Burkitt Lymphoma Specific Zinc Finger Protein ZNF385B Is Involved in Regulation of B Cell Apoptosis

Abstract 1584

Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) are common types of childhood mature B-cell non-Hodgkin's lymphoma (NHL). Both are postulated to be derived from GC B-cells but they are independent disease entities of NHL; however, they have overlapping morphologic and immunophenotypic features. A characteristic translocation t(8;14) of BL also occurs in 5 to 10 percent of cases of DLBCL. It is not uncommon to have difficulty in differentiating BL from DLBCL at initial diagnosis. Therefore, the latest WHO classification of tumors of the lymphoid tissue has recognized a category with overlapping features between BL and DLBCL. Considering this background, gene-expression profiling using microarray should provide an accurate, quantitative method for distinguishing the NHL. Actually, other researchers performed gene-expression profiling of mature B-cell lymphoma at all ages and showed that BL could be reliably distinguished from DLBCL. It has also been reported that DLBCL can be classified into two categories of patients with very different five-year overall survival rates on the basis of gene-expression profile.

In an attempt to investigate the molecules specifically expressed in each type of NHL, we performed the screening of genes expressed characteristically in each type of childhood hematologic malignancy by employing a microarray system using clinical specimens from pediatric patients, consisting of 3 patients with DLBCL, 16 with BL, 6 with BCP-ALL and 6 with T-ALL. Consistent with previous reports, CD40, EBI3, FGD6, LMO2, and SERPINA9 were over-expressed in DLBCL. In addition, over-expression of IL21R, STAT3, BCL6, CD58, JAK3 was observed in DLBCL. In BL, on the other hand, BMP7 and SOX11 were over-expressed as reported previously. Furthermore, we found that ZNF385B, BMP3, PEG10, MUC4, AICDA, SMAD1, C13orf15, CD24 were over-expressed in BL.

In these candidate genes, ZNF385B, also called ZNF533, was expressed in vast majority of BL cases as well as BL cell lines but not in other hematopoietic malignancies, including DLBCL, B-cell precursor ALL, and mature B-cell malignancies originate from another developmental stages of B cells. Therefore, we concluded that ZNF385B is a BL-specific zinc-finger protein and can be used as a diagnostic marker for BL.

Currently three isoforms (IFs) of ZNF385B have been identified and IF-1 is the longest transcript variant, which possesses 4 ZF domains, while IF-2/3 are shorter transcript variants with 3 ZF domains. Although the function and biological significance of this protein have not been clarified at all, ZNF385A has high homology with ZNF385B IF-2/3 is known to be involved in apoptosis regulation. Therefore, we intended to elucidate the functional role of ZNF385B in B cells.

First we analyzed intracellular localization of ZNF385B. When ZNF385B IF-1 proteins fused with EGFP at the N-terminus were expressed transiently in DLBCL BJAB cells, we observed the nuclear localization by obtaining fluorescent images.

Next, to examine the characteristics of ZNF385B, we established a protein inducible system in a tetracycline-dependent manner for both ZNF385B IF-1 and its deletion mutant ZNF385B-DEL corresponds to IF-2/3 lacking ZF domain in N-terminus in BJAB cells. Interestingly, ectopic expression of ZNF385B IF-1 induced up-regulation of PERP (p53 apoptosis effector related to PMP-22) and activation of caspase-3 and −8, resulting in apoptosis induction, whereas ZNF385B-DEL did not. Furthermore, ZNF385B-DEL inhibited apoptosis induced by CD20 cross-linking and BCR stimulation. Immunoprecipitation by anti-p53 antibody indicated the binding of ZNF385B isoform 1 with p53. Since PERP is known to be a p53 transcriptional target, these results suggest the involvement of ZNF385B in B-cell apoptosis by modulating p53 transactivation. Considering our observation of ZNF385B expression in a portion of cells in peripheral lymphoid organs, ZNF385B possibly is involved in the regulation of death and survival that specifically occurs in germinal center B cells.