Keisuke Teshigawara

Inhibition of proliferation and induction of apoptosis by abrogation of heat-shock protein (HSP) 70 expression in tumor cells

Tumor cells often express elevated levels of heat-shock protein (HSP) 70. The present study was designed to invesitgate the role of HSP70 in the proliferation and survival of tumor cells in the human system. When Molt-4 and other tumor cells were treated in vitro with HSP70 antisense oligomer, they displayed propidiumiodide-stained condensed nuclei (intact or fragmented). A ladder-like pattern of DNA fragments was observed with HSP70 antisense-oligomer-treated tumor cells in agrose gel electrophoresis, which was consistent with internucleosomal DNA fragmentation. Flow cytometry analysis revealed the hypodiploid DNA peak of propidium-iodide-stained nuclei in the antisense-oligomer-treated cells. The apoptosis induced by HSP antisense oligomer was dose- and time-dependent. The antisense oligomer induced apoptosis mainly in tumor cells at G1 and S phase, resulting in an inhibition of cell proliferation. HSP70 antisense oligomer caused DNA-sequence-specific inhibition of HSP70 expression, which preceded apparent apoptosis. These results indicate that HSP70 antisense treatment inhibits the expression of HSP70, which in turn inhibits cell proliferation and induces apoptosis in tumor cells and suggest that HSP70 is required for tumor cells to proliferate and survive under normal condition.

Transglutaminase activity during the differentiation of macrophages.

Summary Transglutaminase (R-glutaminyl-peptide:amine γ-glutamyltransferase, E.C. 2,3,2,13) activity during the differentiation of murine leukemia cell lines (M1 cells) was investigated. Ml cells contained a significant transglutaminase activity of the tissue type. During the course of differentiation into mature macrophage-like M1 + cells induced with a protein inducer, the enzymatic activity was stimulated more than ten times as much as in the original undifferentiated Ml − cells. A remarkable enhancement of enzymatic activity was also observed when lipopolysaccharide was utilized as an inducer of differentiation. The enzymatic activity of undifferentiated M1 − cells was eluted at the region of M.W. ca. 80,000 as a single and symmetrical peak on Sepharose 4B column chromatography. By contrast, most of the activity in differentiated Ml + cells was eluted at the void volume under the same condition, though some activities were eluted at the same region as in Ml − cells. These data suggest that most of transglutaminase activity exists in the form of a high-molecular-weight complex with some cellular components in differentiated cells. The possible physiological significance of the enzyme in macrophage functions was discussed.

Accumulation of γ/δ T Cells in Human Dysgerminoma and Seminoma: Roles in Autologous Tumor Killing and Granuloma Formation

The precise biological function of a subset of T cells bearing gamma/delta T cell receptor (TCR) remains poorly understood. The present study demonstrated the presence of gamma/delta T cells in tumor-infiltrating lymphocytes (TIL) and peripheral blood lymphocytes (PBL) of human patients with dysgerminoma and seminoma when determined by flow cytometry and in situ immunohistochemical staining. TIL contained a high percentage of gamma/delta T cells, ranging from 17.3 to 35.1%. gamma/delta T cells often accumulated within the granulomatous inflammation of tumor tissues. The majority of gamma/delta T cells were V gamma 9/V delta 2+ cells. Freshly isolated PBL, TIL and purified gamma/delta T cells showed autologous tumor killing (ATK) activity, which could be inhibited by monoclonal antibodies (mAb) against V delta 2. Furthermore, two gamma/delta T cell clones established from TIL showed cytotoxicity against autologous and allogeneic dysgerminoma, while they had low or no lytic effects on other cell types including carcinomas of ovary and tumor cell lines such as K562, Daudi and Molt-4. Lysis of autologous tumor cells by the clone was inhibited completely by anti-V delta 2 mAb and partially by mAb against CD3, LFA-1 alpha and ICAM-1 molecules, while it was resistant to anti-CD8, anti-HLA-ABC and anti-HLA-DR mAb. Supernatants produced by gamma/delta T cell clones induced adhesion, aggregation and increased DNA synthesis of monocytes and some characteristics of activated macrophages or epithelioid cells. Tumor necrosis factor (TNF)-alpha, granulocyte-macrophage colony stimulating factor (GM-CSF) and interferon (IFN)-gamma were detected in the supernatants of gamma/delta T cell clone. These results suggest that gamma/delta T cells accumulating in dysgerminoma and seminoma exhibit ATK activity through V gamma 9/delta 2 TCR and these gamma/delta T cells also play a role in the formation of granulomatous inflammation, which is associated with human dysgerminoma and seminoma.

A reporter system using a flow cytometer to detect promoter/enhancer activity in lymphoid cell lines

We have devised an experimental system using a flow cytometer to examine the promoter/enhancer activity of DNA fragments in human lymphoid cell lines. Murine CD8 alpha gene cDNA used as a reporter gene was inserted in the reporter constructs under the control of various promoter/enhancers. Furthermore, the Epstein-Barr virus (EBV) OriP, which supports a high transient expression, was also included in the reporter constructs. Cell lines expressing EBV nuclear antigen-1 (EBNA-1) were transfected with the reporter constructs by electroporation. The expression of the reporter gene was measured by a flow cytometric analysis. This experimental system is quite simple and may be especially useful for the analysis of transcriptional elements functioning in lymphocytes.

Inhibition of human immunodeficiency virus replication in a human T cell line by antisense RNA expressed in the cell

The effect of the expression of antisense RNA against the human immunodeficiency virus (HIV) genome in a human T-cell line CEM on HIV replication was investigated. A 2.7 kilobase (kb) fragment of the HIV genome, includingtat and a part ofrev andenv, was cloned into the retroviral vector WB in the antisense orientation under the SV40 or H-2K promoter. CEM cells transduced with this antisense gene via recombinant retrovirus expressed the RNAs of three different molecular sizes containing the antisense construct. CEM cells and these transduced cells were infected with HIV. HIV replication was evaluated 4–10 days later by an immunofluorescense assay and by determining the reverse transcriptase activity in the culture supernatant. The results indicate that although the recombinant retrovirus WB strongly enhanced the HIV replication in CEM cells, the expression of antisense RNA in the cells was highly effective in impeding the replication of HIV. The inhibitory effect was especially high in CEM cells transduced with the antisense gene under the control of SV40 promoter. In this case, HIV antigen-positive cells and reverse transcriptase activity in the culture supernatant of transduced cells were reduced to 30–50% and 5–10% of those in CEM cells and in the CEM cells transduced with WB, respectively.

Possible Involvement of Transglutaminase in Endocytosis and Antigen Presentation

Experiments were carried out to determine as to whether or not internalization of antigen is necessary for subsequent antigen presentation by accessory cells using monoamines which are known as transglutaminase (TGase) inhibitors. It was found that endoeytosis for immune complexes via Fc receptors such as sheep erythrocytes coated with IgG class antibody (EA) was different from receptor‐independent endoeytosis for soluble protein such as horse radish peroxidase (HRP) in the sensitivity to monoamines; methylamine inhibited the receptor‐dependent endoeytosis of immune complexes at a concentration of over 20 mM and the receptor‐independent endoeytosis of HRP at 2 mM, while dansylcadaverine (DC) inhibited both at a concentration of 100 μM. It was noteworthy that antigen‐specific T cell proliferation to splenic adherent cells pulsed with DNP9,6‐ovalbumin (DNP9,6‐OVA) was blocked strongly by DC as well, but weakly by methylamine. These results suggest the possibility that antigen presentation requires internalization of antigen by a mechanism such as receptor‐dependent endoeytosis for the subsequent reexpression of antigen on membranes. Furthermore, it was confirmed that TGase activity is high in peritoneal exeudate and spleen adherent cells, both of which have accessory cell activities for lymphocytes, suggesting the possibility that TGase might be involved intimately in receptor‐dependent endoeytosis and subsequent antigen presentation.

IL-2 Receptor Gene Activation by ATL-Derived Factor (ADF)

The interaction of Interleukin 2 (IL-2) and its specific receptor, Interleukin 2 receptor (IL-2R) (1), plays an important role for the growth and activation of lymphoid cells. One of the unique features of lymphocytes in immune system is the selective proliferation of the clones specific for variety of the antigens. In mature T lymphocytes (T-cells) which express antigen-specific T-cell receptors, the cell growth driven by IL-2/IL-2-R interaction appears to be strictly regulated by the initiative signals elicited by the interaction between T-cell receptors and the corresponding antigens.

TCGF (IL-2) Receptor (Tac Antigen) on ATL and Non-ATL Leukemic Cells

Cell Lines. Hut-102 and MT-1 cells were supplied by Dr. R. C. Gallo and Dr. I. Miyoshi, respectively. ATL-2, ATL-6, and ATL-7 cell lines were established from Japanese patients with typical ATL (Yodoi et al., 1974; Uchiyama et al., 1977). A non-ATL leukemic cell line (YT cells) was established from a 14-year-old boy with acute lymphoblastic lymphoma with thymoma (in preparation).

BCL-2 Inhibits B7-Induced MHC-Unrestricted Cytolysis Mediated by a Human NK Cell Line

Recent work demonstrated that B7 expression by tumor cells can enhance antitumor immune responses. However, the B7 molecule is expressed abundantly on most non-Hodgkins B-cell lymphomas and solid lymphoid tumors. How these tumor cells escape from immune surveillance mechanisms remains unclear. Lately, it has become clear that bcl-2 oncogene is overexpressed in a wide variety of human cancers and renders tumor cells more resistant to cytolytic T-cells (CTL) mediated cytotoxicity. We cloned B7 and B7/Bcl-2 transfectants and compared their susceptibilities to a human natural killer (NK) cell line and normal NK cells. The results demonstrate that Bcl-2 oncoprotein in tumor cells blocks B7-induced cytolysis mediated by a NK cell line and NK cells. Thus, they suggest that Bcl-2 oncoprotein plays a role in tumor avoidance of effective antitumor cytotoxicity.