Abstract
IntroductionOur goal was to specifically render tumor cells susceptible to natural cytolytic anti-αGal antibodies by using a murine α1,3galactosyltransferase (mαGalT) transgene driven by a designed form of HER2/neu promoter (pNeu), the transcription of which is frequently observed to be above basal in breast tumors. Indeed, the αGalT activity that promotes Galα1,3Galβ1,4GlcNAc-R (αGal) epitope expression has been mutationally disrupted during the course of evolution, starting from Old World primates, and this has led to the counter-production of large amounts of cytotoxic anti-αGal antibodies in recent primates, including man.MethodExpression of the endogenous c-erbB-2 gene was investigated in various cell lines by northern blotting. A mαGalT cDNA was constructed into pcDNA3 vector downstream of the original CMV promoter (pCMV/mαGalT) and various forms of pNeu were prepared by PCR amplification and inserted in the pCMV/mαGalT construct upstream of the mαGalT cDNA, in the place of the CMV promoter. These constructs were transferred into HEK-293 control and breast tumor cell lines. Stably transfected cells were analyzed by northern blotting for their expression of αGalT and c-erbB-2, and by flow cytometry for their binding with fluorescein isothiocyanate-conjugated Griffonia simplicifolia/isolectin B4.ResultsWe show that expression of the mαGalT was up- or down-modulated according to the level of endogenous pNeu activity and the particular form of constructed pNeu. Among several constructs, two particular forms of the promoter, pNeu250 containing the CCAAT box and the PEA3 motif adjacent to the TATAA box, and pNeu664, which has three additional PEA3 motifs upstream of the CCAAT box, were found to promote differential αGalT expression.ConclusionOur results strengthen current concepts about the crucial role played by the proximal PEA3 motif of pNeu, and may represent a novel therapeutic approach for the development of targeted transgene expression.
Highlights
Our goal was to render tumor cells susceptible to natural cytolytic anti-αGal antibodies by using a murine α1,3galactosyltransferase transgene driven by a designed form of HER2/neu promoter, the transcription of which is frequently observed to be above basal in breast tumors
Our results strengthen current concepts about the crucial role played by the proximal PEA3 motif of pNeu, and may represent a novel therapeutic approach for the development of targeted transgene expression
Expression of the c-erbB-2 gene in various cell lines Expression of the endogenous c-erbB-2 gene was investigated in various cell lines by northern blotting. c-erbB-2 was very weakly expressed in HEK-293 cells and was differentially transcribed in human breast cancer cell lines (Figs 1 and 2C): it was absent in MDA-MB-231, moderately expressed in MCF7 and MDA-MB-453, and strongly expressed in SK-BR-3 cells
Summary
Our goal was to render tumor cells susceptible to natural cytolytic anti-αGal antibodies by using a murine α1,3galactosyltransferase (mαGalT) transgene driven by a designed form of HER2/neu promoter (pNeu), the transcription of which is frequently observed to be above basal in breast tumors. Given the broad circulation of αGal carbohydrate antigens, humans, apes and Old World monkeys produce large amounts of anti-αGal antibodies, which represent approximately 1% of total IgG in humans [4] These antibodies are responsible for the hyperacute rejection of xenografts and prevent trials on transplantation of pig organs to humans [5,6]. They represent a potential constitutive tool for therapeutic applications because their highly efficient ad5 = adenovirus type 5; AP-2 = activator protein-2; bp = base pair; FCS = fetal calf serum; FITC-GS-I-B4 = fluorescein isothiocyanate-conjugated Griffonia simplicifolia/isolectin B4; αGal = galactose-α1,3galactose-β1,4N-acetylglucosamine-R; αGalT = α1,3galactosyltransferase; GAPDH = glyceraldehyde 3-phosphate-dehydrogenase; LTR = long terminal repeat; mαGalT = murine αGalT; NF-kB = nuclear factor kB; pCMV = CMV promoter; PCR = polymerase chain reaction; pNeu = HER2/neu promoter; VSV-G = vesicular stomatitis viral G glycoprotein
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