Yuriko Matsuzaki

Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae

SummaryThe GAL4 gene positively regulating the expression of the gene cluster GAL7-GAL10-GAL1 in the yeast Saccharomyces cerevisiae was isolated for its ability to suppress a recessive mutation in that gene. When the isolated gene was incorporated into a multi-copy plasmid, the GAL cluster genes in the host chromosome partially escaped the normal control; a yeast that harbors the plasmid bearing the GAL4 gene synthesized the galactose-metabolizing enzymes encoded by the GAL cluster genes at a low but significant level in the absence of galactose. If the GAL7 gene was amplified along with GAL4 on the multi-copy plasmid, the constitutive synthesis of Gal-1-P uridylyl transferase encoded by GAL7 was further pronounced and the enzyme activity reached the level of the fully induced wild-type yeast. Such an escape synthesis of the GAL enzymes was not detected if GAL4 or both GAL4 and GAL7 were carried by a single-copy plasmid. The results suggest that the escape synthesis of GAL enzymes observed in the GAL4-amplified yeast was a consequence of overproduction of the GAL4 protein. The GAL80 gene negatively regulating the GAL cluster genes was also isolated, and when amplified together with GAL4, no escape synthesis of the GAL enzymes was observed, suggesting that the balanced synthesis of two regulatory proteins was essential to maintain the repressed state of the GAL cluster genes.

A New Melanoma Antigen Fatty Acid-Binding Protein 7, Involved in Proliferation and Invasion, Is a Potential Target for Immunotherapy and Molecular Target Therapy

The identification of molecules that are preferentially expressed in melanoma cells and involved in their malignant phenotypes is important for understanding melanoma biology and the development of new diagnostic and therapeutic methods. By comparing the expression profile of a melanoma cell line with those of various normal tissues using GeneChip and by confirming the actual expression of the selected genes by reverse transcription-PCR and Northern and Western blot analyses, fatty acid-binding protein 7 (FABP7), which is frequently expressed in melanomas, was identified. Immunohistochemical examination revealed that FABP7 was expressed in 11 of 15 melanoma tissues. By down-regulating the FABP7 expression with FABP7-specific small interfering RNAs, in vitro cell proliferation and Matrigel invasion were suppressed in two of six melanoma cell lines. Overexpression of FABP7 in a FABP7-negative embryonic kidney cell line 293T by transfecting with the FABP7 cDNA resulted in enhanced cell proliferation and Matrigel invasion, indicating that FABP7 plays a role in the malignant phenotype of some melanoma cell lines. IgG antibodies specific for the phage or bacterial recombinant FABP7 protein were detected in 14 of 25 (56%) or in 8 of 31 (26%) sera from melanoma patients, respectively, but not in sera from healthy individuals, indicating that FABP7 is an immunogenic antigen in melanoma patients. These results showed that FABP7 is frequently expressed in melanoma, may be involved in cell proliferation and invasion, and may be a potential target for development of diagnostic and therapeutic methods.

Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae - II. The isolation and dosage effect of the regulatory gene GAL80

SummaryThe galactose analogue 2-deoxygalactose was found to inhibit the growth of a mutant strain of Saccharomyces cerevisiae constitutively producing the set of galactose utilization enzymes. Based on this fact, the yeast GAL80 gene negatively regulating the expression of the genes encoding those enzymes was isolated for its ability to confer 2-deoxygalactose resistance on a strain carrying a recessive mutation in that gene. The GAL80 gene was located within a 3.0 kb fragment in the cloned DNA. When the isolated gene was incorporated into a multi-copy plasmid, the induced level of three enzymes encoded by the gene cluster GAL7-GAL10-GAL1 in the host chromosome was lowered. Such a gene dosage effect of GAL80 was further pronounced if sucrose, a sugar causing catabolite repression, was added to the growth medium. The ratio of the enzyme activity of the yeast bearing multiple copies of GAL80 to that of the yeast bearing its single copy significantly varied with the enzyme. From these results we suggest that the intracellular inducer interacts with the GAL80 product and that GAL80 molecules directly bind the GAL cluster genes with an affinity different from one gene to another.

A Novel Cancer Testis Antigen That Is Frequently Expressed in Pancreatic, Lung, and Endometrial Cancers

Purpose: To isolate cancer testis antigens that are expressed in pancreatic cancers and may be useful in clinical applications. Experimental Design: To efficiently isolate cancer testis antigens, a testis cDNA library was immunoscreened (SEREX) with serum from a patient with pancreatic ductal adenocarcinoma. The expression of isolated antigens in various cancer cell lines and tissues was evaluated by reverse transcription-PCR and Northern blot analyses. The immunogenicity of the antigen in cancer patients was evaluated by detection of the IgG antibody in sera from patients with various cancers. Results: Of the three clones isolated through screening of a total of 2 × 106 cDNA library clones, one clone (KU-CT-1) was found to be expressed in various cancers but only in testis among normal tissues, indicating that it was a novel cancer testis antigen. The KU-CT-1 gene is located on chromosome 10p12 and produces two splice variants, which encode proteins of 397 and 872 amino acids, respectively. KU-CT-1 was expressed in pancreatic cancer tissues (3 of 9, 33%), lung cancer tissues (9 of 24, 38%), and endometrial cancer tissues (7 of 11, 64%). Specific serum IgG antibodies were detected in 3 of 20 pancreatic cancer patients, 2 of 12 endometrial cancer patients, 1 of 18 colon cancer patients, and 1 of 10 prostate cancer patients but not detected in 30 healthy individuals. Conclusions: KU-CT-1 is a new cancer testis antigen that is expressed in pancreatic, lung, and endometrial cancers and may be useful for diagnosis and immunotherapy for patients with various cancers.

Novel melanoma antigen, FCRL/FREB, identified by cDNA profile comparison using DNA chip are immunogenic in multiple melanoma patients

We applied a strategy that utilized a combination of systematic gene expression analysis with various tissues and immunological detection with sera from melanoma patients to identify melanoma antigens expressed preferentially in melanoma and melanocytes. We selected 101 genes by comparing cDNA profiles obtained by GeneChip analysis of a highly pigmented melanoma cell line, SKmel23, primary cultured melanocytes, HUVECs cultured endothelial cells, keratinocytes, liver and stomach. After the additional selection with criterion of high registered frequency of each cDNA in melanocyte‐related cDNA libraries in the NCBI database, 15 genes including 12 known melanocyte specific genes were identified. One of the remaining 3 genes, FCRL/FREB, encoding a member of the Fc receptor family that was previously reported to express in germinal center B cells, was found to express preferentially in melanocytes and melanoma tissues by RT‐PCR and Northern blot analysis. The FCRL/FREB protein was detected in the cytoplasm of melanoma cells by staining with the murine polyclonal antibody and by transfection with GFP‐fused FCRL/FREB cDNA. The bacterial recombinant protein was recognized by serum IgG antibody obtained from some patients with melanoma. These results suggest that FCRL/FREB may function in melanocytes and melanoma and may be useful for development of diagnostic methods for various pigment disorders and immunotherapy of melanoma.

Immune responses to DNA mismatch repair enzymes hMSH2 and hPMS1 in patients with pancreatic cancer, dermatomyositis and polymyositis.

To identify tumor antigens useful for diagnosis and immunotherapy of patients with pancreatic ductal adenocarcinoma, we applied a SEREX approach with a cDNA library made from 5 pancreatic cancer cell lines and sera obtained from 8 patients with pancreatic cancer, and isolated total 32 genes, including 14 previously characterized genes and 18 genes with unknown functions. Among these isolated antigens, serum IgG antibodies for 2 isolated DNA mismatch repair enzymes, Homo sapiens mutS homolog 2 (hMSH2) and Homo sapiens postmeiotic segregation increased 1 (hPMS1), were detected in patients with pancreatic ductal adenocarcinoma and dermatomyositis (DM), and polymyositis (PM), but not in sera from healthy individuals. Immunohistochemical study demonstrated that hMSH2 and hPMS1 were over‐expressed in pancreatic ductal adenocarcinoma compared to normal pancreatic ducts. These results suggested that hMSH2 and hPMS1 may be useful as CD4+ helper T cell antigens for immunotherapy of pancreatic cancer patients and that serum IgG antibodies may be useful for diagnosis of patients with pancreatic ductal adenocarcinoma and DM/PM.

Frequent immune response to a melanocyte-specific protein KU-MEL-1 in patients with Vogt-Koyanagi-Harada disease

Aim: To isolate autoantigens possibly involved in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease. Methods: Autoantigens recognised by immunoglobulin G antibodies (IgG Ab) in sera from VKH patients were isolated by screening the lambda phage cDNA libraries made from melanocytes and a highly pigmented melanoma cell line with the patients’ sera. Presence of IgG specific for the autoantigens in sera from patients with various panuveitis and healthy individuals was evaluated. Relation between the specific IgG and various clinicopathological features was examined. Results: KU-MEL-1 was found to be one of the 81 isolated positive clones representing 35 distinct genes, which is a previously isolated melanoma antigen preferentially expressed in melanocytes. The IgG Ab specific for KU-MEL-1 was detected in sera from patients with VKH in significantly higher amounts than in sera from patients with Behçet’s disease, sarcoidosis, and from healthy individuals. Positive serum KU-MEL-1 Ab was significantly associated with HLA-DRB1*0405 and male VKH patients. Conclusion: KU-MEL-1 was identified as a new autoantigen for VKH. The highly frequent induction of IgG Ab for KU-MEL-1 in HLA-DRB1*0405 positive VKH patients may suggest the possible involvement of KU-MEL-1 specific CD4+ T cells in the pathogenesis of VKH, suggesting the possible use in the development of diagnostic and therapeutic treatments for VKH patients.

Identification of bladder cancer antigens recognized by IgG antibodies of a patient with metastatic bladder cancer

To identify tumor antigens useful for the diagnosis and treatment of patients with bladder cancer, a lambda phage cDNA library constructed from a high‐grade bladder cancer cell line was screened with autologous serum from a patient with metastatic bladder cancer. Forty‐eight distinct antigens were isolated. By evaluating the immunogenicity and the tissue‐specific expression, KU‐BL‐1 and KU‐BL‐2 were identified as immunogenic antigens with restricted tissue expression. KU‐BL‐1 was found to be a putative human lipoic acid synthetase with a metal‐binding site, CXXXCXXC, that was expressed in bladder cancer cell lines and most bladder cancer tissues, as well as normal bladder mucosa and testis tissues. Immunoglobulin (Ig)G antibody to KU‐BL‐1 was detected in 2 of 28 patients with bladder cancer, but not in 30 healthy individuals. KU‐BL‐2 was found to be a putative human kelch‐like protein that was homologous to Drosophila kelch, with a BTB/POZ domain and kelch repeats. KU‐BL‐2 was expressed in bladder cancer cell lines, most bladder cancer tissues, testis and heart, but not in normal bladder mucosa. IgG antibody to KU‐BL‐2 was detected in 8 of 28 patients with bladder cancer, but not in 16 healthy individuals. Tumor reactive T cells were induced from peripheral blood mononuclear cells (PBMC) by stimulation with one of the HLA–A24 binding KU‐BL‐2 peptides. Therefore, KU‐BL‐1 and KU‐BL‐2, which showed preferential expression in bladder cancer with restricted expression in normal tissues, as well as immunogenicity in multiple patients with bladder cancer, may be useful for the development of diagnostic and therapeutic methods for patients with bladder cancer.

Identification of a Novel Cancer-Testis Antigen CRT2 Frequently Expressed in Various Cancers Using Representational Differential Analysis

Purpose: Cancer-testis antigens are promising targets for cancer immunotherapy. Identification of additional cancer-testis antigens with frequent expression in various cancers was attempted using representational differential analysis (RDA) and immunogenicity evaluation. Experimental Design: cDNAs preferentially expressed in testis were enriched using RDA by subtraction between testis and normal tissues. Thirty clones showing cancer-testis–like expression based on EST database analysis were evaluated by reverse transcription-PCR. A potential antigen, CRT2, was identified and its expression was analyzed with a newly generated anti-CRT2 antibody. The immunogenicity of CRT2 was examined based on reactivity with serum immunoglobulin G (IgG) from cancer patients, using Western blot and ELISA analysis, and on in vitro induction of tumor-reactive CTLs from HLA-A24 transgenic mice and human peripheral blood lymphocytes. Results: CRT2 was expressed in elongated spermatids of testis among normal tissues and in various cancer cell lines and tissues. The recombinant CRT2 protein was recognized by serum IgG from patients with various cancers in Western blot and ELISA analyses. A CRT2-derived peptide was identified as an HLA-A24–restricted T-cell epitope that induced tumor-reactive CTLs. Conclusion: CRT2 was identified as a new cancer-testis antigen expressed in elongated spermatids of testis and in cancer tissues (particularly melanoma) that is recognized by serum IgG from cancer patients. An HLA-A24–restricted T-cell epitope capable of inducing tumor-reactive CTLs was identified, suggesting that CRT2 may be useful for cancer diagnosis and immunotherapy.

Immunological detection of altered signaling molecules involved in melanoma development

To understand immune responses to human cancer and develop more effective immunotherapy, human tumor antigens has been isolated using various immunological methods with tumor reactive T cells or antibodies obtained from patients with melanoma. During the process of tumor antigen isolation, various molecules with genetic alterations or over-expression in tumor cells, which may be involved in proliferation, differentiation, or survival of various cancer cells, were identified. In melanoma, abnormal molecules with mutations including β -catenin, CDK4, and BRAF, and molecules with increased expression including Survivin, were immunologically detected. Therefore, immunological isolation of human tumor antigens contributes to the identification of important molecules including altered signaling molecules involved in melanoma formation.