Shuji Takahashi

CELL ADHESION KINASE BETA FORMS A COMPLEX WITH A NEW MEMBER, HIC-5, OF PROTEINS LOCALIZED AT FOCAL ADHESIONS

Cell adhesion kinase β (CAKβ/PYK2) is the second protein-tyrosine kinase of the focal adhesion kinase subfamily. We identified a cDNA that encodes a CAKβ-binding protein. This cDNA clone encodes the human homologue of Hic-5, the cDNA of which was cloned in 1994 as transforming growth factor β1- and hydrogen peroxide-inducible mRNA. We found that Hic-5 exclusively localized at focal adhesions in a rat fibroblast line, WFB. This localization of Hic-5 was confirmed in WFB cells expressing Myc-tagged Hic-5. The amino acid sequence of Hic-5 is highly similar to that of paxillin in the four LD motifs as well as in the four contiguous LIM domains. The Hic-5 N-terminal domain directly associated in vitrowith the extreme C-terminal region (residue 801 to the end) of CAKβ. CAKβ was coimmunoprecipitated with Hic-5 from the WFB cell lysate. The coimmunoprecipitation of CAKβ with Hic-5 was markedly inhibited by the addition of the extreme C-terminal region of CAKβ. Coimmunoprecipitation of Hic-5 with CAKβ, which was shown in COS-7 cells doubly transfected with cDNA constructs of CAKβ and Myc-tagged Hic-5, was lost when the CAKβ amino acid residues 741–903 were deleted. Hic-5 was tyrosine-phosphorylated in Src-transformed 3Y1 cells and in cells treated with pervanadate. Hic-5 associated with CAKβ was selectively tyrosine-phosphorylated in WFB cells exposed to hypertonic osmotic stress. These results indicate that Hic-5 is a paxillin-related component of focal adhesions and binds to CAKβ, implying possible involvement of Hic-5 in the downstream signaling of CAKβ.

Prolonged survival of rat skin allograft by treatment with FK506 ointment

BACKGROUNDnWe studied the effect of FK506 ointment on rat skin allograft survival.nnnMETHODSnLewis rat (RT1(1)) skin allografts were histologically evaluated on day 7 after transplantation to recipient ACI (RT1a) rats. We set histological gradings for rejection as follows: grade 1, intraepidermal blister formation; grade 2, incomplete epidermal separation from the dermis; and grade 3, complete epidermal separation from the epidermis. According to these histological criteria, the immunosuppressive effect of FK506 ointment was assessed.nnnRESULTSnOur data indicated that all recipient rats without FK506 ointment showed grade 3 rejection on day 7 after transplantation. In contrast, the allografts treated with FK506 ointment showed grade 1 rejection. Furthermore the blood concentration of FK506 was below 0.5 ng/ml, minimizing the systemically adverse effects of FK506.nnnCONCLUSIONSnThus, the present study suggests that the topical administration of FK506 on the skin allografts may be useful and effective in the suppression of skin allograft rejection.

Demonstration of selective protein complexes of p53 with 73 kDa heat shock cognate protein, but not with 72 kDa heat shock protein in human tumor cells.

It has been demonstrated that p53, especially, mutant p53 (mp53), makes protein complexes with major heat shock proteins hsp72/hsc73. However, there is no direct evidence showing whether hsp72 or hsc73 could bind preferentially to p53. In the present study, using TYKnu human ovarial carcinoma cells and monoclonal antibodies reacting specifically to hsp72/hsc73, we were able to find the selective protein complex formation with p53, presumably mp53, and hsc73, but not in the case of p53 and hsp72. The p53-hsc73 protein complexes dissociate with the addition of ATP, indicating that the dissociation is dependent upon the ATP-hydrolysis. These data suggest that hsc73 rather than hsp72 plays an important role in the yet undefined mechanism of disregulated cell growth control by mp53.

Induction of apoptosis by the p53‐273L (Arg→Leu) mutant in HSC3 cells without transactivation of p21Waf1/Cip1/Sdi1 and bax

Codon 273 is one of the hot spots of missense mutation of the p53 tumor suppressor gene found in human cancers. We have previously reported that a mutation at codon 273, p53‐273L (Arg→Leu), suppresses cell growth despite its having no p53‐specific transactivation activity. To further elucidate the mechanism of growth suppression caused by p53‐273L, we used squamous cell carcinoma cell line HSC3 to isolate subclones containing Zn2+‐inducible wild‐type (wt) p53, p53‐175H, and p53‐273L. Northern blot hybridization of the HSC3 cells possessing an inducible function of p53 as well as a luciferase assay for the p21Waf1/Cip1/Sdi1 promoter showed that only wt p53 could induce p21Waf1/Cip1/Sdi1 transcription. Meanwhile, the expression of bax remained unchanged between, before, and after the induction of any analyzed p53s. When wt p53 was induced in HSC3 cells cultured in medium containing 5% fetal bovine serum, cell growth was suppressed through G1 arrest. On the other hand, in medium with 0.1% fetal bovine serum, the growth of HSC3 cells expressing p53‐273L was suppressed to a greater degree than that of cells expressing wt p53. Flow cytometric analysis and DNA ladder formation revealed that, unlike wt p53‐SN3– and p53‐175H–expressing HSC3 cells, p53‐273L–expressing cells contained a larger sub‐G1 fraction under this culture condition. These findings suggest that p53‐273L can induce apoptosis in HSC3 cells without transactivation of p21Waf1/Cip1/Sdi1 and bax. Mol. Carcinog. 26:44–52, 1999.

Immunohistochemical Detection of Estrogen Receptor in Invasive Human Breast Cancer: Correlation with Heat Shock Proteins, pS2 and Oncogene Products

The authors immunohistochemically studied the expression of the estrogen receptor (ER), 27-kD heat shock protein (HSP27) and pS2 in 118 invasive primary human breast cancers. Positive nuclear staining of the ER was detected in 64% of the cases and was closely correlated with the biochemical assay (p < 0.0001). ER-positive tumors were significantly decreased with tumor size and stage (p < 0.001 each), but not with lymph node status. Positivity of the ER was correlated with the cytoplasmic expression of HSP27 (p < 0.005), pS2 (not significant) and HSP70 (not significant). ER negativity was significantly correlated with the expression of p53, epidermal growth factor receptor (EGFR) and c-erbB-2 (p < 0.05 each). Thus, it was concluded that ER-positive breast carcinomas, relatively small in size, preferentially expressed HSP27, HSP70 and pS2 and that ER-negative tumors, relatively large in size, were predisposed to express p53, EGFR and c-erbB-2.

Cytotoxicity of Histocompatibility Leukocyte Antigen–DR8–restricted CD4+ Killer T Cells against Human Autologous Squamous Cell Carcinoma

Although CD8+ killer T cells reacting against human autologous tumor cells have recently been studied in detail, little is known about the cytotoxic mechanism of CD4+ T cells against such tumor cells. In order to investigate this, we have established CD4+ cytotoxic T lymphocyte TcOSC–20 lines. TcOSC–20 showed selective cytotoxic activity against autologous OSC–20 cells, derived from a cancer of the tongue, in an HLA–DR–restricted fashion. HLA–DR8 (DRB1* 08032) is the only DR molecule expressed on OSC–20 cells, and anti–DRS monoclonal antibody could inhibit the Cytotoxicity, suggesting that HLA–DRB1★ 08032 is the tumor rejection antigen–presenting moleculeto TcOSC–20. The Fas ligand was expressed on TcOSC–20 lines, and its expression was induced upon mixed lymphocyte–tumor cell culture of autologous peripheral blood lymphocytes. Furthermore, the Cytotoxicity of TcOSC–20 was inhibited by anti–Fas ligand antibody.These data imply that TcOSC–20 lines recognize the tumor antigenic peptide presented by HLA–DR8, and exert Cytotoxicity against autologous tumor cells via a Fas–mediated cytotoxic pathway.

Human CD8 and CD4 T cell epitopes of epithelial cancer antigens

Abstract Recent human tumor immunology research has identified several genes coding immunogenic peptides recognized by CD8 cytotoxic T lymphocytes (CTLs) in melanoma tumors. Very recently, CD4u2009T 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 CD4u2009T 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 α-enolase, suggesting that it was derived from the processed parental α-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.

Anisakidosis: Global Point of View

Anisakidosis, a human parasitic disease, is one of the zoonoses caused by certain types of anisakid nematodes. More than 30 000 cases have been reported in the world, and most of the cases have occurred in Japan because of the custom of eating raw fish that are the intermediate (paratenic) host. On Hokkaido Island in Japan, a relatively high incidence of anisakidosis is caused by Pseudoterranova decipiens as well as Anisakis simplex. It became apparent that Pseudoterranova decipiens was carried by Steller’s sea lions (Eumetopias jubata) from their natural habitats, Komandor Island and Moneron Island of Sakhalin. According to these observations, we speculated that the incidence of anisakidosis and the infection patterns of the responsible worms depend on the distribution of the intermediate (paratenic) and final hosts of the anisakid worms, which is influenced by sea current and climate.

In vitro proliferation and the cytotoxic specificity of a cryopreserved cytotoxic T cell clone reacting against human autologous tumor cells.

Proliferation and functional maintenance of CTL after cell cryopreservation often proves to be quite difficult. We developed an improved method for proliferating cryopreserved CTL, and for gaining their specific cytotoxic function. T cells were cryopreserved at -180 degrees C in RPMI 1640 containing 50% FCS and 10% DMSO. The cryopreserved T cells were well recovered by culturing in a medium containing the supernatant of primary cultures with TIL and autologous tumor cells, in addition to a high concentration (350 U/ml) of rIL-2. Furthermore, these cells were proliferated more efficiently when MMC-treated autologous tumor cells were used in vitro as a feeder and an antigenic stimulant. However, such a high dose IL-2 cultivation resulted in the loss of cytotoxic reactivity of CTL clone. In contrast, the withdrawal of rIL-2 from in vitro cultivation for 24 h prior to the cytotoxic assays conferred the specificity of cytotoxicity on CTL. By these methods, one can obtain a large number of CTL, and pursue the physiologic detail of the specific cytotoxic mechanism of CTL against autologous human tumor cells.

ECRG4 is a negative regulator of caspase-8-mediated apoptosis in human T-leukemia 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.