Human A1 is induced in leukemic cells by tumor necrosis factor-α and A1 overexpression protects against its toxicity

Human A1 a Bcl-2 related gene, has been reported to be induced by TNF-α in endothelial and hematopoietic cells. In this study, we investigate the effects of up- or down-regulation of A1 expression on TNF-α induced apoptosis. We use U937 leukemia cell line that was transfected with a retroviral vector containing A1 cDNA in the sense or antisense orientation, as well as U937 cells that overexpress Bcl-2 or manganese superoxide dismutase (MnSOD). The effects of TNF-α on A1 mRNA (by Northern analysis) and toxicity (by apoptosis using Annexin V/flow cytometry and colony assay) were measured. Our results show that TNF-α (10-1000 ng/mL) induces A1 mRNA in all experimental groups except for U937 cells overexpressing Bcl-2, and, to a lesser degree, cells overexpressing A1 antisense. Chronic exposure of wild type U937 cells to low concentration of TNF-α (2-3 ng/mL) lead to overexpression of A1 mRNA and promote full protection against TNF-α induced apoptosis. Cells overexpressing A1 in the sense orientation are resistant to TNF-α induced apoptosis while those expressing antisense are more sensitive. U937 cells overexpressing Bcl-2 or MnSOD were also protected against TNF-α toxicity despite total suppression of A1 mRNA expression in the Bcl-2 cells. The addition of 25-50 μg/mL cycloheximide during TNF-α incubation reverses the protective effects of A1 and MnSOD but not that of Bcl-2. These results suggest that A1 may be induced by TNF-α as part of a protective mechanism against TNF-α toxicity in certain cell types. Furthermore, our results suggest a negative regulatory mechanism for A1 may be activated in the presence of high levels of Bcl-2.