Genomic and proteomic analysis of NOXA in B-NHL. (A) Array CGH analysis of Elijah cells shows homozygous deletion of chromosome band 18q21.3. Within the region, NOXA gene expression shows null expression in the Lymphochip microarrays. (B) Western blot and QRT-PCR analyses show expression variation of NOXA in different B-NHL cell lines; in Elijah, NOXA protein expression is absent according to DNA and RNA data. (C) NOXA gene sequence changes in B-NHL cell lines; the nucleotide positions of NOXA are based on GenBank accession sequence NM_021127. Sequencing analysis of the nondeleted alleles reveals a splice site mutation in Karpas 1106 and a missense mutation in the BH3-domain in Namalwa. (D) Immunohistochemistry analysis for NOXA protein on tissue microarrays from 367 biopsies from patients with different B-NHL. In tonsils, NOXA was shown to be expressed only in a fraction of germinal center B cells, whereas mantle, marginal, and interfollicular lymphocytes were negative (i). In lymphoma samples, NOXA protein expression was restricted to diffuse large B-cell lymphoma (32 of 67, 48%) and in a subset of follicular lymphoma (20 of 103, 19%), whereas MCL, splenic marginal zone lymphoma, and B-cell chronic lymphocytic leukemia samples were mostly negative. In the figure, lymph node biopsies from patients with diffuse large B-cell lymphoma (ii), follicular lymphoma (iii), and splenic marginal zone lymphoma (iv) are shown. (E) Proteasome inhibitor, bortezomib (PS-341) in B-NHL cell lines with and without NOXA mutation. Cell lines were incubated for 24 and 48 hours with increasing concentrations of bortezomib. Apoptosis was calculated by measuring phosphatidylserine (PS) externalization as determined using Annexin V. The results correlated with loss of mitochondrial membrane potential (data not shown). Both the induction and rate of apoptosis were comparable among all the cell lines, irrespective of the presence of genetic alterations in NOXA.