Yusuke Makiguchi

Expression of c-kit and kit Ligand in Human Colon Carcinoma Cells

To determine whether c-kit and kit ligand (KL) mRNAs could be expressed in human epithelial tumors, reverse transcriptase-polymerase chain reaction and Northern blot analysis were performed. KL mRNA was shown to be expressed in a variety of epithelial tissues and cell lines. The expression of c-kit mRNA was then examined in hepatocellular and colon carcinoma cell lines. While hepatocellular carcinoma cell lines did not express c-kit mRNA as far as we could ascertain, 2 of 5 colon carcinoma cell lines showed the expression of both c-kit and KL mRNAs. Furthermore, the expression of c-kit in these cells was demonstrated at the protein level by flow cytometry. These data suggest that c-kit and KL may play an important role as an autocrine loop in the proliferation of some colon carcinoma cells.

Detection of circulating anti-MUC1 mucin core protein antibodies in patients with colorectal cancer.

Abstract: MUC1 mucin has a unique immunogenic peptide epitope in the extracellular domain, which has been shown to induce humoral and cellular immune response. In this study, we evaluated the pathophysiological significance of circulating anti-MUC1 mucin core protein IgG antibodies (anti-MUC1 antibodies) in colorectal cancer by Western blot analysis and 51Cr release assay. Anti-MUC1 antibodies were detected in 5 of 31 (16.1%) healthy subjects and in 27 of 56 (48.2%) patients with colorectal cancer. The presence of circulating anti-MUC1 antibodies was not significantly correlated with the level of circulating antigen MUSE11 or with other clinicopathological parameters tested. The incidence of positivity for anti-MUC1 antibodies in stage I and II (staged according to the General Rules for Clinical and Pathological Studies on Cancer of the Colon and Rectum of the Japanese Research Society for Cancer of the Colon and Rectum) cancers was 45.5% and 58.8%, respectively, suggesting that positivity for these antibodies may be of use as an adjunct for the diagnosis of colorectal cancer in the early stages in the absence of serious complications such as liver diseases. Because of the epitope similarity, anti-MUC1 antibodies in the serum may function in a manner similar to that of anti-MUC1 peptide monoclonal antibodies (mAbs). We therefore observed antibody-dependent cell mediated cytotoxicity with anti-MUC1 peptide mAb using MUC1 cDNA-transfected colon cancer CHC-Y1 cells as the target. The decreased sensitivity of MUC1 transfectants to effector cells was restored to a level equivalent to that in control cells. These data suggest that the detection of circulating anti-MUC1 antibodies may be a useful adjunct for the early diagnosis and immunological analysis of colorectal cancer.

Effect of MUC1 Mucin, an Anti‐adhesion Molecule, on Tumor Cell Growth

MUC1 mucin is expressed in a wide variety of tumors and is considered to function as an anti‐adhesion molecule which inhibits cell‐to‐cell interactions. To reveal the biological significance of this activity in tumor cells, MUC1 cDNA was transfected into EJNIH3T3 cells and human colon cancer cell lines, CHCY1 and DLD1. The in vivo growth rate of MUC1+ (MUC1‐transfected) EJNIH3T3, CHCY1 and DLD1 cells in SCID mice was clearly lower than that of MUC1− (mock transfectant) cells. Several in vitro experiments using MUC1+ EJNIH3T3 cells were performed to analyze the mechanisms for the decreased in vivo tumor growth. It was found that (i) the in vitro growth rate of MUC1+ EJNIH3T3 cells was also decreased compared to that of MUC1− cells, (ii) the DNA synthesis of MUC1+ EJNIH3T3 cells after stimulation with either growth factor (fetal calf serum or bombesin) or extracellular matrix (collagen or fibronectin) was lower than that of MUC1− cells, and (iii) MUC1+ EJNIH3T3 cells grew more slowly than MUC1− cells on both collagen‐ and fibronectin‐coated dishes. These data suggest that MUC1 mucin may regulate tumor cell growth through inhibition of cell‐to‐cell, growth factor‐to‐receptor and cell‐to‐matrix interactions.

Increased invasiveness of MUC1 and cDNA-transfected human gastric cancer MKN74 cells

MUC1 mucin is an anti‐adhesion molecule expressed in a wide variety of tumors. To examine whether MUC1 mucin is involved in tumor invasion, we have prepared MUC1 transfectants using the human gastric cancer cell line MKN74 and performed an in vivo tumor assay by transplanting these into nude mice. Tumor weight at 71 days after s.c. injection of transfectants was measured, showing that the in vivo growth of MUC1 transfectants was increased compared to that of mock transfectants. Furthermore, MUC1‐transfectant tumors invaded into the muscle layer, whereas mock‐transfectant tumors did not. In vitro invasion, adhesion to extracellular matrix components and phagokinetic track motility were then evaluated to analyze the mechanisms for the in vivo invasiveness of the transfectants. MUC1 transfectants exhibited an increased in vitro invasiveness, decreased binding to laminin, fibronectin, type I collogen and type IV collagen and increased motility. These effects of MUC1 mucin over‐expression in MKN74 cells were abolished by the treatment of transfectants with an inhibitor of O‐glycan biosynthesis, benzyl‐α‐GalNAc. Our data suggest that MUC1 mucin could be related to the increased invasive ability of MKN74 cells, whereas O‐glycan might play an essential role. Int. J. Cancer 76:377–382, 1998.© 1998 Wiley‐Liss, Inc.

ENHANCEMENT OF REACTIVITY OF ANTI-MUC1 CORE PROTEIN ANTIBODY AND KILLING ACTIVITY OF ANTI-MUC1 CYTOTOXIC T CELLS BY DEGLYCOSYLATION OF TARGET TISSUES OR CELLS

Abstract: MUC1 mucin core protein contains an important tumor-associated peptide antigen that can induce cytotoxic T cells (CTLs) in vivo, although this antigen is generally masked by mucin-type glycans. To reveal the precise expression pattern of MUC1 protein in normal and neoplastic gastric tissues, we performed immunohistochemical staining of periodic acid-treated tissue sections with an anti-MUC1 core protein monoclonal antibody (mAb), MUSE11. In non-cancerous tissues, the deep portion of fundic glands and the luminal surface were predominantly immunostained in normal and metaplastic glands, respectively. In cancerous tissues, the incidence of positivity for MUC1 protein varied from 67% to 88%, depending on histological type. This frequent expression of MUC1 protein in cancer tissues after periodic acid treatment suggested that deglycosylation may be of use for exposing the target antigen of anti-MUC1 CTLs. Accordingly, we then examined the effect of benzyl-α-GalNAc, an inhibitor of O-glycan biosynthesis, on the expression of MUC1 protein and sensitivity to an anti-MUC1 CTL line, designated TS, in gastric cancer JRST cells. After incubation with benzyl-α-GalNAc, the reactivity of mAb MUSE11 with JRST cells and their sensitivity of TS were clearly increased. These findings suggest that deglycosylation may offer an important strategy for enhancing anti-tumor immunity in patients with gastric cancer.

Expression of cytoskeletal-associated protein tyrosine phosphatase PTPH1 mRNA in human hepatocellular carcinoma

We investigated the mRNA expression of cytosolic protein tyrosine phosphatase (PTPH1), which has a homologous domain to cytoskeletal-associated proteins, in human hepatocellular carcinomas (HCCs) by using reverse transcriptase-polymerase chain reaction (RT-PCR). PTPH1 mRNA was detected in all HCC cell lines (n=6), and HCC and adjacent noncancerous tissues (n=8) examined, indicating that PTPH1 was expressed in HCCs and hepatocytes. There was no remarkable difference in the level expression of PTPH1 mRNA between HCC and adjacent noncancerous tissues. We also performed RT-PCR single-strand conformation polymorphism (SSCP) analysis in HCC cell lines and tissues in the C-terminal region of the catalytic domain of PTPH1. In the cHc4 cell line and a HCC tissue specimen, a shifted band was detected, although it was not found in the noncancerous tissue of the HCC specimen. Nucleotide sequence analysis showed a common mutation from T to C at the third letter of codon 919 which did not lead to amino acid substitution. These results suggest that another mutation leading to the development of HCC could occur in some region of PTPH1 other than that investigated in this study.

MUC1 as a human tumor marker

Abstract MUC1 mucin serves as a tumor-associated antigen in various carcinomas. We have reported the expression of MUC1 in multiple myeloma (MM) cells and the establishment of an HLA-unrestricted cytotoxic T lymphocyte (CTL) that recognized MUC1 from peripheral lymphocytes in a patient with MM. In this study, we successfully induced MUC1-specific CTLs from bone marrow cells in two MM patients. Both CTLs lysed MUC1-positive cell lines with different kinds of HLA, and the cytotoxicity was inhibited by anti-CD3, anti-αβ T cell receptor and anti-MUC1 monoclonal antibodies. The treatment with O-glycosylation inhibitor augmented the cytotoxicity of the CTLs. These data showed that HLA-unrestricted CTLs that recognize the underglycosylated form of MUC1 could be induced from MM patients. MUC1 is also an important factor in cancer metastasis. We previously reported that MUC1 mucin has anti-adhesion effects that contribute to the separation of tumor cells from the primary site. We next investigated the involvement of MUC1 in tumor invasion that is a second step of metastasis. MUC1-transfectants transplanted subcutaneously into nude mice invaded the muscular layer. The treatment with O-glycosylation inhibitor abolished these effects of MUC1. Our data indicated that MUC1 mucin increases the invasive ability of tumor cells, while O-glycan plays an essential role.

A case of Wegener’s granulomatosis with intracranial involvement

We report a case of Wegener’s granulomatosis (WG) associated with intracranial involvement and brain magnetic resonance imaging (MRI) findings. A 44-year-old woman was diagnosed as having WG with involvement of an ear, lungs and kidneys. A brain computed tomography scan showed a space-occupying lesion with a ring-like enhancement in the left temporal lobe. Findings on a brain MRI, different from those characteristic of a brain abscess, supported an intracerebral granulomatous inflammation due to WG. Immunosuppression, which included corticosteroids, was an effective treatment.