A novel nuclear DnaJ protein, DNAJC8, can suppress the formation of spinocerebellar ataxia 3 polyglutamine aggregation in a J-domain independent manner.

Polyglutamine (polyQ) diseases comprise neurodegenerative disorders caused by expression of expanded polyQ-containing proteins. The cytotoxicity of the expanded polyQ-containing proteins is closely associated with aggregate formation. In this study, we report that a novel J-protein, DNAJ (HSP40) Homolog, Subfamily C, Member 8 (DNAJC8), suppresses the aggregation of polyQ-containing protein in a cellular model of spinocerebellar ataxia type 3 (SCA3), which is also known as Machado-Joseph disease. Overexpression of DNAJC8 in SH-SY5Y neuroblastoma cells significantly reduced the polyQ aggregation and apoptosis, and DNAJC8 was co-localized with the polyQ aggregation in the cell nucleus. Deletion mutants of DNAJC8 revealed that the C-terminal domain of DNAJC8 was essential for the suppression of polyQ aggregation, whereas the J-domain was dispensable. Furthermore, 22-mer oligopeptide derived from C-termilal domain could suppress the polyQ aggregation. These results indicate that DNAJC8 can suppress the polyQ aggregation via a distinct mechanism independent of HSP70-based chaperone machinery and have a unique protective role against the aggregation of expanded polyQ-containing proteins such as pathogenic ataxin-3 proteins.

HSP DNAJB8 controls tumor-initiating ability in renal cancer stem-like 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.

ECRG4 is a negative regulator of caspase-8-mediated apoptosis in human T-leukemia 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.

The feasibility of Cep55/c10orf3 derived peptide vaccine therapy for colorectal carcinoma.

In our previous study, we demonstrated that a peptide derived from the novel centrosome residing protein Cep55/c10orf3 can be targeted by the cytotoxic T lymphocytes (CTLs) in peripheral blood mononuclear cells (PBMCs) of breast carcinoma patients. In this report, we evaluated the feasibility of cancer immunotherapy using Cep55/c10orf3 peptide for colorectal carcinoma (CRC). To evaluate the expression of Cep55/c10orf3 in CRC tissues, we performed immunohistochemical staining of using anti-Cep55/c10orf3 monoclonal antibody. Sixty-three percent cases showed weak positive for Cep55/c10orf3 in total 70 CRC cases. The Cep55/c10orf3 expression intention was collated with high histological grade of CRC. Thus, we hypothesized that Cep55/c10orf3 can also be the target of CTLs in CRC cases. We generated CTLs from PBMCs of human leukocyte antigen (HLA)-A24-positive colorectal carcinoma patients using HLA-A24-restricted Cep55/c10orf3 peptides. Two of 6 colorectal cancer patients were reactive for the Cep55/c10orf3_193(10) peptide, which was the only immunogenic peptide in breast carcinoma patients. CTL clone specific for Cep55/c10orf3_193(10) recognized and lysed HLA-A24 (+) and Cep55/c10orf3 (+) colorectal carcinoma cell lines. In addition, 1 of 6 colorectal carcinoma patients was reactive for the Cep55/c10orf3_402(11) and Cep55/c10orf3_283(12) peptides, but not for Cep55/c10orf3_193(10) with the ELISPOT assay. These observations suggest that the antigenic peptide repertoire presented by HLA-A24 in colorectal carcinoma might be different from that in breast carcinoma. Thus, these peptide vaccination peptide mixture of Cep55/c10orf3_193(10), Cep55/c10orf3_402(11) and Cep55/c10orf3_283(12) might be more effective than a single peptide in the treatment of colorectal carcinoma patients.

Inhibition of osteopontin reduces liver metastasis of human pancreatic cancer xenografts injected into the spleen in a mouse model.

PURPOSE: Pancreatic cancer is associated with the poorest prognosis of any digestive cancer due to the high incidence of liver metastasis. This study evaluated the possibility that osteopontin (OPN) RNA interference (RNAi) and anti-OPN antibody (Ab) could have antimetastatic effects. METHODS: The differential gene expression was measured in a parental cell line, HPC-3, and an established highly liver metastatic cell line, HPC-3H4. This study investigated the effect of OPN RNAi and anti-OPN Ab on the metastatic ability of HPC-3H4 to the liver. An OPN RNAi-expressing vector was introduced into HPC-3H4 cells (HPC-3H4/miOPN), in which OPN production was reduced to the level of the parental HPC-3 cells. Finally, the ability of anti-OPN Ab to suppress liver metastasis was investigated. RESULTS: Osteopontin was upregulated 11.1-fold in HPC-3H4 in comparison to HPC-3. The metastatic rate of HPC-3H4/miOPN was significantly reduced to 25% in comparison to the 100% metastatic rate of HPC-3H4 and control HPC-3H4/miNeg cells (P < 0.01). The metastatic rate of the group given anti-OPN Ab was 50%. CONCLUSION: OPN RNAi and anti-OPN Ab had remarkable inhibitory effects against liver metastasis by the pancreatic cancer cell line.

Establishment of shared antigen reactive cytotoxic T lymphocyte using co-stimulatory molecule introduced autologous cancer cells.

Cytotoxic T lymphocytes (CTLs) play an essential role in immunological responses for tumor rejection. In the past decade, many tumor-associated antigens (TAAs) have been identified predominantly in melanomas. Several clinical trials based on such antigenic peptides with or without adjuvants brought about partially favorable results, suggesting that identification of more immunogenic TAAs is needed. We show here the successful establishment of human leukocyte antigen (HLA)-A24-restricted CTL (TcLHK2 line1) from a pleural effusion of lung cancer patient, using B7.1 (CD80) transduced autologous lung cancer cells as an antigen-presenting cell (APC). TcLHK2 line1 recognized autologous lung adenocarcinoma cell line LHK2 in an HLA-A24-restricted fashion. Moreover, this CTL line also recognized allogeneic HLA-A24-positive lung adenocarcinoma cell line, gastric carcinoma cell line and melanoma cell line. These data raise the possibility that co-stimulatory molecule B7.1 (CD80) plays important role to overcome the immunological tolerance. Furthermore, TcLHK2 line1 is a useful tool for the identification of widely expressed shared antigens restricted by HLA-A24. Further analysis of this CTL and autologous cancer cell line will bring about novel TAAs.

Targeting to static endosome is required for efficient cross-presentation of endoplasmic reticulum-resident oxygen-regulated protein 150-peptide complexes.

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.

Cep55/c10orf3, a tumor antigen derived from a centrosome residing protein in breast carcinoma.

Identification of tumor-associated antigens may facilitate vaccination strategies to treat patients with malignant diseases. We have found that the centrosomal protein, Cep55/c10orf3 acts as a novel breast carcinoma-associated tumor-associated antigen. Cep55/c10orf3 mRNA was detectable in a wide variety of tumor cell lines. Expression was barely detectable in normal tissues except for testis and thymus. Moreover, Cep55/c10orf3 protein could be detected by a monoclonal anti-Cep55/c10orf3 antibody (# 11-55) in 69.8% of breast carcinoma, 25% of colorectal carcinoma, and 57.8% of lung carcinoma tissues. The expression of Cep55/c10orf3 protein did not show any relationship with the hormone receptors such as estrogen receptor and progesterone receptor or expression patterns of p185 HER2/neu. We designed 11 peptides which displayed a human leukocyte antigen-A24 binding motif. One Cep55/c10orf3-peptide, Cep55/c10orf3_193(10) (VYVKGLLAKI), induced cytotoxic T lymphocytes (CTLs) in 3 of 3 patients with Cep55/c10orf3 (# 11-55)-positive breast carcinoma. A Cep55/c10orf3_193(10)-specific CTL clone could also recognize Cep55/c10orf3 (+) displayed on human leukocyte antigen-A24 (+) cancer cell lines. These data indicate that Cep55/c10orf3 peptides were naturally presented by breast cancer cells and can cause CTL clonal expansion in vivo. Monoclonal antibody # 11-55 and the Cep55/c10orf3_193(10) peptides may be useful as part of a therapeutic strategy for hormonal therapy or anti-p185 HER2/neu monoclonal antibody therapy-resistant breast carcinoma patients.

Novel spliced form of a lens protein as a novel lung cancer antigen, Lengsin splicing variant 4.

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.

Molecular pathological approaches to human tumor immunology.

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.

Clonal diversity of cytotoxic T lymphocytes that recognize autologous oral squamous cell carcinoma.

Cytotoxic T lymphocytes (CTLs) play an essential role in immunologic responses for tumor rejection. In the past decade, various melanoma tumor-associated antigens (TAAs) have been identified, and several clinical trials of vaccination immunotherapy and adoptive immunotherapy using such antigens with or without adjuvants have had fascinating results. However, this has not been the case with oral squamous cell carcinoma (OSCC) because of the difficulty of establishing oral cancer cell lines and CTLs against autologous oral cancer cells. Therefore, few oral cancer antigens have been identified with such CTLs. We herein present the successful establishment of an oral squamous cell carcinoma cell line, POT-1, and an HLA-A24-restricted CTL line (TcPOT-1) from a patient's autologous peripheral blood lymphocytes. TcPOT-1 recognized autologous POT-1 cells in an HLA-A24-restricted manner, and also allogeneic HLA-A24 (+) OSCC cell lines OSC-70 and HSC-2. We also succeeded in isolating two distinct CTL clones from TcPOT-1, HLA-A24-restricted CTL clone 4F11 and HLA-A33-restricted clone 4A11. Both of these clones recognized autologous POT-1 but not allogeneic OSSC cell lines. These data imply that the TcPOT-1 CTL line may include several CTL subpopulations with distinct antigen specificities, such as an HLA-A24-restricted POT-1-specific clone, HLA-A33-restricted POT-1-specific clone, and HLA-A24-restricted allogeneic OSCC-recognizing clone. Therefore, precise analysis of TcPOT-1-recognizing antigens may provide us with important information on as-yet-unknown tumor rejection antigens in OSCC.

Polyamine compound deoxyspergualin inhibits heat shock protein-induced activation of immature dendritic cells.

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.

Disruption of the association of 73 kDa heat shock cognate protein with transporters associated with antigen processing (TAP) decreases TAP-dependent translocation of antigenic peptides into the endoplasmic reticulum.

Major histocompatibility complex class I-bound antigenic peptides generated in the cytosol are translocated into the ER by TAP. In the present study, the physical association of HSC73 with TAP in human lymphoblastoid T1 cells was demonstrated. The dissociation was induced in the presence of 10 mM ATP, indicating that the ADP-binding form of HSC73 might be associated with TAP. We found that HSC73-binding immunosuppressant, MeDSG disrupted the HSC73-TAP association, whereas it did not affect the binding of HSC73 to a substrate protein. MHC class I expression on the cell surface was also downregulated. Then, the effect of MeDSG on the TAP-mediated ER translocation was examined using two homologous model peptides, NGT-Bw4 and NGT-Bw6, which had distinct binding affinity to HSC73. Although high-affinity peptide NGT-Bw4 was translocated by TAP, low-affinity peptide NGT-Bw6 was not. The TAP-dependent translocation of NGT-Bw4 was abolished in the presence of MeDSG. Decreased presentation on the cell surface was shown for the human leukocyte antigen (HLA)-A31-restricted natural antigenic peptide F4.2, which had high affinity to HSC73, in the presence of MeDSG. It was indicated that disruption of the HSC73-TAP association resulted in inhibition of TAP-dependent translocation of HSC73-bound peptides. Our findings highlighted an important role of HSC73 for feeding antigenic peptides to TAP, and suggested a possibility that a synthetic polyamine might inhibit the function of HSC73, thereby suppressing MHC class I-restricted presentation of HSC73-bound antigenic peptides.

Biological heterogeneity of the peptide-binding motif of the 70-kDa heat shock protein by surface plasmon resonance analysis.

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.

Inhibitory effect of endothelin A receptor blockade on tumor growth and liver metastasis of a human gastric cancer cell line.

BACKGROUND: With metastatic progression, gastric cancer is incurable. Using a DNA microarray, we performed differential gene expression analysis of established highly metastatic gastric cancer cell lines and compared the findings with those from a low-metastatic parental cell line. The results demonstrated that the endothelin A receptor (ET-A) gene was the only one from the highly metastatic cell lines that was generally up-regulated. METHODS: To investigate the role that ET-A plays in gastric cancer metastasis, we studied the effect of an ET-A-selective antagonist, YM598, on cell proliferation, tumor growth, and liver metastasis of the highly liver metastatic cell line AZ-H5c, established from the low metastatic human gastric cancer cell line AZ-521. RESULTS: An in vivo study using nude mice demonstrated that YM598 had a significant growth inhibition effect on AZ-H5c at doses of 0.5-10.0 mg/kg. The liver metastatic rate was also significantly reduced by YM598: control, 83.3%; 1 mg/kg dosage, 16.7%; 10 mg/kg, 20%; and pretreatment at 1 mg/kg, 16.7%. There was no evidence of gross toxicity resulting from the YM598 treatment. CONCLUSION: The ET-A blockade by YM598 had a strong inhibitory effect against tumor growth and liver metastasis of the gastric cancer cell lines. These data suggest that YM598 has potential as a novel therapeutic agent for inhibiting liver metastasis of gastric cancer.

Survivin expression is regulated by coexpression of human epidermal growth factor receptor 2 and epidermal growth factor receptor via phosphatidylinositol 3-kinase/AKT signaling pathway in breast cancer cells.

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.

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.

Aberrant expression and potency as a cancer immunotherapy target of inhibitor of apoptosis protein family, Livin/ML-IAP in lung cancer.

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.

A calcium binding protein, S100A4, mediates T cell dependent cytotoxicity as a transformation-associated antigen.

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.

Localization and function in endoplasmic reticulum stress tolerance of ERdj3, a new member of Hsp40 family protein.

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.