No differences in proliferation were observed between WT and Noxa−/− for any fraction of B cells (Figure 2C, supplemental Figure 2A). (C) Quantification of the ratio of Noxa−/−/WT cells in the FB versus GC B cell fractions as shown in panel B. Noxa−/− cells are enriched within the antigen-specific pool. In this report, five melanoma cell lines and a myeloma cell line are treated with three different proteasome inhibitors (MG-132, lactacystin, and bortezomib), and the mechanism underlying the apoptotic pathway is defined. Interestingly, Noxa-deficient animals showed a reduction in GC B cells that bound PE with high intensity (Figure 4A-B). Again, no significant differences in hypermutation frequencies were found (data not shown). Proteasome inhibitors induce apoptosis in cultured melanoma cells but not in normal melanocytes. The induction of Noxa in activated B cells suggests that the inability of Noxa−/− mice to generate high-affinity antibody responses is B-cell intrinsic. Binding to TNP-BSA of captured antibodies was tested by duplicate injections of 0, 5, 10, 50, 200, and 500nM TNP-BSA. 22); and inactivation of effector caspases regulated by apoptotic protease activating factor-1 ( WT (CD45.2) recipients (n = 10) were lethally irradiated and transferred with mixed WT (CD45.2) and Noxa−/− (CD45.1) BM in a 4:1 ratio. The neighbor-joining method: a new method for reconstructing phylogenetic trees. 55) as well as a therapeutic perspective ( Germinal centers in human lymph nodes contain reactivated memory B cells. Thus, differences in the timing of caspase activation can be appreciated when various melanoma cell lines are exposed to proteasome inhibitors, although NOXA induction was one of the earliest detectible changes in all melanoma cell lines tested. On reinfection with identical pathogens, clearance is primarily executed by high-affinity antibodies.44  Thus, to reestablish itself in a host, there will be selection for pathogens that have acquired mutations to avoid recognition by neutralizing antibodies. Fig. NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

However, knockdown of Noxa attenuated the apoptosis … To confirm and extend these findings, Northern blot analysis was initially done using RNA extracted from C8161 melanoma cells before and after (2, 4, 6, and 8 hours) bortezomib (1 μmol/L) exposure ( Defining the mechanism of apoptotic action for proteasome inhibitors has been previously elusive, and the current results clearly highlight a key role for NOXA in triggering apoptosis in melanoma and myeloma cells. Fig. and you may need to create a new Wiley Online Library account.

On day 20, mice were euthanized and tumors dissected from surrounding tissue and weighed. (B) Histograms of FB cells and GC B cells in BM chimeric mice 12 days after PE/Alum immunization. Enter your email address below and we will send you your username, If the address matches an existing account you will receive an email with instructions to retrieve your username. The efficiency of humoral immune responses depends on the selective outgrowth of B cells and plasmacells that produce high affinity antibodies. The 18-hour response of a myeloma cell line to proteasome inhibitors revealed a concentration-dependent induction of NOXA by bortezomib (0.01-10 μmol/L) and by MG-132 (10 μmol/L), accompanied by activation of caspases 9, 3, and 8 and poly(ADP)-ribose polymerase cleavage (

3A We now show that apoptosis involving Noxa sets an important threshold for expanding B cells, which is distinct from hypermutation-driven affinity maturation. Fig. Thus, normal elimination of low affinity cells favors outgrowth of the remaining high-affinity clones, and this is mandatory for the generation of proper antibody responses. Qin, B. Nickoloff, and L. Miele, unpublished observations. C, proteasome inhibitors MG-132 (10 μmol/L) and lactacystin (10 μmol/L) trigger apoptosis in melanoma cell lines with minimal killing of proliferating melanocytes (24-hour continuous exposure).

Studies using B cells from BCR transgenic mice showed that under noncompetitive conditions BCR affinity had little effect on the number of cells contributing to the overall response, nor on the amount of antibody produced. Fig. These proteins include opposing factions of antiapoptotic and proapoptotic members. designed and performed experiments, analyzed data, and corrected the paper; A.M.d.B. These data indicate proteasome inhibitors are unable to trigger NOXA induction in normal melanocytes, and that p53 is not absolutely required for NOXA induction. Unlimited viewing of the article/chapter PDF and any associated supplements and figures. When C57BL/6 mice are immunized with nitrophenylacetyl linked to chicken γglobulin (NP-CGG), GC cells primarily use the VH186.2 gene and a large fraction of these cells displays an affinity-enhancing (W33L) mutation.28  When Noxa-deficient mice were immunized with NP-CGG, no difference in the average number of mutations per sequence was observed compared with WT animals. This fraction represents the most activated B-cell subset, as demonstrated by the high number of dividing cells compared with CD38+ B cells (Figure 2C, supplemental Figure 2A). NOXA is a pro-apoptotic member of the BCL-2 family, which mediates induction of apoptosis via activation of mitochondria and the intrinsic apoptosis signaling pathway. Cells were washed with PBS and transfection mix (1 mL) was added. Next, average antigen affinity of the immune sera was measured by Biacore analysis. However, addition of either MG-132 or bortezomib still triggered prominent NOXA induction after serum withdrawal (Supplementary Fig. 40– This decrease was especially apparent under limiting concentrations of PE, implying that high-affinity PE-specific B cells were specifically absent in Noxa−/− mice (supplemental Figure 4A). Cells were stimulated with anti-IgM/LPS alone (−IL4), or in combination with IL-4 (+IL4). Antigen-specific plasma cell numbers were again not reduced, showing that there is no defect in differentiation, but in the amount of high-affinity antibodies produced (Figure 7C). We chose to further investigate Noxa, because the ablation of Noxa alters the selection process of antigen-reactive T cells, and moreover conditional deletion of its antagonist Mcl-1 in B cells blocks GC formation and memory B-cell generation.17. No differences in activation markers, such as MHC-II induction (supplemental Figure 5A) were observed. Noxa expression in GC B cells was lower than in vitro activated cells, suggesting that Noxa expression in vivo may be higher early after B-cell activation. ). 3E). As previously reported in nonmelanoma cells, Bcl-xL overexpression reduced LZ-TRAIL–mediated apoptosis in RJ002L cells ( Products were run through Genescan and analyzed with GeneMapper (ABI) for spectratyping. Kinetics of NOXA induction and caspase processing in proteasome inhibitor–mediated apoptosis of melanoma cells. (B) FACS plots of B220+CD38dimGL7+ cells stained with annexin V and DNA dye after 2 days of stimulation with anti-IgM/LPS and IL-4. Molecular processes regulating selection of B cells in the GC have been studied extensively,2-5  but the mechanisms which control the access of B cells to the GC are largely unclear. Both WT and Noxa−/− cells were capable of generating PE-specific B cells. 29).