NOXA: A critical p53-independent determinant of specificity for proteasome inhibitor–mediated killing of melanoma cells. Tumors of C8161 melanoma cells injected with PBS did not contain detectable NOXA, but injection of bortezomib (2.5 mg/kg) did induce NOXA in these treated tumors ( B and C, proliferating RJ002L (B) and C8161 (C) melanoma cells were subjected to a mitochondrial isolation procedure before and after various time intervals following exposure to bortezomib (1 μmol/L). During the initial 6 hours of exposure, minimal changes in viability of the culture were observed, but prominent apoptosis was detected at 18- and 24-hour time points ( There was a significant (P < 0.01) reduction in tumor weight comparing PBS-injected tumor versus tumor injected with 1.25 mg/kg bortezomib, and no further reduction was apparent at 2.5 mg/kg dose. 4). RJ002L melanoma cells were plated in six-well plates at a density of 2 × 105 cells per well and transfection was accomplished using Oligofectamine in Opti-MEM medium following the protocol of the manufacturer (Invitrogen). Mouse IgG or IgM was captured from sera diluted 1:50 in flow buffer before injection. Mean and SEM are shown (*P < 0,05, **P < .005; Student t test). The factors responsible for affinity maturation of B cell clones in the germinal center (GC) have been well established but selection mechanisms that allow clones to enter the GC are largely unknown. Mean and SEM are shown (*P < .05, **P < .005, and ***P < .0001; Student t test). A novel biologically based treatment approach uses agents targeting the proteasome, a universal and broadly active cellular complex responsible for regulating protein degradation and maintenance of normal cell function ( Noxa ablation provides a survival advantage for activated B cells in vivo. Histograms of FB and GC B cells in the spleen, stained intracellularly for BrdU are shown (n = 5 per genotype). After 1 week, mice were assigned to each of the following tumor bearing groups (5 mice/group) and injected with either (a) PBS as control, (b) bortezomib −1.25 mg/kg, or (c) bortezomib −2.5 mg/kg. We now show that apoptosis involving Noxa sets an important threshold for expanding B cells, which is distinct from hypermutation-driven affinity maturation. Asterisks, statistically significant differences between untreated and treated melanoma cells. Noxa, a proapoptotic member of the Bcl-2 protein family, triggers Bax-mediated mitochondrial dysfunction via indirect inhibition of Bcl-2 protein family members (Oda et al., 2000) . However, addition of either MG-132 or bortezomib still triggered prominent NOXA induction after serum withdrawal (Supplementary Fig. B220+ (yellow) and PNA+ (purple) cells were stained. As of yet it is unclear how exactly BCR affinity controls the balance between pro and antiapoptotic molecules, but a plausible parallel can be drawn between B cells and T cells. Complete but curtailed T-cell response to very low-affinity antigen. Blood 2012; 119 (6): 1440–1449. At the peak of the GC response after PE/Alum immunization, antigen-specific B cells were isolated and analyzed for clonal diversity within the VH186.229  family (supplemental Figure 8B for spectratyping). This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Leucine zipper-Apo2Ligand/tumor necrosis factor–like apoptosis-inducing ligand (i.e., LZ-TRAIL) was obtained from Genentech, and used as previously described ( As substrates for the proteasome include regulatory proteins involved in cell cycle progression, apoptosis, and angiogenesis, targeting the proteasome represents an attractive therapeutic approach for cancer treatment ( Flow buffer was PBS; 0.005% P20 at 30 μL/min. Targeting molecular pathways that regulate diverse cellular responses has emerged as a promising therapeutic strategy ( After immunization or influenza infection, Noxa−/− mice display enlarged GCs, in which B cells with reduced antigen affinity accumulate. Using RJ002L melanoma cells (left), a kinetic analysis was done in which either control antisense oligonucleotide or NOXA antisense oligonucleotide–pretreated cells were exposed to bortezomib (1 μmol/L; 3, 6, 18, and 24 hours). 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. Bortezomib triggered significant apoptosis in all three melanoma cells (P < 0.05). Defining a dose-response effect for proteasome inhibitors such as bortezomib using normal proliferating melanocytes or early-passage melanoma cells has not been well documented. Fig. Cells were stimulated in RPMI with 10% fetal calf serum (FCS) with soluble anti-IgM (HB88) plus lipopolysaccharide (LPS; Escherichia coli 0111:B4, Sigma-Aldrich). B, proliferating melanoma cell lines (RJ002L, MUM2B, and C8161) were exposed (18 hours) to MG-132 or lactacystin as noted in (A), revealing accumulation of ubiquitinated isoform p53 in RJ002L and C8161 cells, but no p53 was detected before or after treatment in MUM2B cells carrying homozygous p53 mutations. This study aimed to elucidate the expression and role of Noxa in salivary gland ACC. When the C8161 melanoma cells, which display a lower apoptotic response to proteasome inhibitors compared with RJ002L cells were studied ( A promising new oncologic approach uses molecularly targeted therapeutics that overcomes apoptotic resistance and, at the same time, achieves tumor selectivity. The reduced number of apoptotic CD38dimGL7+ cells in cultures with Noxa−/− cells correlated with a significant increase in IgG1 secretion (Figure 2D). 34, The withdrawal of growth factors in the melanoma cells maintained in a serum-free environment indicates that proteasome inhibitors can induce NOXA and apoptosis in nonproliferating cells in an equivalent fashion as rapidly proliferating melanoma cells (Supplementary Fig. After confirming overexpression by Western blot analysis (insets), the apoptotic susceptibility to either bortezomib (1 μmol/L) or LZ-TRAIL (100 ng/mL) was determined. 4-1BB promotes the survival of CD8+ T lymphocytes by increasing expression of Bcl-xL and Bfl-1. Antibody levels were determined using biotinylated detection antibodies (Southern Biotech), using isotype specific antibodies (Southern Biotech) as standards. (A) Representative FACS plots of activated B cells. Fig. and R.A.W.v.L. Fig. Again, TNP-specific IgM levels were similar between Noxa−/− and WT mice, but after secondary immunization, IgG1 and especially IgG2b levels were significantly reduced in Noxa−/− sera compared with WT (supplemental Figure 9). A, proliferating RJ002L melanoma cells were infected with either empty retrovirus (i.e., linker) or with a FADD DN–containing retrovirus or a Bcl-xL–containing retrovirus. As Bcl-2 family members can significantly control mitochondrial integrity, thereby contributing to the apoptosis resistance to conventional chemotherapeutic agents, it is remarkable that proteasome inhibitors can overcome the relatively high levels of survival factors present within melanoma cells ( The full text of this article hosted at iucr.org is unavailable due to technical difficulties. 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. From the perspective of vaccine design, an important finding of our study is that Noxa does not only affect primary B-cell responses, but also greatly impairs recall responses. Nonetheless, these data indicate an important role for NOXA induction in mediating the killing of melanoma cells achieved by the use of proteasome inhibitors. Together, these findings indicate that antigenic stimulation in the absence of Noxa results in the generation of low affinity IgG+ B cells and plasma cells, possibly as a result of persistent survival of low-affinity B-cell clones. Knockdown of p53 levels using p53 siRNA not only reduced constitutive and bortezomib-inducible p53 protein levels but also significantly reduced bortezomib-induced MDM2 and GADD45 levels accompanied by only a slight reduction in bortezomib-induced NOXA levels.