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PDF file - 23K, List of Primers

Supplementary Figure 1 from A Novel Isoform of TUCAN Is Overexpressed in Human Cancer Tissues and Suppresses Both Caspase-8– and Caspase-9–Mediated Apoptosis

Caspase-associated recruitment domains (CARD) are protein-protein interaction modules found extensively in proteins that play important roles in apoptosis. One of the CARD-containing proteins, TUCAN (CARD8), was reported previously as an antiapoptotic protein with a molecular weight of 48 kDa, which was up-regulated in colon cancer cells. We identified a novel isoform of TUCAN with a molecular weight of 54 kDa. The new variant of TUCAN, termed TUCAN-54, was expressed in gastric, colon, and breast cancer tissues but was barely detected in normal noncancerous tissues, whereas 48-kDa TUCAN was detected in tumor tissues and noncancerous tissues. To know the function of TUCAN-54 in the apoptosis of cancer cells, TUCAN-54 was overexpressed in tumor cells by gene transfection. Its overexpression inhibited pro-caspase-9 activation, leading to the suppression of the cell death induced by a protein kinase inhibitor, staurosporine, or a chemotherapeutic reagent, etoposide (VP-16). In contrast, specific small interfering RNA–mediated suppression of TUCAN-54 expression in tumor cells increased the VP-16–induced cell death rate, indicating that expression of TUCAN-54 might be associated with chemoresistance of tumor cells. In addition, it inhibited caspase-8 activation as well, thereby suppressing Fas-induced cell death. It was revealed that Fas-associated death domain was physically associated with TUCAN-54 but not with 48-kDa TUCAN. Thus, TUCAN-54 might be a novel tumor-specific antiapoptotic molecule expressed in a variety of human cancer tissues, which might aggravate malignant potential of cancer cells, such as chemoresistance and immunoresistance.

PDF file - 68K, Isolation of CSCs/CICs from human RCC cells

PDF file - 74K, RT-PCR analysis

PDF file - 91K, SP analysis of DNAJB8- transduced cells

PDF file - 67K, Western blot analysis

PDF file - 142K, Protein expression of DNAJB8 in SP and MP cells

PDF file - 92K, Serine-rich region of DNAJB8 has role for induction of SP cells

Cancer stem–like cells (CSC) are a small population of cancer cells with superior tumor initiating, self-renewal, and differentiation properties. In this study, we show that the cancer-testis antigen and HSP40 family member DNAJB8 contributes to the CSC phenotype in renal cell carcinoma (RCC). DNAJB8 overexpression increased the percentage of side population (SP) cells representing CSCs in RCC cells, enhancing their tumor-initiating ability. Conversely, attenuation of DNAJB8 decreased SP cells and reduced tumor-initiating ability. The utility of DNAJB8 as an immunologic target was established in DNA vaccination experiments. Compared with immunization with the tumor-associated antigen survivin, which was expressed in both CSCs and non-CSCs in RCC, immunization with Dnajb8 expression plasmids yielded stronger antitumor effects. Together, our findings suggest that DNAJB8 plays a role in CSC maintenance and that it offers a candidate for CSC-targeting immunotherapy in RCC. Cancer Res; 72(11); 2844–54. ©2012 AACR.

Heat shock proteins (HSPs) such as Hsp70, gp96, and Hsp90 have been shown to elicit intriguing, efficient CTL responses by cross-presentation via an as yet entirely unknown mechanism. Oxygen-regulated protein 150 (ORP150), also known as grp170, is an endoplasmic reticulum-resident HSP and is up-regulated by hypoxia. It has been demonstrated that ORP150 binds tumor-associated Ag peptides within cancer cells. Immunization with an ORP150-tumor Ag complex has been shown to generate tumor-specific CTLs. Most recently, it has been shown that exogenous ORP150 induces cross-presentation of a chaperoned Ag, thereby stimulating Ag-specific CTLs. However, the mechanism underlying this efficient cross-presentation is still unsolved. In this study, we show that the ORP150-precursor peptide complex can elicit CTL response through cross-presentation as well as the CD4+ T cell response by dendritic cells. Furthermore, we observed that the internalized ORP150-peptide complex, but not OVA protein, which was not cross-presented, was sorted to the Rab5+, EEA1+ static early endosome, followed by translocation to a recycling endosome, where the ORP150-chaperoned peptide was processed and bound to MHC class I molecules. Moreover, we observed that immunization of mice with ORP150-peptide complexes elicited strong peptide-specific CTLs and antitumor effects in vivo. Our data indicate that targeting of the Ag to a “static” early endosme by ORP150 is required for the efficient cross-presentation.

A glutamine synthetase I family protein, Lengsin, was previously identified as a novel lens-specific transcript in the vertebrate eye. In this report, we show for the first time that Lengsin is a novel tumor-associated antigen expressed ectopically in lung cancer. Interestingly, a novel spliced form of human Lengsin termed 'splicing variant 4', gaining exon 3 that codes extra 63 amino acids, is the dominant transcript form in lung cancer cells. Lengsin mRNA could be detected in 7 of 12 (58%) lung cancer cell lines and 7 of 7 (100%) surgically resected lung cancer tissues. On the other hand, Lengsin transcripts could not be detected in normal major tissues or in other cancer cell lines, including melanoma, colorectal carcinoma, breast carcinoma and hepatocellular carcinoma. In addition, knockdown of Lengsin mRNA with RNAi caused cell death and a decrease of cell viability, suggesting that Lengsin has some essential role in cell survival. Since the lens is an immune-privileged site, we regard Lengsin as a highly immunogenic cancer antigen. Anti-Lengsin autoantibodies were detectable in sera of lung cancer patients, although these patients did not show any lens-related disturbances. Hence, Lengsin splicing variant 4 might be an immunogenic lung cancer-specific antigen that is suitable as a diagnostic marker and for molecular targeting therapy, including immunotherapy.

70-kDa heat shock protein family is a molecular chaperone that binds to a variety of client proteins and peptides in the cytoplasm. Several studies have revealed binding motifs between 70-kDa heat shock protein family and cytoplasmic proteins by conventional techniques such as phage display library screening. However, little is known about the binding motif based on kinetic parameters determined by surface plasmon resonance analysis. We investigated the major inducible cytosolic 70-kDa heat shock protein (Hsp70)-binding motif with the human leukocyte antigen B*2702-derived peptide Bw4 (RENLRIALRY) by using a Biacore system based on surface plasmon resonance analysis. The K(D) value of Hsp70-Bw4 interaction was 1.8 x 10(-6) m. Analyses with truncated Bw4 variant peptides showed the binding motif of Hsp70 to be seven residues, LRIALRY. To further study the characteristics of this motif, 126 peptides derived from Bw4, each with single amino acid substitution, were synthesized and analyzed for Hsp70 binding affinity. Interestingly, the Hsp70 binding affinity was abrogated when the residues were substituted for by acidic (Asp and Glu) ones at any position. In contrast, if the substitute residue was aromatic (Trp, Tyr, and Phe) or an Arg residue at any position, Hsp70 binding affinity was maintained. Thus, this study presents a new binding motif between Hsp70 and peptides derived from the natural protein human leukocyte antigen B*2702 and may also elucidate some characteristics of the Hsp70 binding characteristic, enhancing our understanding of Hsp70-binding determinants that may influence diverse cellular and physiological processes.

Several mechanisms have been described by which malignant cells escape from the immune system and/or chemotherapy. Recently we isolated TUCAN-54 gene that is over-expressed in a wide variety of epithelial cancers but not in normal counterparts. TUCAN-54 inhibited both death receptor-mediated and mitochondria/cytochrome C-mediated apoptosis. Moreover, down-regulation of TUCAN-54 in colon cancer cells by siRNA treatment could increase the sensitivity of the cells to an anti-cancer drug. Finally, we revealed that Fas-associated death domain protein (FADD) was physically associated with TUCAN-54, but not with TUCAN-48. Thus, TUCAN-54 might be a novel tumor-specific anti-apoptotic molecule, which might aggravate malignant potential of cancer cells, such as immunoresistance and chemoresistance.

Survivin, a member of the inhibitor of apoptosis protein family, is widely expressed in a variety of human cancer tissues. Survivin inhibits activation of caspases, and its overexpression can lead to resistance to apoptotic stimuli. In this study, survivin protein expression was assessed by immunohistochemical staining of 195 invasive breast cancer specimens. Overall, 79.5% of the tumors were positive for survivin. The expression of epidermal growth factor receptor (EGFR) family, human epidermal growth factor receptor 2 (HER2) and EGFR, was also examined in 53 cases, and consequently, it was indicated that survivin positivity might be correlated with the coexpression of HER2 and EGFR. To clarify the regulatory mechanism of survivin expression in breast cancer cells, the effect of HER2 and/or EGFR expression on the survivin levels was examined. It was revealed that the survivin protein level was up-regulated by the coexpression of HER2 and EGFR, leading to the increased resistance against etoposide-induced apoptosis in breast cancer cells. Conversely, survivin levels and apoptosis resistance were decreased when cells were treated with HER2-specific inhibitor, Herceptin. Although Herceptin could down-regulate both phosphatidylinositol 3-kinase (PI3K)/AKT signal and mitogen-activated protein/extracellular signal-related kinase (ERK) kinase 1 (MEK1)/ERK signal in HER2-positive breast cancer cells, PI3K-specific inhibitor but not MEK1-specific inhibitor could decrease the survivin levels. The present study clarified the regulatory mechanism of HER2 in the expression of survivin protein in breast cancer cells.

Caspase-associated recruitment domains (CARD) are protein-protein interaction modules found extensively in proteins that play important roles in apoptosis. One of the CARD-containing proteins, TUCAN (CARD8), was reported previously as an antiapoptotic protein with a molecular weight of 48 kDa, which was up-regulated in colon cancer cells. We identified a novel isoform of TUCAN with a molecular weight of 54 kDa. The new variant of TUCAN, termed TUCAN-54, was expressed in gastric, colon, and breast cancer tissues but was barely detected in normal noncancerous tissues, whereas 48-kDa TUCAN was detected in tumor tissues and noncancerous tissues. To know the function of TUCAN-54 in the apoptosis of cancer cells, TUCAN-54 was overexpressed in tumor cells by gene transfection. Its overexpression inhibited pro-caspase-9 activation, leading to the suppression of the cell death induced by a protein kinase inhibitor, staurosporine, or a chemotherapeutic reagent, etoposide (VP-16). In contrast, specific small interfering RNA–mediated suppression of TUCAN-54 expression in tumor cells increased the VP-16–induced cell death rate, indicating that expression of TUCAN-54 might be associated with chemoresistance of tumor cells. In addition, it inhibited caspase-8 activation as well, thereby suppressing Fas-induced cell death. It was revealed that Fas-associated death domain was physically associated with TUCAN-54 but not with 48-kDa TUCAN. Thus, TUCAN-54 might be a novel tumor-specific antiapoptotic molecule expressed in a variety of human cancer tissues, which might aggravate malignant potential of cancer cells, such as chemoresistance and immunoresistance.

CD8+ CTLs have an essential role in immune response against tumor. Although an increasing number of tumor-associated antigens that can be recognized by CTLs have been identified from human tumors, a limited number of tumor-associated antigens is known in lung cancer. In addition, because some of them are expressed in noncancerous tissues, there exist limitations in their application to tumor immunotherapy. Livin/ML-IAP is one of recently identified inhibitor of apoptosis protein (IAP) family, which is overexpressed in melanoma cells. In this report, we show that Livin/ML-IAP is aberrantly expressed in many lung cancer cell lines and primary lung cancer tissues, whereas it is not detectable in normal tissues, including lung by reverse transcription-PCR methods. To identify HLA-A24-restricted T-cell epitopes of Livin/ML-IAP, eight peptides were selected from the amino acid sequence of this protein and screened for their binding affinity to HLA-A24. It was revealed that Livin7 peptide (amino acid sequence, KWFPSCQFLL) had the highest affinity to HLA-A24. By stimulating peripheral blood lymphocytes of HLA-A24-positive lung cancer patients with Livin7 peptide in vitro, the peptide-specific CTLs were successfully induced from four of five patients with Livin/ML-IAP-positive lung cancer but not from any of four patients without Livin/ML-IAP expression in their cancer tissues. Furthermore, the CTLs induced by Livin7 peptide showed cytotoxicity against Livin/ML-IAP+ lung cancer cell lines in an HLA-A24-restricted manner. Our data suggest that Livin/ML-IAP may be an excellent target antigen in immunotherapy for lung cancer and Livin7 peptide may serve as a potent peptide vaccine for HLA-A*2402+/Livin+ lung cancer patients.

To investigate the effects of anchor substitutions in SYT-SSX junction peptide, an HLA-A24 anchor residue (position 9) of the SYT-SSX B peptide (GYDQIMPKK) was substituted to more favorable residues according to the HLA-A24-binding motif. Among four substitutes constructed, a substitute with isoleucine (termed K9I peptide) most apparently enhanced the affinity for HLA-A24 molecule. Subsequent in vitro CTL induction analysis using PBMCs of 15 HLA-A24+ synovial sarcoma patients revealed that the original B peptide allowed to induce synovial sarcoma-specific CTLs from 7 patients (47%), whereas such CTLs were inducible from 12 patients (80%) with K9I peptide. Moreover, the extent of cytotoxicity against HLA-A24+ synovial sarcoma cell lines was higher in K9I peptide-induced CTLs than B peptide-induced CTLs. Influence of anchor substitution on peptide/TCR interaction was evaluated by cytotoxicity assays against autologous cells and tetramer analysis. CTLs induced from a synovial sarcoma patient using K9I peptide did not lyse autologous PHA blasts or EBV-infected B cells. In vitro stimulations of PBMCs from 5 HLA-A24+ synovial sarcoma patients with K9I peptide increased the frequency of T cells reacting with both HLA-A24/K9I peptide tetramer and HLA-A24/B peptide tetramer. In contrast, the frequency of T cells reacting with HLA/HIV-derived peptide tetramer remained low. These findings support the validity in design of anchor residue substitution in SYT-SSX fusion gene-derived peptide, and provide a potential clue to the current stagnation in vaccination trials of fusion gene-derived natural junction peptides.

The nature of target molecules of natural killer (NK) cell-mediated lysis remains to be elucidated. As we previously reported, mAb 109 recognizes one of the tumor-associated antigens, designated as 109 antigen (Ag), expressed on the cell surface of rat fibrosarcomas W31 and W14, which are transformants of WFB (rat fetal fibroblast cell line) with H-ras oncogene. 109Ag was thought to be a target structure of NK cells since mAb 109 inhibited NK cell-mediated lysis against W31 and W14. Here, we demonstrate by molecular cloning that 109Ag is identical to rat CD44. Immunoprecipitation and immunoblotting studies also showed that mAb 109 and anti-rat CD44 mAb OX-50 recognize the same protein of W31 cell lysates with an 86 kDa molecular size. CD44 was suggested to be a target structure of NK cell-mediated lysis; however, rat CD44 cDNA transfection alone into CD44 null cell lines did not result in up-regulation of target cell susceptibility to NK cell-mediated lysis. Our results therefore indicated that CD44 may play a crucial role as one of the target structures in our rat fibrosarcoma system though the cell surface expression of CD44 alone does not affect NK susceptibility of the target cells.

We established a monoclonal antibody, 3G12 (IgG1), with antiproliferative effects on a human T-cell leukaemia cell line, SUP-T13. Among haematolymphoid cell lines, 3G12 reacted with most T-cell lines, Epstein-Barr transformed B-cell lines, some myelomonocytic cell lines and, most strongly with an anaplastic large cell lymphoma (ALCL) cell line, Karpas 299, The cell panel reactive with 3G12 was similar, but not identical, to that of the anti-CD30 antibody Ber-H2. 3G12, induced Fas-independent apoptosis in SUP-T13 and it also induced growth-inhibition in a limited number of other cell lines, but not Karpas 299, Immunohistochemical studies on paraffin-embedded tissue specimens demonstrated that 3G12 reacted with most CD30-positive ALCL cases and some T-cell lymphomas and some Hodgkin's lymphomas, but not with B-cell lymphomas or non-haematogeneic tumours. The immunoprecipitation study with 3G12 demonstrated a major band of 200 kD and a minor band of 100 kD, which were different from CD30, Thus 3G12. defines a novel antigen that shares a similarity to CD30 in terms of distribution among haemopoietic cells. The data suggest that the 3G12-defined antigen, designated Hal-l. is important as a marker for ALCL and may play a role in its pathogenesis.

Hydrogen peroxide-inducible clone (Hic)-5 is induced during the senescent process in human fibroblasts, and the overexpression of Hic-5 induces a senescence-like phenotype. Structurally, Hic-5 and paxillin, a 68-kDa cytoskeletal protein, share homology such as the LD motifs in the N-terminal half and the LIM domains in the C-terminal half. Here we show that Hic-5 binds to focal adhesion kinase (FAK) by its N-terminal domain, and is localized to focal adhesions by its C-terminal LIM domains. However, Hic-5 is not tyrosine phosphorylated either by the coexpressed FAK in COS cells or by integrin stimulation in 293T cells. Furthermore, overexpression of Hic-5 results in a decreased tyrosine phosphorylation of paxillin. These findings suggest that putative functions of Hic-5 are the recruitment of FAK to focal adhesions and a competitive inhibition of tyrosine phosphorylation of paxillin.

Integrin-ligand binding induces the tyrosine phosphorylation of various proteins including focal adhesion kinase (pp125FAK) and Crk-associated substrate (Cas). FAK is activated and autophosphorylated by the ligation of integrins, although the substrate of FAK has not been revealed. We show here that p130Cas and Cas-L are FAK substrates. FAK directly phosphorylates Cas proteins primarily at the YDYVHL sequence that is conserved among all Cas proteins. Furthermore, the phosphorylated YDYVHL sequence is a binding site for Src family protein-tyrosine kinases, and the recruited Src family kinase phosphorylates the other tyrosine residues within Cas. The Cas-L YDYVHL sequence is phosphorylated upon integrin-ligand binding, and this integrin-mediated tyrosine phosphorylation is inhibited by the cotransfection of the FAK COOH-terminal domain that does not contain a kinase domain. These findings strongly suggest that FAK initiates integrin-mediated tyrosine phosphorylation of Cas proteins; then, Src family tyrosine kinases, which are recruited to phosphorylated Cas and FAK, further phosphorylate Cas proteins.

Certain MHC class I molecules on target cells are known to inhibit the cytotoxic action of NK cells. By using monoclonal antibody (mAb) Cho-1, we have found inhibitory non-MHC class I cell surface molecules that are noncovalently-associated with 200 kDa and 40 kDa antigens. Poly I-C-induced rat NK cells were not cytotoxic to rat fetus-derived fibroblast WFB cell line. In contrast, NK cells were cytotoxic to H-ras oncogene-induced transformants of WFB, W14 and W31. FACS analysis indicated that mAb Cho-1 reacts with WFB, but not with W14 and W31 cells. Thus, this antigen may disappear concomitantly with cell growth and transformation. Cho-1 antigens were also expressed on other NK-resistant lines, such as mouse BALB3T3 fibroblast, EL-4 lymphoma and human fibroblast HEPM. However, they were not expressed on NK-sensitive mouse YAC-1 and H-ras transformant (Brash) of BALB3T3 cells. Furthermore, treatment of target cells with IFN-gamma clearly induced the cell surface expression of Cho-1 antigens, and conferred a resistance to NK cytolysis on target cells. These data strongly suggest that Cho-1 antigen expression may correlate with target cell susceptibility to NK cells. Indeed, treatment of NK-resistant WFB as well as HEPM cells with F(ab')2 fragments of mAb Cho-1 resulted in the acquisition of susceptibility to NK cytolysis. Cho-1 antigens may be novel molecules that regulate the NK resistance of cells.

We previously established Fas-resistant variant clones from the human T-cell leukemia lines Jurkat and SUP-T13. Comparative gene expression analysis of the Fas-resistant and Fas-sensitive clones revealed several genes that were aberrantly expressed in the Fas-resistant clones. One of the genes, esophageal cancer-related gene 4 (ECRG4), contained a VDAC2-like domain that might be associated with apoptotic signals. In the present study, we examined the subcellular localization and function of ECRG4 in Fas-mediated apoptosis. By confocal fluorescence microscopy, ECRG4-EGFP fusion protein was detected in mitochondria, endoplasmic reticulum and the Golgi apparatus in gene-transfected HeLa cells. Overexpression of ECRG4 in Fas-sensitive Jurkat cells inhibited mitochondrial membrane permeability transition, leading to resistance against Fas-induced apoptosis. Tumor necrosis factor-alpha-induced apoptosis was also suppressed in ECRG4-overexpressing Jurkat cells. Immunoprecipitation assay demonstrated that ECRG4 is associated with procaspase-8. The inhibitory mechanism included the inhibition of caspase-8 activity and Bid cleavage. Since ECRG4 expression is downregulated in activated T cells, our results suggest that ECRG4 is a novel antiapoptotic gene which is involved in the negative regulation of caspase-8-mediated apoptosis in T cells.

Research on human tumor immunology has greatly advanced in the past two decades. Many immunogenic tumor antigens have been identified, and some of these antigens entered in clinical trials. Consequently, it has been shown that these antigens can inhibit tumor growth in patients to some extent, indicating that they act as potent immunogenic therapeutic vaccines in cancer patients with malignancies originating from various tissues. These patients had antigen-specific cytotoxic T-lymphocyte (CTL) responses when assessed on tetramer, enzyme-linked immunospot (ELISPOT), T-cell clonotype and CTL induction efficiency. Thus, it has become clear that human tumor vaccines can evoke clinical and immunological anti-tumor responses in patients. The tumor regression effects of tumor vaccines, however, are generally low, and it is obvious that current vaccination protocols are generally too weak to provide substantial and satisfactory clinical benefits. This means that other drastic and more potent clinical and immunological protocols are required in cancer immunotherapy. To find such efficient protocols the basic immunological and biological properties of cancers must be investigated. In the present review the identification of human tumor antigens recognized on CTL and the clinical trials are introduced. Next, the most recent analysis of human cancer-initiating cell (cancer stem cell)-associated antigens is described. These antigens might be able to act as ‘universal, general and fundamental’ tumor antigens. Also present is the authors' recent study for increasing cross-presentation efficiency in dendritic cells and subsequent enhancement of human leukocyte antigen (HLA)-class I-restricted peptide antigenicity by using HSP90 and ORP150 molecular chaperones that act as endogenous Toll-like receptor ligands. In addition to the aforementioned manipulation of the positive loop of tumor immunity, it is necessary to regulate and intervene in the negative loop. In particular, the potential of the expression of HLA class I molecule regulation by epigenetic mechanisms will be discussed. Finally, the type of basic and clinical tumor immunology research highly required currently, and in the very near future, are described.

Polyamine compound deoxyspergualin (DSG) is a potent immunosuppressive agent that has been applied clinically for protecting graft rejection and treatment of Wegener's granulomatosis. Though DSG can bind to heat-shock proteins (HSPs) in cells, its mechanism of immunosuppressive action remains unknown. It is widely accepted that extracellular HSPs are capable of stimulating dendritic cells (DC) through cell surface receptors, leading to DC activation and cytokine release. In this study, we examined if DSG analogs could inhibit HSP70-induced DC activation. Bone marrow derived immature mouse DCs and peripheral blood mononuclear cell-derived immature human DCs were generated and incubated with Alexa 488-labeled Hsp70 in the presence of methoxyDSG (Gus-1) that had comparable HSP70-binding affinity to DSG or DSG analog GUS-7, which had much more reduced binding affinity for HSP70. The binding of HSP70 to immature DCs was analyzed by laser microscopy and flow cytometry. HSP70-induced DC activation was assessed by TNF-alpha release by enzyme-linked immunosorbent assay. Binding of Hsp70 to the cell surface of immature DCs was inhibited under the presence of Gus-1, but not under the presence of Gus-7. Immature DCs were activated and released TNF-alpha by the stimulation with HSP70 for 12 hours; however, the HSP70-induced TNF-alpha release was suppressed under the presence of Gus-1, and partially suppressed under the presence of Gus-7. Similar results were observed when immature human DCs were stimulated under the same conditions. Immunosuppressive mechanism of DSG may be explained, at least in part, by the inhibition of extracellular HSP70-DC interaction and HSP70-induced activation of immature DCs.

The nature of the target molecule of TCR gamma delta T cell-mediated lysis remains to be determined. As we previously reported, #067 monoclonal antibody (mAb) recognizes one of the transformation-associated antigens, designated as #067 antigen. This antigen is expressed on the cell surface of rat fibrosarcoma W31 cells, which are established by transformation of fetal fibroblastic WFB cells with H-ras oncogene. It has been suggested that the #067 antigen is a target molecule for TCR gamma delta T cells since #067 mAb inhibited TCR gamma delta T cell-mediated lysis against #067 positive cells. In this study we attempted to identify the protein sequence of the #067 antigen. By using molecular cloning techniques, we demonstrated that a calcium binding protein, S100A4, was possibly one and the same molecule as the #067 antigen. It was shown that the expression of S100A4 was higher in W31 cells than in WFB cells at transcription and protein level. Flow cytometry and immunocytochemical studies showed that #067 antigen partially co-localized with S100A4 on the cell surface as well as the cytoplasm of W31 cells. Moreover, rabbit anti-S100A4 polyclonal antibodies (pAb) inhibited TCR gamma delta T cell-mediated lysis against #067 positive cells. Our results indicated that S100A4 may play a role as a possible target molecule for TCR gamma delta T cell-mediated lysis although how S100A4 is involved in TCR gamma delta T cell-mediated lysis remains to be determined.

Heat shock protein 40 (Hsp40) family proteins are known to bind to Hsp70 through their J-domain and regulate the function of Hsp70 by stimulating its adenosine triphosphatase activity. In the endoplasmic reticulum (ER), there are 5 Hsp40 family proteins known so far, 3 of which were recently identified. In this report, one of the novel Hsp40 cochaperones, ERdj3, was characterized in terms of its subcellular localization, stress response, and stress tolerance of cells. By using ERdj3-specific polyclonal antibody, endogenous ERdj3 protein was shown to reside in the ER as gene transfer-mediated exogenous ERdj3. Analysis of the expression level of endogenous ERdj3 protein revealed its moderate induction in response to various ER stressors, indicating its possible action as a stress protein in the ER. Subsequently, we analyzed whether this molecule was involved in ER stress tolerance of cells, as was the case with the ER-resident Hsp70 family protein BiP. Although overexpression of ERdj3 by gene transfection could not strengthen ER stress tolerance of neuroblastoma cells, reduction of ERdj3 expression by small interfering ribonucleic acid decreased the tolerance of cells, indicating that ERdj3 might have just a marginal role in the ER stress resistance of neuroblastoma cells. In contrast, overexpression of ERdj3 notably suppressed vero toxin-induced cell death. These data suggest that ERdj3 might have diverse roles in the ER, including that of the molecular cochaperone of BiP and an as yet unknown protective action against vero toxin.

To date an increasing number of T-cell epitopes derived from various tumor-associated antigens have been reported, and they proved to play significant roles for tumor rejection both in vivo and in vitro. Survivin was originally identified as a member of the inhibitor of apoptosis protein family. Expression of this gene is developmentally regulated. Although survivin is expressed during normal fetal development, the expression is barely detected in terminally differentiated adult tissues except for testis, thymus, and placenta. In contrast, it is abundantly expressed in a wide variety of malignant tissues. We examined the expression of survivin and the two splicing variants survivin-2B and survivin-DeltaEx3 in various cancer cells, immortalized cells, and normal adult tissues. It was demonstrated that two splicing variants were detected in various types of cancer cells as well as survivin, and their expression was more restricted to cancer cells as compared with survivin expression. To identify HLA-A24-restricted T-cell epitopes from survivin and the variant proteins, three peptides were selected from amino acid sequence of these proteins, based on the HLA-A24-binding motif. Peptide binding assay to HLA-A24 revealed that only one peptide designated as survivin-2B80-88 (AYACNTSTL) was capable of binding to HLA-A24. By stimulating peripheral blood lymphocytes with the peptide-pulsed antigen-presenting cells, CTLs were successfully induced in vitro from five of five HLA-A24-positive cancer patients. The CTLs showed significant cytotoxicity against HLA-A24-positive survivin-2B-positive cancer cells. These data suggest that survivin-2B80-88 may be a potent T-cell epitope eliciting CTL response against a splicing variant survivin-2B, which is specifically expressed in many kinds of cancer cells.

We previously reported the cytotoxic negative regulatory molecule, Cho-1, that was expressed on the cell surface of rat fetal fibroblast cells in the cytotoxicity by natural killer (NK) cells. This molecule was IFN-ƒA-inducible, but appeared to be different from MHC class I. It was expressed on NK-resistant cells but not on NK-sensitive murine target cells such as YAC-1. In this paper, first we determined whether Cho-1 could also act as the negative regulatory molecule in a human NK-resistant HEPM line. Our data strong-ly suggested that Cho-1 could act as such a negative regulatory molecule in human NK cytotoxicity. The immunoprecipitates made with HEPM cell lysate and anti-MHC class I monoclonal antibody (mAb) did not react against anti-Cho-1 mAb, indicating that Cho-1 was different from MHC class I. Second, an assessment was made as to whether or not this molecule is involved in the cytotoxicity of CD8 (+) cytotoxic T lym-phocytes (CTL) against human autologous tumor cells. The data indicated that although this cell surface molecule was expressed on certain tumor lines, it was not involved in the cytotoxic mechanism of CTL. Thus, Cho-1 appeared to be the novel regulatory molecule in the NK cytotoxic mechanism . Key words: Natural killer cell inhibitory ligand, Cho - 1

Cas-L (pp105), a Crk-associated substrate (p130(Cas))-related protein, was first identified as a 105-kDa protein that is tyrosine-phosphorylated following beta1 integrin cross-linking in T cells. Cas-L contains possible multiple binding sites for the Src homology (SH) 2 domains of various signaling molecules, and appears to be involved in signal transduction through phosphorylated tyrosine-mediated protein-protein interaction. Since Cas-L is preferentially expressed in lymphocytes, it is conceivable that Cas-L plays an important role in lymphocyte-specific signals. Here, we show the involvement of Cas-L in the T cell receptor (TCR)/CD3 signaling pathway. Cas-L is transiently phosphorylated following CD3 cross-linking, and tyrosine-phosphorylated Cas-L binds to Crk and C3G. Furthermore, a Cas-L mutant that lacks the SH3 domain, the binding site for focal adhesion kinase (FAK), is also tyrosine-phosphorylated upon CD3 cross-linking, but not upon beta1 integrin crosslinking, suggesting that FAK is not involved in CD3-dependent Cas-L phosphorylation. Taken together, the present study indicates a novel signaling pathway mediated by tyrosine-phosphorylated Cas-L upon the TCR/CD3 stimulation.