Abstract
BackgroundWe previously reported the usefulness of the αGal epitope as a target molecule for gene therapy against cancer. To induce cancer cell specific transcription of the αGal epitope, an expression vector which synthesizes the αGal epitope under the control of a promoter region of the human telomerase reverse transcriptase (hTERT), NK7, was constructed.MethodsNK7 was transfected into a human pancreatic carcinoma cell line, MIA cells, and telomerase-negative SUSM-1 cells served controls. Expression of the αGal epitope was confirmed by flow cytometry using IB4 lectin. The susceptibility of transfected MIA cells to human natural antibodies, was examined using a complement-dependent cytotoxic cross-match test (CDC) and a flow cytometry using annexin V.ResultsThe αGal epitope expression was detected only on the cell surfaces of NK7-transfected MIA cells, i.e., not on naive MIA cells or telomerase negative SUSM-1 cells. The CDC results indicated that MIA cells transfected with NK7 are susceptible to human natural antibody-mediated cell killing, and the differences, as compared to NK-7 transfected telomerase negative SUSM-1 cells or telomerase positive naïve MIA cells, were statistically significant. The flow cytometry using annexin V showed a higher number of the apoptotic cells in NK-7 transfected MIA cells than in naïve MIA cells.ConclusionsThe results suggest that αGal epitope-expression, under the control of the hTERT-promoter, may be useful in cancer specific gene therapy.
Highlights
One of the common difficulties commonly encountered in cancer gene therapy is manipulation of a gene which acts exclusively in cancer cells, without affecting normal cells
A1–3 GT driven by the human telomerase reverse transcriptase (hTERT) promoter was functional in human pancreatic cancer cells As described in Methods, SUSM-1 cells were shown to be negative for the telomerase-activity
NK7-transfected MIA cells showed a high positive log shift for the aGal epitope (Fig. 3D). These results indicate that the hTERT promoter acts only on MIA cells, which are telomerase-positive, i.e., not on SUSM-1 cells which are telomerase-negative
Summary
Construction of NK7 The full length bovine a1–3 GT cDNA was cut at Apa- and BamH-sites of N3GA [15] and ligated to an pEGFP-1 vector (CLONTECH, Palo Alto, CA) to form an E1GA vector. Individual neomycin resistant clones were picked up and transferred to a 24 well tissue culture plate, and DNA extracted from these cells was subjected to PCR to confirm integration of the a1–3 GT cDNA. Ten neomycin-resistant clones from MIA cells or SUSM-1 cells, which were positive for the 1355 bp-PCR product, were transferred to 10cm tissue culture plates to obtain a sufficient number of cells for flow cytometry and complement dependent cross-match test (CDC). Flow cytometry The promoter activity of the hTERT gene was expected to operate in the endogenously telomerase-positive MIA cells, but not in the telomerase-negative SUSM-1 cells. At least 1 ́ 106 cells prepared by human serum and rabbit complement described in CDC were incubated with FITC-conjugated annexin V at room temperature for 15 min.
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