The use of α(1,3)galactosyltransferase (αGT) as a method of inducing hyperacute rejection of tumors has been gaining interest recently. However, the approach is based in part on the sensitivity of each tumor line to the effects of complement lysis. Tumors expressing complement resistance factors such as membrane cofactor (CD46), decay accelerating factor (CD55) and protectin (CD59) have been shown to be more resistant to complement mediated lysis. Anchored to the membrane by a glycosylphosphoinositol moiety (GPI-anchored), CD55 and CD59 can be cleaved by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PIPLC). Complement resistant A549 human lung carcinoma cells were engineered to express both the murine αGT gene and the B. thuringiensis PIPLC gene to alleviate complement resistance and enhance αgal-mediated cancer killing. The PIPLC native signal sequence was replaced with the human epidermal growth factor signal sequence, EGFssPIPLC, to induce secretion from A549. Expression of EGFssPIPLC resulted in complete removal of CD55 and CD59 while sparing the non-GPI-anchored CD46. Results demonstrated that A549 cells transduced with two recombinant retroviral vectors carrying the αGT and EGFssPIPLC genes expressed high levels of αgal epitope and exhibited a 5-fold increase in sensitivity to anti-αgal mediated complement lysis.

Proc Amer Assoc Cancer Res, Volume 45, 2004 3819 Ataxia-telangeictasia (AT) is an autosomal recessive disorder characterized by immune dysfunction, genomic instability, chronic oxidative damage, and increased cancer incidence. Compared to normal cells, AT cells exhibit unusual sensitivity to exogenous oxidants, including t-butyl hydroperoxide. Since ferritin releases labile iron under oxidative stress (which is chronic in AT) and labile iron mediates the toxic effects of t-butyl hydroperoxide, we hypothesized that chelation of intracellular labile iron would increase the genomic stability of AT cells, with and without exogenous oxidative stress. At first, we found that the ferrous iron chelator desferrioxamine was shown to increase resistance to exogenous oxidative stress of AT, but not normal cells, in the colony forming-efficiency assay. This data suggested that iron metabolism is dysregulated in AT. Since desferrioxamine alters gene expression, as well as chelates iron, we tested the effect of apoferritinn and the iron chelating flavonoid quercetin on AT cell genomic stability. The data shown that apoferritin and quercetin increase the genomic stability of AT cells. To further analyze the role of labile iron in AT, we measured labile iron in the sera of AT knockout mice and found significantly elevated iron levels compared to syngeniec wild type mice. Our data supports a role for increased labile iron acting as a Fenton catalyst in AT, thus contributing to the chronic oxidative stress seen in this disease. Our data further suggests that iron chelators as a class might promote genomic stability in AT cells.

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by genomic instability, chronic oxidative damage, and increased cancer incidence. Compared to normal cells, AT cells exhibit unusual sensitivity to exogenous oxidants, including t-butyl hydroperoxide (t-BOOH). Since ferritin releases labile iron under oxidative stress (which is chronic in AT) and labile iron mediates the toxic effects of t-butyl hydroperoxide, we hypothesized that chelation of intracellular labile iron would increase the genomic stability of AT cells, with and without exogenous oxidative stress. Here we report that desferrioxamine treatment increases the plating efficiency of AT, but not normal cells, in the colony forming-efficiency assay (a method often used to measure genomic stability). Additionally, desferrioxamine increases AT, but not normal cell resistance, to t-butyl hydroperoxide in this assay. Last, AT cells exhibit increased sensitivity to the toxic effects of FeCl2 in the colony forming-efficiency assay and fail to demonstrate a FeCl2-induced G2 checkpoint response when compared to normal cells. Our data indicates that: (1) chelation of labile iron increases genomic stability in AT cells, but not normal cells; and (2) AT cells exhibit deficits in their responses to iron toxicity. While preliminary, our findings suggest that AT might be, in part, a disorder of iron metabolism and treatment of individuals with AT with desferrioxamine might have clinical efficacy.