Takashi Fukuyama

Cytokine production of lung cancer cell lines: Correlation between their production and the inflammatory/immunological responses both in vivo and in vitro

Cytokines produced by tumor cells may have various effects on antitumor immune responses and tumor growth. In the present study, the cytokine production of 31 lung cancer cell lines was evaluated, while any correlation with the histological type, the induction of tumor‐specific cytotoxic T lymphocytes (CTL) in vitro, and angiogenesis and the infiltration of inflammatory cells in tumor tissues were also examined. Production of interleukin (IL)‐1α, IL‐1β, IL‐4, IL‐6, IL‐8, IL‐10, tumor necrosis factor (TNF)‐α, granulocyte macrophage colony stimulating factor (GM‐CSF), granulocyte colony stimulating factor, transforming growth factor (TGF)‐β and vascular endothelial growth factor (VEGF) in the culture supernatant was measured using enzyme‐linked immunosorbent assay. Each cytokine was produced in a substantial number of the tumor cell lines. In particular, IL‐6, IL‐8, TGF‐β and VEGF were produced in 18 (55%), 29 (94%), 31 (100%) and 28 (90%) of 31 cell lines, respectively. However, neither IL‐4 nor TNF‐α was produced at all by any tumor cell line. TGF‐β production was significantly higher in adenocarcinoma than in squamous cell carcinoma (P = 0.03). Immunohistochemical staining revealed the magnitude of macrophage infiltration, and angiogenesis in surgically resected tumor tissue specimens correlated well with GM‐CSF and IL‐8 production from the corresponding cell lines. Among six lung cancer cell lines, CTL were induced in the three lung cancer cell lines that produced a lower amount of TGF‐β (<100 pg/mL). These findings suggested that TGF‐β produced by tumor cells could inhibit the induction of CTL in vitro. The present results suggest that the production of various cytokines from tumor cells might exert various paracrine effects both in vivo and in vitro. (Cancer Sci 2007; 98: 1048–1054)

Identification of a New Cancer/Germline Gene, KK-LC-1, Encoding an Antigen Recognized by Autologous CTL Induced on Human Lung Adenocarcinoma

The purpose of our present study is to identify a tumor-specific antigen capable of inducing a specific cellular immune response in lung cancer patients. We established a lung adenocarcinoma cell line, designated as F1121L, and induced tumor-specific CTL clone H1 from regional lymph node lymphocytes of patient F1121. CTL clone H1 lysed autologous tumor cells in an HLA-B*1507-restricted manner, but not autologous EBV-B, phytohemagglutinin-blast cells, and K562. The CTL clone also recognized allogeneic HLA-B*1501- or 1507-positive lung cancer cell lines in the HLA-restricted manner. Using the CTL clone, we identified an antigen-coding gene by cDNA expression cloning technique. The gene consisted of 556 bp, including an open reading frame consisted of 113 amino acids, designated as Kita-kyushu lung cancer antigen 1 (KK-LC-1). A 9-mer peptide (KK-LC-1(76-84); RQKRILVNL) was identified as an epitope peptide. The genomic DNA of this antigen was located in chromosome Xq22. A reverse transcription-PCR analysis revealed that the mRNA of this gene was only expressed in the testis among normal tissues. It was expressed in 9 of 18 (50%) allogeneic non-small-cell lung cancer cell lines and in 40 of 100 (40%) non-small-cell lung cancer tissues. We thus identified a new tumor antigen-coding gene categorized as a cancer/germline gene by an autologous lung cancer and CTL system. The new cancer/germline gene was located in Xq22, which is apparently different from the locations of previously reported cancer/germline genes.

Identification of the HLA-Cw*0702-Restricted Tumor-Associated Antigen Recognized by a CTL Clone from a Lung Cancer Patient

Purpose: A large number of tumor-associated antigens have been used in vaccination trials for mainly melanomas. Our purpose of this study is to identify a novel tumor antigen useful for immunotherapy of lung cancer patients. Experimental Design: Analysis of an autologous tumor-specific CTL clone F2a that was established from regional lymph node lymphocytes of a patient with lung cancer (A904) by a mixed lymphocyte-tumor cell culture. Results: F2a recognized and killed autologous tumor cells (A904L), whereas it did not respond to autologous EBV-transformed B cells, phytohemagglutinin-blastoid T cells, and K562 cells. cDNA clone 31.2 was isolated by using cDNA expression cloning method as a gene encoding antigen. This gene was identical to the reported gene whose function was unknown. The antigen encoded by the cDNA was recognized by the CTL in a HLA-Cw*0702-restricted manner. Furthermore, a 9-mer peptide at positions 659 to 685 in cDNA clone 31.2 was identified as a novel epitope peptide. The CTL recognized some allogeneic cancer cell lines with HLA-Cw*0702 as well as some HLA-Cw*0702-negative cell lines when transfected with HLA-Cw*0702, thus indicating that the identified antigen was a cross-reactive antigen. Conclusions: Although exact mechanism to process the encoded protein and present the antigen in the context of HLA class I remains to be elucidated, the CTL recognized some of tumor cells in the context of HLA-Cw*0702 but did not recognize a variety of normal cells and also autologous EBV-transformed B cells. These results indicated that the antigen identified in this study may therefore be a possible target of tumor-specific immunotherapy for lung cancer patients.

Identification of HLA‐A24 restricted shared antigen recognized by autologous cytotoxic T lymphocytes from a patient with large cell carcinoma of the lung

The aim of the present study was to elucidate the tumor‐specific cellular immunological responses occurring in a patient with large cell carcinoma of the lung who had no evidence of recurrence following surgical resections of both a primary lung lesion and a metastatic adrenal lesion. We analyzed an autologous tumor‐specific cytotoxic T lymphocytes (CTL clone F2b), which were HLA‐A*2402 restricted from regional lymph node lymphocytes. The F2b possessed T cell receptor (TCR) using the Vα5 and Vβ7 gene segment. The existence of precursor CTL (pCTL) against autologous tumor cells (A904L) was analyzed using CTL clone‐specific PCR. Lymphocytes with the same TCR as F2b were detected in the primary tumor tissue, regional lymph node and the peripheral blood collected from the patient 3 years after the operation. Using the F2b, we identified a cDNA clone encoding the tumor antigen using cDNA expression cloning method. The gene was found to encode splicing variant of the Tara gene. Finally, we identified the 9‐mer Ag peptide, using constructions of mini‐genes. The F2b recognized 3 out of 7 HLA‐A24 positive allogeneic tumor cell lines and in 1 out of 7 HLA‐A24 negative allogeneic tumor cell lines when transfected with HLA‐A24. This peptide is therefore considered to be potentially useful for performing specific immunotherapy in a significant proportion of lung cancer patients bearing HLA‐A24.

Reevaluation of erythropoietin production by the nephron.

Erythropoietin production has been reported to occur in the peritubular interstitial fibroblasts in the kidney. Since the erythropoietin production in the nephron is controversial, we reevaluated the erythropoietin production in the kidney. We examined mRNA expressions of erythropoietin and HIF PHD2 using high-sensitive in situ hybridization system (ISH) and protein expression of HIF PHD2 using immunohistochemistry in the kidney. We further investigated the mechanism of erythropoietin production by hypoxia in vitro using human liver hepatocell (HepG2) and rat intercalated cell line (IN-IC cells). ISH in mice showed mRNA expression of erythropoietin in proximal convoluted tubules (PCTs), distal convoluted tubules (DCTs) and cortical collecting ducts (CCDs) but not in the peritubular cells under normal conditions. Hypoxia induced mRNA expression of erythropoietin largely in peritubular cells and slightly in PCTs, DCTs, and CCDs. Double staining with AQP3 or AE1 indicated that erythropoietin mRNA expresses mainly in β-intercalated or non α/non β-intercalated cells of the collecting ducts. Immunohistochemistry in rat showed the expression of HIF PHD2 in the collecting ducts and peritubular cells and its increase by anemia in peritubular cells. In IN-IC cells, hypoxia increased mRNA expression of erythropoietin, erythropoietin concentration in the medium and protein expression of HIF PHD2. These data suggest that erythropoietin is produced by the cortical nephrons mainly in the intercalated cells, but not in the peritubular cells, in normal hematopoietic condition and by mainly peritubular cells in hypoxia, suggesting the different regulation mechanism between the nephrons and peritubular cells.

Identification of tumor associated antigens recognized by IgG from tumor-infiltrating B cells of lung cancer: correlation between Ab titer of the patient's sera and the clinical course.

We previously demonstrated that TIB recognize tumor antigens and produce antibodies against them. In the present study, we identified three tumor antigens recognized by TIB in lung cancer and evaluated whether changes in the antibody titer against these antigens correlated with the patients clinical course. A lung cancer cell line, G603L, was established from a primary lung tumor of a patient, G603. Seven months later, adrenal metastasis was detected and surgically resected. The latter tumor was mildly infiltrated with B cells and xenotransplanted into SCID mice to obtain human IgG. A cDNA library was constructed from G603L and SEREX was carried out using TIB‐derived IgG. The seroreactive clones were sequenced and one of these antigens was revealed to be MAGE‐B2 whereas the others were novel antigens. In the immunomonitoring of the patients sera, high antibody titer against MAGE‐B2 was observed before operation and the titer decreased after resection of the primary tumor. It was elevated again at the time of adrenal metastasis, but then decreased after resection. The change in antibody titer against the second antigen was similar to MAGE‐B2, and the antibody titer against the third antigen was low before the primary operation but increased at the time of recurrence. Our results suggest that TIB recognized tumor antigens and the antibody titers against these antigens were changed along with the patients clinical course. Therefore, these antibodies could be used as tumor markers for the patient. (Cancer Sci 2005; 96: 882–888)

Antitumor Effect of Antibody against a SEREX-Defined Antigen (UOEH-LC-1) on Lung Cancer Xenotransplanted into Severe Combined Immunodeficiency Mice

We previously reported the humoral immune response of tumor-infiltrating B lymphocytes in a lung cancer patient and 22 genes coding tumor-associated antigens identified using the serological identification of antigens by recombinant expression cloning method. In this study, we investigated one of these genes, designated University of Occupational and Environmental Health-Lung cancer antigen-1 (UOEH-LC-1), which has an extracellular domain. Quantitative reverse transcription-PCR revealed that UOEH-LC-1 was expressed ubiquitously in the normal tissues tested. However, it was overexpressed in 5 of 11 (45.5%) lung cancer cell lines and also in 9 of 15 (60%) lung cancer tissues compared with the paired normal lung tissues. A sequence analysis revealed that UOEH-LC-1 has a transmembrane domain. Flow cytometry analysis using a polyclonal antibody against UOEH-LC-1 revealed positive staining on lung cancer cell lines that were positive for expression of mRNA of UOEH-LC-1. Phage plaque assay showed the specific reactivity of anti-UOEH-LC-1 antibody against UOEH-LC-1 protein derived from the antigen encoding phage. By immunohistochemical staining with the anti-UOEH-LC-1 antibody, 7 of 28 (25.0%) lung cancer specimens showed positive staining on the cell surface. The administration of anti-UOEH-LC-1 antibody inhibited the growth of the UOEH-LC-1-positive tumors that were xenotransplanted into severe combined immunodeficiency mice. Complement-dependent cytotoxicity was one of the mechanisms to suppress the tumor growth. These results suggest that the antibody against UOEH-LC-1 therefore seems to have a promising therapeutic potential as a treatment for lung cancer.

Helicobacter pylori, a carcinogen, induces the expression of melanoma antigen-encoding gene (Mage)-A3, a cancer/testis antigen

Cancer/testis antigens (CTAs) are known to be expressed in various cancer types but are minimally or not expressed in normal tissues except for germline tissues. CTAs are attractive targets for cancer immunotherapy and diagnosis because of their restricted expression. The mechanisms of CTAs expression are unclear because the inducers of CTAs expression remain to be elucidated. We hypothesized that carcinogens may induce the cellular expression of CTAs. To prove this, we attempted to inoculate Helicobacter pylori, a known carcinogen, in Meth-A cells, normal gastric cells, and normal splenocytes and induce the expression of a CTA. Melanoma antigen-encoding gene (Mage)-A3, one of the CTAs, was not expressed in both normal cells but in Meth-A cells inoculated with H. pylori. Furthermore, we performed limiting dilution using Meth-A cells inoculated with H. pylori and established derivative clone from Meth-A designated as Meth-A/pylori/3C3 which permanently express Mage-A3 after excluding H. pylori. We herein report the first successful induction of a CTA in a cell line via exposure to a carcinogenic agent. Furthermore, the establishment of Meth-A/pylori/3C3, which is Meth-A expressing Mage-A3, is considered to contribute to the resolution of the mechanism of CTAs expression.

Early gastric cancer frequently has high expression of KK-LC-1, a cancer-testis antigen

AIM To assess cancer-testis antigens (CTAs) expression in gastric cancer patients and examined their associations with clinicopathological factors. METHODS Eighty-three gastric cancer patients were evaluated in this study. Gastric cancer specimens were evaluated for the gene expression of CTAs, Kitakyushu lung cancer antigen-1 (KK-LC-1), melanoma antigen (MAGE)-A1, MAGE-A3 and New York esophageal cancer-1 (NY-ESO-1), by reverse transcription PCR. Clinicopathological background information, such as gender, age, tumor size, macroscopic type, tumor histology, depth of invasion, lymph node metastasis, lymphatic invasion, venous invasion, and pathological stage, was obtained. Statistical comparisons between the expression of each CTA and each clinicopathological background were performed using the χ2 test. RESULTS The expression rates of KK-LC-1, MAGE-A1, MAGE-A3, and NY-ESO-1 were 79.5%, 32.5%, 39.8%, and 15.7%, respectively. In early stage gastric cancer specimens, the expression of KK-LC-1 was 79.4%, which is comparable to the 79.6% observed in advanced stage specimens. The expression of KK-LC-1 was not significantly associated with clinicopathological factors, while there were considerable differences in the expression rates of MAGE-A1 and MAGE-A3 with vs without lymphatic invasion (MAGE-A1, 39.3% vs 13.6%, P = 0.034; MAGE-A3, 47.5% vs 18.2%, P = 0.022) and/or vascular invasion (MAGE-A1, 41.5% vs 16.7%, P = 0.028; MAGE-A3, 49.1% vs 23.3%, P = 0.035) and, particularly, MAGE-A3, in patients with early vs advanced stage (36.5% vs 49.0%, P = 0.044), respectively. Patients expressing MAGE-A3 and NY-ESO-1 were older than those not expressing MAGE-A3 and NY-ESO-1 (MAGE-A3, 73.7 ± 7.1 vs 67.4 ± 12.3, P = 0.009; NY-ESO-1, 75.5 ± 7.2 vs 68.8 ± 11.2, P = 0.042). CONCLUSION The KK-LC-1 expression rate was high even in patients with stage I cancer, suggesting that KK-LC-1 is a useful biomarker for early diagnosis of gastric cancer.

Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase

To study the impact of epigenetic changes on biological functions, the ability to manipulate the epigenetic status of certain genomic regions artificially could be an indispensable technology. “Epigenome editing” techniques have gradually emerged that apply TALE or CRISPR/Cas9 technologies with various effector domains isolated from epigenetic code writers or erasers such as DNA methyltransferase, 5-methylcytosine oxidase, and histone modification enzymes. Here we demonstrate that a TALE recognizing a major satellite, consisting of a repeated sequence in pericentromeres, could be fused with the bacterial CpG methyltransferase, SssI. ChIP-qPCR assays demonstrated that the fusion protein TALMaj-SssI preferentially bound to major chromosomal satellites in cultured cell lines. Then, TALMaj-SssI was expressed in fertilized mouse oocytes with hypomethylated major satellites (10–20% CpG islands). Bisulfite sequencing revealed that the DNA methylation status was increased specifically in major satellites (50–60%), but not in minor satellites or other repeat elements, such as Intracisternal A-particle (IAP) or long interspersed nuclear elements-1 (Line1) when the expression level of TALMaj-SssI is optimized in the cell. At a microscopic level, distal ends of chromosomes at the first mitotic stage were dramatically highlighted by the mCherry-tagged methyl CpG binding domain of human MBD1 (mCherry-MBD-NLS). Moreover, targeted DNA methylation to major satellites did not interfere with kinetochore function during early embryonic cleavages. Co-injection of dCas9 fused with SssI and guide RNA (gRNA) recognizing major satellite sequences enabled increment of the DNA methylation in the satellites, but a few off-target effects were also observed in minor satellites and retrotransposons. Although CRISPR can be applied instead of the TALE system, technical improvements to reduce off-target effects are required. We have demonstrated a new method of introducing DNA methylation without the need of other binding partners using the CpG methyltransferase, SssI.