Bfl-1S, a novel alternative splice variant of Bfl-1, localizes in the nucleus via its C-terminus and prevents cell death.

Bfl-1 is an antiapoptotic Bcl-2 family member and a mouse A1 homologue. The mouse A1 has been reported to have three isoforms, but little is known about human Bfl-1. By reverse-transcriptase polymerase chain reaction analysis, we have identified Bfl-1S (short form), an alternative splice variant of Bfl-1. The Bfl-1S primary sequence contains four conserved Bcl-2 homology (BH) domains and a positive-charged C-terminus containing KKRK amino acids. The expression of Bfl-1S mRNA was detected predominantly in normal lymph nodes and in B-lymphoid leukemia cells. Confocal microscopic analysis using green fluorescence protein fusion proteins demonstrated that Bfl-1S is localized in the nucleus by its C-terminus as an intrinsic nuclear localization sequence. Bfl-1S acts as an antiapoptotic agent in coexpression experiments with Bax, a proapoptotic molecule. The expression of Bfl-1S provided significant resistance against staurosporine (STS) treatments in Molt-4 human T-leukemia cells. Bfl-1S also significantly inhibited the cleavage of Bid, and of caspases 3 and 8 against STS treatment. These results indicate that Bfl-1S is a novel human Bcl-2 family member that possesses antiapoptotic function.

Exosomes: a new delivery system for tumor antigens in cancer immunotherapy.

Exosomes are small membrane vesicles that are released into the extracellular environment during fusion of multivesicular bodies with plasma membrane. Exosomes are secreted by various cell types including hematopoietic cells, normal epithelial cells and even some tumor cells. They are known to carry MHC class I, various costimulatory molecules and some tetraspanins. Recent studies have shown the potential of using native exosomes as immunologic stimulants. Here, we demonstrate a novel means of using exosomes engineered to express a specific tumor antigen to generate an immune response against tumors. We expressed a target tumor antigen, human MUC1 (hMUC1), in 2 MHC type-distinct mouse cell lines, CT26 and TA3HA. Analysis of exosomes purified from these cells revealed that exosomes contained the target MUC1 antigen on their surfaces as well as other well-described exosomal proteins, including Hsc70 and MHC class I molecules. In addition, both autologous and allogenic exosomes were able to stimulate the activation of immune cells and suppress hMUC1-expressing tumor growth in a MUC1-specific and dose-related manner. Therefore, these data suggest that exosomes can be engineered from tumor cell lines to deliver a target immunogen capable of inducing an effective immune response and that they may represent a new cell-free tumor vaccine.