Engineering malignant cells to express a heterologous α-gal antigen can induce heterograft hyperacute rejection, resulting in complement-dependent cytolysis (CDC) of tumor cells, which has been considered as a novel strategy for antitumor therapy. A549 cells engineered to express Galα1-3Galβ1-4GlcNAc-R (α-gal) epitope exhibited strong resistance to CDC treated by normal human serum (NHS) in a previous study. We hypothesized that the expression of membrane-bound complement regulatory proteins (mCRPs) decay accelerating factor (CD55) and protectin (CD59) influenced the efficacy of the α-gal/NHS-mediated antitumor effect to tumor cells in vitro. The present study confirmed that A549 cells expressed high levels of CD55 and CD59, whereas Lovo cells expressed relatively low levels of these proteins. A549 and Lovo cells transfected with plasmids containing or lacking the α-gal epitope were evaluated for their susceptibility to CDC by NHS and detected using a trypan blue exclusion assay. α-gal-expressing Lovo (Lovo-GT) cells were almost completely killed by α-gal-mediated CDC following incubation with 50% NHS, whereas no cytolysis was observed in α-gal expressing A549 (A549-GT) cells. Abrogating CD55 and CD59 function from A549-GT cells by various concentrations of phosphatidylinositol-specific phospholipase C (PI-PLC) or blocking antibodies increased the susceptibility of cells to CDC, and the survival rate decreased significantly comparing to the controls (P<0.05). The findings of the present study indicated that using the α-gal/NHS system to eliminate tumor cells via inducing the complement cascade reaction might represent a feasible approach for the treatment of cancer. However, high levels of mCRP expression may limit the efficacy of this approach. Therefore, an improved efficacy of cancer cell killing may be achieved by combining strategies of heterologous α-gal expression and mCRP downregulation.

Engineering cancer cells to express heterologous antigen α-gal and induce the destruction of tumor cells depending on the complement cascade may be a promising strategy of tumor therapy. However, the feasibility and effect of using α-gal to induce colorectal adenocarcinoma cell line cytolysis is not yet known. In this study, we evaluated α-gal expression's ability to sensitize human colorectal adenocarcinoma cell lines to complement attack in cell lines LoVo, SW620, and Ls-174T. Nearly all α-gal-expressing LoVo and SW620 cells were killed by normal human serum (NHS), but α-gal-expressing Ls-174T cells showed no significant lysis. We analyzed the expression levels of membrane-bound complement regulatory proteins (mCRPs) on the three cell lines, and their protective role in α-gal-mediated activation of the complement. LoVo showed no expression of any of the three proteins. CD59 was strongly expressed by SW620 and Ls-174T. CD46 and CD55 varied between the two cell lines. CD46 on SW620 was only half the intensity of CD46 on Ls-174T. Ls-174T showed a notable expression of CD55, while expression of CD55 on SW620 was not detected. The sensitivity of Ls-174T expressing α-gal to NHS greatly increased following the downregulation of CD46 and CD55 with short hairpin RNA (shRNA). However, there is no increase in cell killing when CD59 expression was diminished. Our findings suggest that the use of α-gal as antigen to induce tumor cell killing may be a potential therapeutic strategy in colon cancer and that CD55 plays a primary role in conferring resistance to lysis.

BACKGROUND AND OBJECTIVE:The absence of α-gal in humans is caused by the inactivity of α-1,3GT gene. However, humans have pre-existing and abundant anti-gal antibodies. Xenotransplantation procedures have indicated the high potential of introducing α-1,3GT gene to synthesize α-gal for cancer gene therapy by mimicking hyper-acute rejection. The aim of this study is to construct a lung cancer A549 cell line that expressed α-gal, and to observe the antitumor mechanisms mediated by human serum.METHODS:A549 cells were transfected with pEGFP-N1-GT plasmids constructed in a previous study. A stable transgenic cell line, A549-GT, was then selected and cultivated. The biological characteristics of A549-GT cells, including morphology and proliferation, were examined. α-1,3GT mRNA expression was detected by RT-PCR. Direct immunofluorescence staining and flow cytometry (FCM) were used to analyze the synthesis of α-gal in A549-GT. The binding of human serum IgM and C3 with A549-GT were also detected.RESULTS:α-1,3GT mRNA was expressed in A549-GT. Direct immunofluorescence staining and FCM indicated a high and stable α-gal expression rate in A549-GT. Compared with parental A549 cells, the biological characteristics of A549-GT were unaltered. α-Gal expression was not detected in the human fetal lung fibroblast cell line MRC-5 even though A549-GT and its culture medium were cultivated with the enzyme. Immunofluorescence staining and FCM also indicated abundant binding between A549-GT treated with human serum and IgM/C3.CONCLUSIONS:α-Gal expression in tumor cells by gene transduction can induce complement-dependent cytototic antitumor effects.

OBJECTIVES To establish a human lung adenocarcinoma cell subline A549 that can stably express the Chinese Banna minipig inbred-line (BMI) alpha1 ,3-galactosyltransferase (alpha1 ,3GT) gene and alpha-galactosyl (Gala1-3Galb1-4GlcNAc-R, alpha-gal) epitopic, providing a cell model which expressed xenotransplantation antigens for the further research on the effect of complement dependent cytotoxic lysis of the tumor cells triggered by human natural serum. METHODS The pEGFP-CMV-GT plasmid containing Banna minipig alpha1 ,3-GT gene was ransfected into A549 cells with lipofectin in vitro. After screened with G418,the single clones were got out and then amplified, the stable transfected cells was named A549-GT. The transcription of alpha1, 3-GT gene in A549-GT cells was detected by RT-PCR. Direct immunofluonrescence methods and flow cytometer were performed to observe the expression of alpha-gal and the binding conditions of IgM and complement C3 in human serum on A549-GT cells. The biological characters of A549-GT cells including morphology, proliferation, and tumorigenesis in nude mice were also examined. RESULTS After G418 screening, A549-GT that stablely transfected with alpha1, 3-GT gene was obtained and has been passaged for 2 years. The expression of alpha1,3-GT mRNA and alpha-gal was detected continuously and stably in A549-GT. The expression rate of alpha-gal positive cells reached 80.1% +/- 3.2%. The binding of human serum IgM and C3 in human serum on A549-GT cells were founded. Compared with parental A549 cells, its biological characteristics did not change. CONCLUSION A549-GT cell line stably and continuously expressing alpha1, 3-GT and alpha-gal was established successfully. It provided a useful cell model for the further study of pig alpha1,3-GT gene in tumor immunotherapy.

Objective:To construct an xenoantigen synthetase α-1,3 galactosyltransferase(α-1,3GT) eukaryotic expression vector regulated by human telomerase catalytic subunit(hTERT) promoter,and to investigate its targeting expression of α-1,3GT in lung cancer cell lines.Methods:Previously prepared and confirmed pig α-1,3GT gene was inserted into pEGFP-hTERTp plasmid to construct eukaryotic expression vector pEGFP-hTERTp-GT.pEGFP-hTERTp-GT and pEGFP-N1-GT(α-1,3GT eukaryotic expression vector under the control of CMV promoter) were transfected into telomerase-positive human lung adenocarcinoma A549 cells and telomerase-negative human embryonic lung fibroblast MRC-5 cells.1,3-GT mRNA expression in the transfected cells was detected by RT-PCR.Expression of α-gal antigen in transfected cells was examined by immunofluorescence and flow cytometry.Results:pEGFP-hTERTp-GT eukaryotic expression vector was successfully constructed.Both A549 and MRC-5 cells transfected with pEGFP-N1-GT showed expression of α-1,3GT mRNA;A549 cells but not telomerase-negative MRC-5 cells expressed α-1,3GT mRNA after transfection with pEGFP-hTERTp-GT.Furthermore,both A549 and MRC-5 cells transfected with pEGFP-N1-GT showed expression of xenoantigen α-gal;A549 but not MRC-5 cells expressed xenoantigen α-gal after transfection with pEGFP-hTERTp-GT(P0.01).Conclusion:α-1,3GT gene under the regulation of hTERT promoter can be specifically expressed in telomerase-positive lung cancer cell lines,which can induce production of xenoantigen α-gal.