Myeloid cell leukemia-1 (Mcl-1) protein is an anti-apoptotic Bcl-2 family protein that plays essential roles in multiple myeloma (MM) survival and drug resistance. In MM, it has been demonstrated that proteasome inhibition can trigger the accumulation of Mcl-1, which has been shown to confer MM cell resistance to bortezomib-induced lethality. However, the mechanisms involved in this unwanted Mcl-1 accumulation are still unclear. The aim of the present study was to determine whether the unwanted Mcl-1 accumulation could be induced by the unfolded protein response (UPR) and to elucidate the role of the endoplasmic reticulum stress response in regulating Mcl-1 expression. Using quantitative RT-PCR and Western blot, we found that the translation of activating transcription factor-4 (ATF4), an important effector of the UPR, was also greatly enhanced by proteasome inhibition. ChIP analysis further revealed that bortezomib stimulated binding of ATF4 to a regulatory site (at position −332 to −324) at the promoter of the Mcl-1 gene. Knocking down ATF4 was paralleled by down-regulation of Mcl-1 induction by bortezomib and significantly increased bortezomib-induced apoptosis. These data identify the UPR and, more specifically, its ATF4 branch as an important mechanism mediating up-regulation of Mcl-1 by proteasome inhibition.

AIM: To investigate the expression pattern of epithelial cell adhesion molecule (Ep-CAM) on normal and malignant colon tissues to evaluate its diagnostic and therapeutic significance. METHODS: cDNA encoding Ep-CAM extracellular domain was cloned by reverse transcription-polymerase chain reaction (RT-PCR) from excised malignant colon tissues and inserted into a glutathione S-transferase (GST)-tagged vector. Ep-CAM-GST fusion protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and purified with glutathione-sepharose. The Ep-CAM-GST fusion protein was mixed with Freund’s adjuvant and Balb/c mice were immunized with it. Sp2/0 myeloma cells were fused with the spleen cells of the immunized mice. After having selected by indirect ELISA, the anti-Ep-CAM monoclonal antibodies (MAbs) were generated and the corresponding ascites were obtained. Finally, the human colon carcinoma tissue array prepared from seventy individual patients was stained with the anti-Ep-CAM MAbs. RESULTS: The isolated Ep-CAM cDNA sequence was identical to the data in GenBank. The expressed fusion protein was almost soluble and had a molecular weight (MW) of 53 ku. Four MAbs against Ep-CAM were obtained and designated as FMU-Ep1, FMU-Ep2, FMU-Ep3 and FMU-Ep4 respectively. Among them, FMU-Ep4 could recognize the natural Ep-CAM on Colo205 and SW480 cells, and all of them could be used for immunohistochemical staining of tissue sections. It was found that Ep-CAM was distributed differently in normal and various malignant colon tissues, including squamous cell carcinoma, signet-ring cell carcinoma and adenocarcinoma. In normal colon gland epithelia, Ep-CAM antigen was mainly distributed on the basolateral membrane and in the region between the basolateral membrane and the cytoplastic part near the nuclei, whereas the expression pattern of colon malignancies was mainly on the whole surface of epithelia and the expression was much higher than the normal colon tissues. The staining pattern of tissue array showed in adenocarcinoma and papillary adenocarcinoma, and the expression of Ep-CAM was increased from grade I to grade III. CONCLUSION: MAbs against Ep-CAM might be useful for research on the structure and function of Ep-CAM and may have diagnostic and therapeutic value to various colon carcinomas.

HAb18G/CD147, a glycoprotein of the immunoglobulin super‐family (IgSF), is a T cell activation‐associated molecule. In this report, we demonstrated that HAb18G/CD147 expression on both activated CD4⁺ and CD8⁺ T cells was up‐regulated. In vitro cross‐linking of T cells with an anti‐HAb18G/CD147 monoclonal antibody (mAb) 5A12 inhibited T cells proliferation upon T cell receptor stimulation. Such co‐stimulation inhibited T cell proliferation by down‐regulating the expression of CD25 and interleukin‐2 (IL‐2), decreased production of IL‐4 but not interferon‐γ. Laser confocal imaging analysis indicated that HAb18G/CD147 was recruited to the immunological synapse (IS) during T cell activation; triggering HAb18G/CD147 on activated T cells by anti‐HAb18G/CD147 mAb 5A12 strongly dispersed the formation of the IS. Further functional studies showed that the ligation of HAb18G/CD147 with mAb 5A12 decreased the tyrosine phosphorylation and intracellular calcium mobilization levels of T cells. Through docking antibody–antigen interactions, we demonstrated that the function of mAb 5A12 is tightly dependent on its specificity of binding to N‐terminal domain I, which plays pivotal role in the oligomerization of HAb18G/CD147. Taken together, we provide evidence that HAb18G/CD147 could act as a co‐stimulatory receptor to negatively regulate T cell activation and is functionally linked to the formation of the IS.

To study the expression of N-myc Downstream Regulated Gene-2 (NDRG2) in prostatic carcinoma (PCA) tissue and in different PCA cell lines, and to investigate its clinical and pathological implicatio...

Optimal T-cell activation requires both an antigen-specific and a costimulatory signal. CD167 is a tyrosine kinase receptor for native type I collagen, its physiologic functions include matrix homeostasis and cell growth, adhesion, branching, and migration, but the specific role of CD167 in T cells has not yet been characterized. In this study, we found that CD167 expression on T cells was up-regulated after activation. Cooperation of CD167 engagement with suboptimal TCR/CD3 signals induced T-cell proliferation, enhanced expression of activation markers such as CD25 and CD69, elevated intracellular calcium mobilization and tyrosine phosphorylation, and introduced a bias toward a TH1/Tc1 immune response. Cooperation of CD167 engagement also enhanced mixed lymphocyte responses to alloantigens. Moreover, CD167 rapidly localized to the aggregated lipid rafts upon T-cell activation, this provided a molecular base for the signaling machinery of CD167. Together these findings, we demonstrate for the first time that CD167 could serve as a novel costimulatory receptor for T-cell activation.

Growth of solid tumors is often associated with the development of an immunosuppressive tumor microenvironment (TME). It has been suggested that the influence of the TME may extend beyond the local tumor and results in systemic immunosuppression. Here, we utilize two murine cancer models to explore the influence of solid tumors on the occurrence of alloreactivity-driven GvHD and graft-versus-solid tumor (GvT) effects following MHC-mismatched allogeneic bone marrow transplantation (allo-BMT). Melanoma- or colon carcinoma–bearing C57BL/6 mice did not develop GvHD after BMT even when the bone marrow inoculum was supplemented with donor-type splenocytes. This protection against GvHD required the presence of tumors because its resection prior to allo-BMT promptly resulted in development of GvHD. In addition, tumor-bearing mice given T-cell–depleted allo-BMT (allo-TCD-BMT) failed to develop GvHD and also showed significantly stronger GvT effects than mice given allo-BMT. The GvT effects in allo-TCD-BMT recipients were associated with profound changes in tumor-infiltrating cells compared with that in allo-BMT recipients, with significantly reduced donor-derived regulatory T cells (Treg), increased cytotoxic effector (IFNγhi) CD8 T cells, and increased M1 macrophages (iNOShi, arginaselo, and IL10lo); the use of macrophage-depleted bone marrow abrogated the GvT effects. Collectively, these results indicate that the presence of M1 macrophages may disrupt the generation of donor-type Treg cells so that the immunomodulatory effect of the TME can affect systemic immunity. Significance: These findings show that cells such as T cells or macrophages in the bone marrow inoculum may interfere with the systemic and local immune reactivity against tumors.