RT is a CRUK Senior Clinical Research Fellow. The online version of this article contains a supplementary appendix. Authorship and Disclosures LM and GR designed research, performed experiments, analyzed data and wrote the paper. secretory stage of differentiation is observed in the presence of defective expression.9,10 XBP1 is a key component of the unfolded protein response (UPR).11 This stress response triggered by accumulation of unfolded protein in the ER, balances adaptive and apoptotic fates.12 During the UPR splicing of 26 nucleotides from mRNA results in a reading frame shift, giving rise to an active form of XBP1 XBP1(S).13,14 The essential role for in PC differentiation, and immunoglobulin synthesis reflects a requirement for XBP1(S)15,16 and expansion of the secretory apparatus.8 XBP1(S) has eluded direct assessment in human tissue, a critical issue for our understanding of the UPR, humoral immunity and malignancies derived (S,R,S)-AHPC hydrochloride from differentiating B-cells and PCs. Design and Methods XBP1(S) monoclonal antibody splicing and Western blotting were as described.19,20 A Bond automated system (Leica) was used for XBP1(S) immunostaining of TMA sections. Double immunoenzymatic labeling was as described.6 In all immunostained paraffin sections, PCs provided an internal positive control. Multi-color immunofluoresence (MCIF) was performed on human tonsil tissue as described21 (mRNA splicing in U937 cells undergoing an UPR after treatment with dithiothreitol or thapsigargin.19 The expected correlation was observed PAX3 with detection of a specific band at 54 kDa by Western blot following mRNA splicing (Figure 1A). Specificity was further (S,R,S)-AHPC hydrochloride confirmed by detection of a specific band in cells transfected with XBP1(S) expression vector and myeloma cell lines (Figure 1B). The OCI-LY3 cell line was used as a negative control. Open in a separate window Figure 1. Characterization of anti-XBP1(S) monoclonal antibody and XBP1(S) expression patterns in normal tissue. (A) XBP1(S) protein is detected during the UPR following induced XBP1 mRNA splicing. U937 cells were left untreated or subject to an UPR with dithiothreitol (DTT) or thapsigargin (Tg) for indicated times, RT-PCR for XBP1 mRNA splicing (top) and Western blot with anti-XBP1(S) or anti-Actin monoclonal antibodies (bottom). In addition to the specific band at 54kDa, a non-specific band at 50kDa was detected in U937 cells (in all B-cell subsets, while splicing and active ER stress. Next the relationship of XBP1(S) to PAX5 and BLIMP1 expression was directly examined. As expected, XBP1(S) was predominantly co-expressed with BLIMP1 (S,R,S)-AHPC hydrochloride in the absence of PAX5. Occasional cells weakly co-expressed PAX5 with both BLIMP1 and XBP1(S). Significantly, a rare but distinct population of cells co-expressed XBP1(S) and PAX5 in the absence of BLIMP1 (Figure 1E and nor loss of is essential to allow XBP1(S) expression in B-cells iexpression.9 Whether such XBP1(S) expressing B cells survive to give rise to functional PCs is uncertain. These patterns are paralleled in DLBCL in which XBP1(S) is restricted to the plasmablastic sub-type. Moreover, our results further delineate heterogeneity amongst these neoplasms. XBP1(S) expression identifies disease with advanced PC differentiation, which may be more closely related to myeloma than DLBCL, 24 raising the question of alternate treatment choices for this sub-group. In the UPR, adaptive and apoptotic pathways are finely balanced.12 splicing mediates a major adaptive pathway and identifies cells undergoing an active stress response. Our antibody provides a direct practical tool for assessing this in patient samples. Existing and novel treatment strategies aimed at myeloma and other secretory tumors in part act through destabilizing the balance of the UPR.25 We propose that application of our antibody in the diagnostic process may help predict response to such treatments. Supplementary Material [Maestre et al. – Supplementary Appendix] Click here to view. Acknowledgments the authors express their gratitude to all members of the Tumor Bank Network of the CNIO for their technical contribution and assistance. Manuscript received September 25, 2008. Revised version arrived on November 5, 2008. Manuscript accepted on November 10, 2008. Footnotes Funding: this work was supported by Ministerio de Sanidad y Consumo (G03/179, PI051623, PI052800, CIBER-ER) and Ministerio de Ciencia y Tecnologa. (SAF2005- 00221, BIO 2005-01078). RT is a CRUK Senior Clinical Research Fellow. The online version of this article contains a supplementary appendix..