The Bcl -2 family member, A1 -a: Studies in localization and function

Abstract

A1 is a member of the Bcl-2 family of anti-apoptotic proteins. These proteins are involved in regulating apoptosis found during normal development, tissue homeostasis and in defense against pathogens. Initial studies have shown that A1 can protect cells from undergoing apoptosis in a manner similar to Bcl-2. Additional studies had also shown that A1 differed from Bcl-2; e.g. A1 might localize to the nucleus, something not seen before in this family of proteins. Our hypothesis was that A1 would localize to the nucleus and function there as an anti-apoptotic regulator. To determine the localization of A1, we developed both monoclonal and polyclonal antibodies against the A1 protein. Using these antibodies for both western blot analysis and immunocytochemistry in a COS-7 overexpression system, we found that A1 localized to the nucleus, compared to Bcl-2 which localized to the cytoplasm as expected. Nuclear A1 in Cos-7 cells protected them from staurosporine induced apoptosis. A1 localization was different in fresh murine leukocytes where it was found primarily in the cytoplasm. In addition, we studied murine macrophages from both wild type and A1 knock out mice. A1 was shown to be partially responsible for protecting the macrophages from nitric oxide induced apoptosis, a phenomenon mimicking inflammation. Studies have shown in RAW264.7 cells, that nitric oxide mediated apoptosis is partially dependent on the transcription factor, p53. Our experiments showed that A1 dependent protection of nitric oxide induced apoptosis was not mediated through levels of p53 nor the p53 induced proapoptotic protein, Bax. A1 did prevent the release of cytochrome C from the mitochondria suggesting that A1 may play its antiapoptotic role in a manner similar to Bcl-2. Additional experiments attempted to look at the role of A1 in host defense against intracellular pathogens. In an attempt to create an in vitro model of pathogen infection, murine macrophages from both wild type and A1 knock out mice were infected with different pathogens. Experiments looking at various apoptotic markers, such as caspase activity and TUNEL staining, showed that our in vitro model did not mimic the conditions seen during an in vivo infection.