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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

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.

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.

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.

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. 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. 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%. OPN RNAi and anti-OPN Ab had remarkable inhibitory effects against liver metastasis by the pancreatic cancer cell line.

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.

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.

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.

To elucidate the mechanisms of metastasis, we established two sublines HPC-1H5 with a highly liver metastatic cell line and HPC-1P5a with a highly peritoneal disseminating cell line, which were sequentially selected from the parental pancreatic cancer cell line HPC-1. Using these three cell lines, we investigated several biological properties and mRNA levels of differentially-expressed genes involved in cancer metastasis by cDNA macroarray. Microscopic findings for the three cell lines were the same. The tumorigenicity, in vitro growth ability, motile activity, adhesive activity and the production of IL-8 of metastatic sublines were higher than those of parental HPC-1 cells. Particularly, HPC-1H5 cells showed clearly higher levels of IL-8 expression and tumors of HPC-1H5 cells grew faster and bigger than those of HPC-1P5a cells. In cDNA macroarray analysis of HPC-1H5 cells, 22 genes were up-regulated and 44 genes were down-regulated compared with parental HPC-1 cells. In HPC-1P5a cells, 9 genes were up-regulated and 28 genes were down-regulated compared with parental HPC-1 cells. This study provides a demonstration of global gene expression analysis of pancreatic cancer cells with liver metastasis and peritoneal dissemination. Furthermore, our results provide a new insight into the study of liver metastasis and peritoneal dissemination of human pancreatic cancer.

We previously showed that the cell surface-expressed Mr 70,000 heat shock cognate (hsc70, a constitutively expressed member of the hsp70 family) protein-like molecule (#067 molecule) interacts with rat CD3+, CD4-, CD8-, T-cell receptor (TCR)alphabeta-, natural killer recetor-P1- T cells. This 70hsc-like molecule was also suggested to present cellular peptide antigens to these T cells. In the present study, we identified the genetic structure of the TCR by establishing T-cell hybridomas between these T cells and mouse BW5147 cells. Our data indicated that these T cells preferentially used TCRs with the Vdelta6 family. Analysis of the nucleotide sequence of the CDR3 junctional portion showed that there are substantial diversities, with insertion of seven to nine amino acid residues. These data provide indirect evidences for our hypothesis that an hsc70-like molecule could be presented together with cellular peptide antigens to particular T cells with TCR gammadelta chains. Since the expression of this hsc70-like #067 antigen on the cell surface is usually induced along with cell transformation by activated oncogenes, T cells with the TCR Vdelta6 family are likely to contribute to host resistance to tumor cells.

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.

The regulatory mechanism of the recognition and cytotoxicity by natural killer (NK) cells in placental tissue remains unclarified. Previous reports indicated that monoclonal antibody Cho-1-defined molecule (Cho-1 molecule) may act as the negative regulator in the cytotoxicity by human NK cells. The Cho-1 molecule is composed of non-covalently associated cell surface molecules of approximately 200 kDa and 40 kDa. In the present study we analyzed the expression of this novel molecule in extravillous cytotrophoblast cells, which are presumed to be exposed to the cytotoxic action by maternal NK cells, from clinical cases of successful pregnancy and spontaneous abortion. By using monoclonal antibody Cho-1, our immunohistochemical data indicated that the Cho-1 molecule is clearly expressed in the cytotrophoblast cells of the early phase of successful pregnancy, but only weakly expressed in those from spontaneous abortion. The cytotrophoblast cells in the late phase (9-10 months) of pregnancy also expressed this molecule. Fluorescence-activated cell sorter analysis also showed that it is expressed on the cytotrophoblast cell surface of successful pregnancy but not on that of spontaneous abortion, suggesting that Cho-1 antigen may act as a negative regulator of the cytotoxicity by NK cells in successful pregnancy of the fetus.

beta1-integrins play crucial roles in a variety of cell processes such as adhesion, migration, proliferation, and differentiation of lymphocytes. For understanding the molecular mechanisms of these various biological effects, it may be particularly important to analyze cell signaling through the beta1-integrins. Our previous study had shown that PLC-gamma, pp125FAK (focal adhesion kinase), pp105, paxillin, p59fyn, p56lck and ERK1/2 are phosphorylated in their tyrosine residues upon engagement of beta1-integrins. We identified pp105 as Cas (Crk-associated substrate)-related protein and successfully cloned its cDNA. pp105 is a Cas homologue predominantly expressed in the cells of lymphoid lineage, which led us to designate it as Cas-L. Like p130Cas, Cas-L contains a single SH3 domain and multiple SH2 binding sites (YXXP motif), which is suggested to bind SH2 domains of Crk, Nck, and SHPTP2. Subsequent studies revealed that pp125FAK binds Cas-L on its SH3 domain and phosphorylates its tyrosine residues upon beta1-integrin stimulation. Since Cas-L is preferentially expressed in lymphocytes, it is conceivable that Cas-L plays an important role in lymphocyte-specific signals. We have shown that Cas-L is involved in the T-cell receptor (TCR)/CD3 signaling pathway as well as the beta1-integrin 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 (Cas-LDeltaSH3), which lacks the binding site for FAK, is still tyrosine-phosphorylated upon CD3 cross-linking, but not upon beta1-integrin cross-linking, suggesting that FAK is not involved in CD3-dependent Cas-L phosphorylation. Finally, we have identified a crucial role of Cas-L in beta1-integrin-mediated T-cell co-stimulation. beta1-integrins have known to provide a co-stimulus for TCR/CD3-driven interleukin-2 production and proliferation of peripheral T-cells. We have found that this co-stimulatory pathway is impaired in the Jurkat T-cell line, and that the expression level of Cas-L is reduced in Jurkat cells compared with peripheral T-cells. The transfection of Cas-L cDNA into Jurkat cells restored the beta1-integrin-mediated co-stimulation, while the transfection of Cas-LDeltaSH3 mutant failed to do so, showing a contrast to the case with CD3-mediated signaling. These results indicate that Cas-L plays a key role through the association and phosphorylation by FAK in the beta1-integrin-mediated T-cell co-stimulation. Taken together, Cas-L might be the bi-modal docking protein that assembles the signals through beta1-integrins and TCR/CD3, and participates in a variety of T-cell functions.

Crk-associated substrate (Cas) lymphocyte-type (Cas-L) is a 105-kDa cytoplasmic protein consisting of Src homology-3 domain and multiple YXXP motifs (substrate domain). Our previous studies showed that Cas-L is tyrosine-phosphorylated following the ligation of TCR and β1 integrins in T lymphocytes. Here we show that Cas-L is involved in T cell motility following the ligation of TCR and β1 integrin. Peripheral T lymphocytes showed a marked increase of migration on fibronectin (FN) after the ligation of TCR. In contrast, the migrating Jurkat cells, in which Cas-L was marginally expressed, were less than one-tenth in number on the same condition. Transfection of wild-type Cas-L into Jurkat cells resulted in restoring CD3 plus FN-induced cell migration. Furthermore, following the ligation of β1 integrin alone, the Cas-L transfectants significantly migrated better than the vector control. Mutational analysis of Cas-L revealed that the substrate domain is required for both FN- and CD3-induced tyrosine phosphorylation of Cas-L and cell migration caused by FN alone and CD3 plus FN. In contrast, the Src homology-3 domain is required only for the FN-induced tyrosine phosphorylation of Cas-L and cell migration, but not for CD3-induced tyrosine phosphorylation or CD3 plus FN-induced cell migration. These data strongly suggest that Cas-L is a key molecule in T cell migration induced by the ligation of CD3 and β1 integrins and that tyrosine phosphorylation of Cas-L is essential for T cell migration.

β1 integrins provide a costimulus for TCR/CD3-driven T cell activation and IL-2 production in human peripheral T cells. However, this β1 integrin-mediated costimulation is impaired in a human T lymphoblastic line, Jurkat. We studied the molecular basis of this impaired costimulation and found that Cas-L, a 105-kDa docking protein, is marginally expressed in Jurkat T cells, whereas Cas-L is well expressed in peripheral T cells. Cas-L is a binding protein and a substrate for focal adhesion kinase and is tyrosine phosphorylated by β1 integrin stimulation. We here show that the transfection of wild-type Cas-L in Jurkat T cells restores β1 integrin-mediated costimulation. However, Cas-L transfection had no effect on CD28-mediated costimulation, indicating that Cas-L is specifically involved in the β1 integrin-mediated signaling pathway. Furthermore, transfection of the Cas-LΔSH3 mutant failed to restore β1 integrin-mediated costimulation in Jurkat cells. Cas-LΔSH3 mutant lacks the binding site for focal adhesion kinase and is not tyrosine phosphorylated after β1 integrin stimulation. These findings strongly suggest that the tyrosine phosphorylation of Cas-L plays a key role in the signal transduction in the β1 integrin-mediated T cell costimulation.

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.

We present a unique case of crisis type chronic adult T-cell leukemia (ATL), in which the majority of leukemic cells had abundant periodic acid-Schiff (PAS)-positive cytoplasmic inclusions. These inclusions were found to be composed of glycogen because the PAS-positivity completely disappeared after digestion with amylase or human saliva. Electron microscopy also revealed that the inclusions consisted of aggregated beta particles of glycogen. The mechanism of glycogen accumulation in leukemic cells remains unknown; however, the presence of such inclusions in leukemic cells may be helpful diagnostically in T-lymphocyte malignancies.

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.

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.

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.

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.

Introduction We have generated HLA-A*2601-restricted CD8+ CTL clones against an autologous pancreatic cancer cell line. Aims To characterize the antigen expressed on the cancer cells. Methodology We assessed cytotoxic activities and cytokine production of these CTL clones reacting against cancer cell lines that stably or transiently expressed the HLA-A*2601 gene. Results These CTL clones recognized 4 of 10 allogeneic pancreatic cancer cell lines and a gallbladder cancer cell line in the context of HLA-A*2601. However, the CTL clones did not recognize three hepatocellular carcinoma cell lines, two esophageal squamous cell carcinoma cell lines, or a lung adenocarcinoma cell line. Conclusions Thus, the CTL clones may recognize a shared, but not ubiquitously expressed, tumor antigen on pancreatic and gallbladder cancer cells.

Purpose: Pancreatic cancer is associated with the poorest prognosis of any digestive cancer due to the high incidence of peritoneal dissemination, which is the cause of death in most cases. To determine the mechanisms of peritoneal dissemination in pancreatic cancer, we established a mouse model of high peritoneal dissemination. Methods: A novel highly peritoneal-disseminating cell line was established from the human pancreatic cancer cell line; CAPAN-1. The new cell line, CAPAN-1P4a, was established from CAPAN-1 by repeated in vivo selection (four times) of the tumor cell line. To clarify the candidate genes implicated in peritoneal dissemination of pancreatic cancer, global gene expression screening was done using a cDNA macroarray. Results: CAPAN-1P4a cells showed 100% metastasis 3 weeks after injection and high reproducibility in the inoculated mice. Twenty-seven genes were upregulated and 14 genes were downregulated in CAPAN-1P4a cells compared with CAPAN-1 cells. The genes differentially expressed in the two cell lines were included as tumor suppressor/apoptosis genes, regulatory transcription factor, membrane receptors, cell adhesion protein, membrane receptors, and so on. Conclusions: Our established CAPAN-1P4a model offers a new means of conducting global gene expression analysis of pancreatic cancer cells with peritoneal dissemination and it has the potential to provide new insights into the mechanism of peritoneal dissemination in human pancreatic cancer.

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.

Introduction Recently, several mice models have been used for investigating cancer metastasis. However, there are no metastatic and peritoneal dominated variants from the same parental cell line. Aim and methodology To elucidate the mechanisms of metastasis, we established highly liver metastatic and peritoneal disseminated models in nude mice, and then characterized several factors related to metastasis in these cells. We established a series of well-characterized sublines that showed metastatic potentials to different organ sites of nude mice. Two sublines were selected sequentially from the parental pancreatic cancer cell line, HPC-4, resulting in a highly liver metastatic cell line, HPC-4H4, and a highly peritoneal disseminated cell line, HPC-4P4a. Using these three cell lines, we investigated several biologic properties and mRNA levels of differentially expressed genes involved in cancer metastasis. Results The tumorigenicity, the motile activity, and the adhesive activity of metastatic sublines were higher than those of parental HPC-4 cells. Macroscopic and microscopic findings and the DNA ploidy pattern were the same among the three cell lines. In addition, HPC-4H4 cells expressed clearly higher levels of vascular endothelial growth factor and IL-8 expression than did HPC-4P4a cells. In fluorescence-activated cell sorter analysis of adhesion molecules, the expression of integrin-alpha2 was enhanced in HPC-4 cells, integrin-alphavbeta5 was enhanced in HPC-4H4 cells, and integrin-alpha3 was enhanced in HPC-4P4a cells. Osteopontin, vascular endothelial growth factor, and hepatocyte growth factor were among the genes that were upregulated in HPC-4H4 cells compared with HPC-4P4a cells. HPC-4P4a cells did not metastasize to the liver by intrasplenic injection. Conversely, HPC-4H4 cells metastasized remarkably to the peritoneum by intraabdominal injection. Conclusion These sublines are the first reported liver metastatic and peritoneal disseminated models derived from the same parental cell lines. The results of our study suggest that the process of hematogenous metastasis is not the same as that of peritoneal dissemination.

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.

The mechanism of the cytotoxicity by natural killer (NK) cells is not known. It is speculated that there exist several positively regulated and negatively regulated target molecules expressed on the target cell surface. Although one of the latter is considered to be major histocompatibility complex antigen (MHC) class I, in this study we described a novel non-MHC class I molecule that may negatively regulate the NK cytotoxicity. This antigen is defined by monoclonal antibody Cho-1 and is composed of noncovalently associated antigens that are 40 and 200 kilodaltons in molecular size. The expression of this antigen is reduced along with the cell growth induced by growth factors and/or oncogenes. Thus, Cho-1-defined antigen appears to be involved as one of the resistant molecules in the cytotoxic mechanism of NK cells.

Recent human tumor immunology research has identified several genes coding immunogenic peptides recognized by CD8 cytotoxic T lymphocytes (CTLs) in melanoma tumors. Very recently, CD4 T cell antigenic epitopes were also determined in certain melanoma tumors. The use of these peptides in conjunction with human immunotherapy could prove to be of great benefit. However, such peptides in clinically common tumors of epithelial cell origin, such as of the stomach, colon, lung, etc., have not yet been determined extensively. We describe for the first time an HLA-A31 (A*31012)-restricted natural antigenic peptide recognized by the CD8 CTL TcHST-2 of gastric signet ring cell carcinoma cell line HST-2. We also identified the HLA-DRB1*08032-restricted peptide recognized by the CD4 T cell line TcOSC-20 of squamous cell carcinoma OSC-20 derived from the oral cavity. The antigenic peptide of HST-2, designated F4.2, is composed of 10 amino acid residues with two anchor motif residues necessary for binding to HLA-A31 molecules. The synthetic F4.2 peptide enhanced the reactivity of TcHST-2 against HST-2 cells. Furthermore, introduction of an expression minigene coding F4.2 peptide to HLA-A31(+) cells conferred cytotoxic susceptibility to TcHST-2 on the cells. Some stomach cancer lines into which the HLA-A31 gene had been introduced, such as MKN28-A31-2, were lysed by TcHST-2, suggesting the presence of F4.2 peptide in at least some HLA-A31(+) stomach cancers. Furthermore, F4.2 peptide induced an F4.2 peptide-specific CTL response in at least 30-40% of HLA-A31(+) peripheral blood lymphocytes from gastric cancer patients, suggesting that F4.2 peptide could be used as a cancer vaccine for gastric tumors. The natural antigenic peptide of OSC-20 was also determined using acid extraction and biochemical separation and by mass spectrometry. Consequently, OSC-20 peptide was designated as the 6-1-5 peptide, an HLA-DRB1*08032-restricted 16-mer peptide with two possible anchor motifs. It has an amino acid sequence identical to that of human alpha-enolase, suggesting that it was derived from the processed parental alpha-enolase protein. We are presently attempting to determine the genes that code tumor rejection antigens recognized by HLA-A24- and A26-restricted T cells, including those of pulmonary and pancreatic carcinomas. The search for these antigenic peptides may lead to the identification of immunogenic peptide antigens that would be suitable for clinical use in commonly occurring epithelial cancers.