Zhijian; Zhao, Zhouzhou; Liu, Hui; Jiang, Dejun; Mao, Xin; Li, Wenxin

doi:10.3892/ijmm.16.3.409

Cited by 4 publications

(4 citation statements)

References 20 publications

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“…11,17,18 Similar speculation has been made in several studies. 15,19,20 However, our present results differ from those of others, 11,16 in that MCP+DAF exerted an inhibitory effect on complement-mediated cytolysis, similar to MCP+CD59 but more potent than DAF+CD59. Because expression levels of hCRPs in cells might influence their function, we used real-time PCR to determine mRNA levels of MCP, DAF, and CD59, which was not done in other studies.…”

Section: Discussioncontrasting

confidence: 57%

“…Results confirmed that IRES could drive double or single gene expression, consistent with previous reports. 15,16 Stable transfection in NIH 3t3 cells The pEGFP-N1 plasmid was used as a positive control for transfection. We investigated the expression of green fluorescent protein (GFP) in NIH 3T3 cells 36 hours after transfection under an inverted fluorescence microscope.…”

Section: Resultsmentioning

confidence: 99%

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Protective Effects of Different Combinations of Human MCP, DAF, and CD59 on Complement-Dependent Cytolysis in NIH 3T3 Cells

Yang¹,

Deng²,

Jiang³

et al. 2012

Exp Clin Transplant

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Objectives: To analyze the protective effects against complement-mediated cytolysis of the MCP, DAF, and CD59 human complement regulatory proteins, alone and in combination, on NIH 3T3 mouse fibroblast cells. Materials and Methods:We constructed 3 doubleand 3 single-human complement regulatory protein plasmids (pIRES-hMCP-hDAF, pIRES-hMCP-hCD59, pIRES-hDAF-hCD59, pIRES-A-hMCP, pIRES-B-hDAF, and pIRES-B-hCD59). The plasmids were transfected into NIH 3T3 cells, and stable transfectants were obtained by treatment with 200 kg/m3 G418 for 2 weeks. Normal human serum (50%) as a source of complement was added to the culture medium of stable transfectants. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to analyze the protective ability of different human complement regulatory protein plasmids on complement-dependent cytolysis. Results: The viability of double-human complement regulatory protein stable transfectants was significantly higher than that of single-human complement regulatory protein stable transfectants (P < .05). Among the double-transfectants, cells expressing pIRES-hMCP-hDAF and pIRES-hMCPhCD59 survived better than cells expressing pIREShDAF-hCD59 (91.75% ± 3.30% and 84.88% ± 2.36% vs 66.19% ± 6.52%; P < .05). Among the singletransfectants, cells expressing pIRES-A-hMCP or pIRES-B-hDAF survived better than cells expressing pIRES-B-hCD59 or pIRES empty vector (53.76% ± 3.84% and 56.32% ± 2.83% vs 43.28% ± 0.96% and 40.27% ± 1.11%; P < .05). Conclusions: These results suggest that the MCP+DAF and MCP+CD59 combinations could be more effective than DAF+CD59 in protecting the NIH 3T3 cells from injury caused by complementdependent cytolysis, whereas MCP or DAF alone is stronger than CD59 alone in inhibiting membrane attack complex formation.

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Section: Discussioncontrasting

confidence: 57%

Section: Resultsmentioning

confidence: 99%

Protective Effects of Different Combinations of Human MCP, DAF, and CD59 on Complement-Dependent Cytolysis in NIH 3T3 Cells

Yang¹,

Deng²,

Jiang³

et al. 2012

Exp Clin Transplant

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“…Xu et al [20] studied the effect of transfection of human complement‐regulatory proteins on protection against human complement. They transfected human decay‐accelerating factor and protectin (CD59) with a dicistronic mammalian expression vector into the NIH/3T3 cell line, separating the two genes by an internal ribosomal entry site.…”

Section: Gene Therapymentioning

confidence: 99%

Xenotransplantation Literature Update September–October, 2005

Baertschiger

Bühler

2006

Xenotransplantation

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“…There is also evidence to support a role for CRegs in influencing cancer progression in humans 13 . The precise impact of CReg expression is not fully understood, as some studies have reported that low levels of CReg expression are associated with disease progression, 14 , 15 , 16 while others indicate that high levels of CRegs are associated with decreased survival 17 , 18 , 19 , 20 . Despite these seemingly conflicting findings, these data are compatible with a role for complement activation in influencing the phenotype of tumour cells and hence their capacity to proliferate and survive.…”

mentioning

confidence: 99%

Complement‐induced protection: an explanation for the limitations of cell‐based tumour immunotherapies

et al. 2012

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Complement is involved in the inflammatory response and clearance of infected or altered cells. It is therefore unexpected that complement-deficient animals are less susceptible to carcinogen-induced tumours and more readily control growth of injected tumour cell lines than their wild-type counterparts, implying that complement promotes tumour development and progression. Conversely, natural killer (NK) and CD8 þ T cells are known to limit progression of the same tumours. Previous studies indicate that sublytic levels of the complement membrane attack complex protect cells against further attack by lytic doses of complement and other pore-formers such as perforin. We hypothesise that inefficient attack by complement in vivo allows tumour cells to avoid lysis by both NK cells and antigen-specific cytotoxic T cells, thereby promoting tumour outgrowth. Complement could thus be limiting the efficacy of NK and T cell-targeted cancer therapies, and the inclusion of complement inhibitors could optimise these immunotherapeutic regimes.

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Co-expression of the complement regulatory proteins human DAF and CD59 with an IRES-mediated dicistronic mammalian vector enhances their cell protective effects

Cited by 4 publications

References 20 publications

Protective Effects of Different Combinations of Human MCP, DAF, and CD59 on Complement-Dependent Cytolysis in NIH 3T3 Cells

Protective Effects of Different Combinations of Human MCP, DAF, and CD59 on Complement-Dependent Cytolysis in NIH 3T3 Cells

Xenotransplantation Literature Update September–October, 2005

Complement‐induced protection: an explanation for the limitations of cell‐based tumour immunotherapies

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