Fujii S

Loss of E-cadherin-dependent cell-cell adhesion due to mutation of the beta-catenin gene in a human cancer cell line, HSC-39.

Detachment of cell-cell adhesion is indispensable for the first step of invasion and metastasis of cancer. This mechanism is frequently associated with the impairment of either E-cadherin expression or function. However, mechanisms of such abnormalities have not been fully elucidated. In this study, we demonstrated that the function of E-cadherin was completely abolished in the human gastric cancer cell line HSC-39, despite the high expression of E-cadherin, because of mutations in one of the E-cadherin-associated cytoplasmic proteins, beta-catenin. Although immunofluorescence staining of HSC-39 cells by using an anti-E-cadherin antibody (HECD-1) revealed the strong and uniform expression of E-cadherin on the cell surface, cell compaction and cell aggregation were not observed in this cell. Western blotting (immunoblotting) using HECD-1 exhibited a 120-kDa band which is equivalent to normal E-cadherin. Northern (RNA) blotting demonstrated a 4.7-kb band, the same as mature E-cadherin mRNA. Immunoprecipitation of metabolically labeled proteins with HECD-1 revealed three bands corresponding to E-cadherin, alpha-catenin, and gamma-catenin and a 79-kDa band which was apparently smaller than that of normal beta-catenin, indicating truncated beta-catenin. The 79-kDa band was immunologically identified as beta-catenin by using immunoblotting with anti-beta-catenin antibodies. Examination of beta-catenin mRNA by the reverse transcriptase-PCR method revealed a transcript which was shorter than that of normal beta-catenin. The sequencing of PCR product for beta-catenin confirmed deletion in 321 bases from nucleotides +82 to +402. Southern blotting of beta-catenin DNA disclosed mutation at the genomic level. Expression vectors of Beta-catenin were introduced into HSC-39 cells by transfection. In the obtained transfectants, E-cadherin-dependent cell-cell adhesiveness was recovered, as revealed by cell compaction, cell aggregation, and immunoflourescence staining. From these results, it was concluded that in HSC-39 cells, impaired cell-cell adhesion is due to mutations in beta-catenin which results in the dysfunction of E-cadherin.

In vivo gene delivery to tumor cells by transferrin-streptavidin-DNA conjugate

To target disseminated tumors in vivo, transgenes [ β‐galactosidase gene, green fluorescence protein (GFP) gene, herpes simplex virus thymidine kinase (HSV‐TK)] were conjugated to transferrin (Tf) by a biotin‐streptavidin bridging, which is stoichiometrically controllable, and Tf receptor (Tf‐R) affinity chromatography, which selects Tf conjugates with intact receptor bindings sites from reacting with the linker. Tf‐β ‐galactosidase plasmid conjugate thus constructed was specifically transfected to human erythroleukemia cells (K562) via Tf‐R without the aid of any lysosomotropic agents. The transfection efficiency of the conjugate was superior to those of lipofection (1% staining) and retroviral vector (5%) and slightly lower than that of adenovirus (70%). The high level of expression with our conjugate was confirmed using other tumor cells (M7609, TMK‐1) whereas in normal diploid cells (HEL), which express low levels of Tf‐R, expression was negligible. When GFP gene conjugates were systemically administered through the tail vein to nude mice subcutaneously inoculated with tumor, expression of GFP mRNA was found almost exclusively in tumors and to a much lesser extent in muscles, whereas GFP revealed by fluorescence microscopy was detected only in the former. To exploit a therapeutic applicability of this method, suicide gene therapy using Tf‐HSV‐TK gene conjugate for massively metastasized k562 tumors in severe combined immunedeficient mice was conducted, and a marked prolongation of survival and significant reduction of tumor burden were confirmed. Thus, this method could also be used for gene therapy to disseminated tumors.—Sato, Y., Yamauchi, N., Takahashi, M., Sasaki, K., Fukaura, F., Neda, H., Fujii, S., Hirayama, M., Itoh, Y., Koshita, Y., Kogawa, K., Kato, J., Sakamaki, S., Niitsu, Y. In vivo gene delivery to tumor cells by transferrin‐streptavidin‐DNA conjugate. FASEB J. 14, 2108–2118 (2000)

Administration of subtumor regression dosage of TNF-α to mice with pre-existing parental tumors augments the vaccination effect of TNF gene-modified tumor through the induction of MHC class I molecule

One obstacle in treating pre-existing parental tumors by vaccination with cytokine gene-modified tumor cells is the impaired expression of immune-related molecules such as MHC class I. In this study, to enhance MHC class I expression on pre-inoculated parental tumors, low dose TNF (300 U, 500 U, 1000 U), that is, TNF at levels shown to cause neither tumor regression nor any severe adverse reaction, was systemically injected into parental tumors bearing mice before vaccination with TNF gene-modified Meth-A cells or B-16 cells. Since the class I expression was confirmed to continue for at least 24 h following administration of TNF, TNF was administered 6 h before vaccination. Complete regression of relatively large parental tumors (M0) (8.0–10.0 mm in diameter) was observed in five of eight mice treated with 1000 U TNF, partial regression was observed in mice treated with 500 U, and a lesser yet significant regression was observed in mice treated with only 300 U. Contrarily, in the mice which had received vaccination without the TNF pretreatment, no complete regression was observed. This effect was inhibited with the anti-class I antibody or anti-CD8 antibody. Growth of a re-established, B16 tumor was significantly suppressed with a combination of TNF preadministration and vaccination of TNF gene-modified B16. These results indicate that pre-administration of low-dose TNF may be promising for enhancing vaccination effects of TNF gene-modified tumor cells.

Augmented systemic immunity in mice implanted with tumor necrosis factor-α, gene-transduced Meth-A cells

Syngeneic BALB/c mice bearing methylcholanthrene‐induced fibrosarcoma (Meth‐A) cells transduced with a tumor necrosis factor (TNF) gene showed a longer life span and tumor regression as compared with mice carrying TNF‐non‐producing Meth‐A cells. To elucidate the mechanism of the reduced tumorigenicity of TNF‐producing Meth‐A, we compared systemic immune responses between mice bearing high TNF producer (C5) and unmodified Meth‐A cells (M0). The results indicated that the cytotoxic activity of lymphokine‐activated killer cells (LAK) induced from spleen cells of mice bearing C5 was higher against both M0 and C5 than that of LAK from mice bearing M0. Also, C5 was more sensitive to LAK induced from spleen cells of C5‐ and M0‐ bearing mice than M0. We also found that cytotoxic T lymphocyte from spleen cells of mice transplanted with C5 was more cytotoxic to M0 than that from mice with M0. In addition, the population of Lyt2 (CD8)‐positive T cells was higher in freshly isolated spleen cells of mice transplanted with C5 than from mice with M0. Finally, we observed a higher expression of MHC class 1 antigen on C5 than on M0. These observations suggest that the augmented host systemic immunity in mice carrying TNF gene‐modified Meth‐A cells is one of the mechanisms of the reduced tumorigenicity of C5 and that the increased systemic immunity can be ascribed to the increased immunogenicity of the tumor cells. Thus, the use of TNF gene‐modified tumor cells as vaccines appears to be promising for therapeutic and/or prophylactic application.

Mechanisms of tumor regression of TNF gene-transduced Meth-A cells transplanted in mice.

We transduced a tumor necrosis factor (TNF) gene into methylcholanthreninduced mouse fibrosarcoma (Meth-A) cells retrovirally to establish a high TNF producer clone (C5). C5 was subcutaneously transplanted into syngeneic mice, resulting in remarkable tumor regression and a much longer life span as compared with subjects transplanted with the parent Meth-A (MO) or NeoR genetransduced Meth-A (ML). Since C5 was resistant to exogenous TNF in vim, it seems unlikely that the observed tumor regression was due to any direct antitumor effect of TNF produced from C5. In this presentation, we studied mechanisms of regression of the C5 tumor. First, we investigated whether augmented systemic immunity was induced in C5-bearing mice. Cytotoxic effects of interleukin-2 (IL-2) activated splenocytes (LAK) from MOand C5-transplanted mice were studied against MO and C5, respectively, by 51Cr release assay; LAK from C5-bearing mice showed higher cytotoxic activity, the level of which was higher in targeting C5 than MO. Cytotoxic effects of CTL induced from splenocytes of MOand C5-bearing mice were also studied against MO, showing increased cytotoxic activity of CTL from mice with C5. SurFace antigens of freshly isolated splenocytes from C5and MO-bearing mice were also analyzed by flow cytometry, resulting in higher expression of T cell (particularly CD8+ cells) in splenocytes from C5-bearing mice than that from MO-bearing ones. Second, we studied whether C5 accompanied any alterations compared with MO. Sensitivities of MO and C5 to IL-2-activated splenocytes from allogenic normal mouse (allogenic LAK) were studied; C5 showed a higher sensitivity to the LAK than MO. Sensitivities of MO and C5 to CTL induced from splenocytes of C5-bearing mice were also studied; C5 showed a higher sensitivity to the CTL. Major histocompatibility complex (MHC) antigen and adhesion molecule expression of MO and C5 were investigated; class 1 expression was higher in C5 than in MO. Third, we analyzed C5 and MO tumor tissue with immunohistochemical staining; C5 tumor revealed higher expression of CD4, CD8, ICAM-1, and VCAM1.

[RS3PE syndrome associated with senile Epstein-Barr virus-positive diffuse large B cell lymphoma of a patient with colon cancer].

A 75-year-old woman consulted her doctor in January 2014 because of pain in the dorsum of the hands, elbows, shoulders, and knees, bilaterally, and was diagnosed as having remitting seronegative symmetrical synovitis with pitting edema (RS3PE) syndrome. Although the joint pain improved with low-dose prednisolone administration, she was referred to our department in April of 2014 because she had become aware of swelling of the right cervical lymph node. Biopsy of the lymph node demonstrated that she had Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) of the elderly, and colonoscopy revealed early colon cancer. Also, both the lymphoma and colon cancer stained positive for vascular endothelial growth factor (VEGF). Complete remission was achieved after two courses of R-CHOP, and RS3PE syndrome did not relapse. This case suggested the involvement of VEGF produced by EBV-positive DLBCL in the pathogenesis of RS3PE syndrome.

The efficacy of early use of recombinant soluble thrombomodulin for disseminated intravascular coagulation complicated with hematologic malignancies

We retrospectively investigated treatment outcomes with soluble recombinant thrombomodulin (rTM) for disseminated intravascular coagulation (DIC) associated with hematological malignancies (acute leukemia and malignant lymphoma) at our hospital. After rTM administration, DIC scores improved in 29 of 39 cases with hematological malignancies (74.36%). Although one case with recurrent and refractory APL died due to cerebral bleeding during rTM administration, no bleeding-associated adverse events were observed in the other 38 cases. DIC improvement was augmented in cases with acute leukemia when rTM was introduced in the pre-DIC state. CRP decreased in 26 of 36 cases with hematological malignancies (72.22%) after rTM introduction, and CRP decreased particularly significantly in cases with malignant lymphoma, suggesting rTM to exert anti-inflammatory activity. Taken together, these observations indicate that rTM, which rarely causes bleeding-associated adverse events, is an excellent agent in terms of both efficacy and safety for treating DIC associated with hematological malignancies, and the potential anti-inflammatory activity of this agent was also suggested.

PEDUNCULATED GASTRIC SIGNET RING CELL CARCINOMA

Signet ring cell carcinoma (SRCC) is characterized by poor ductal formation and a diffuse progression pattern and generally presents as a depressed lesion in the majority of cases. We describe here an extremely rare case of gastric SRCC that presented as a pedunculated lesion. We hypothesize that the major factor responsible for the elevation of this lesion was proliferation of signet ring cells that did not lose their mutual connections. Among the two cases of early elevated‐type gastric SRCC that have been reported in the literature, this is the first case of pedunculated intramucosal SRCC. Pathological examination revealed no cancer cells in the basal part of the elevated lesion. The cellular morphology was consistent with SRCC, although the ductal structure was well preserved and more similar to well‐differentiated adenocarcinoma. Endoscopic examination showed a smooth‐surfaced lesion with no depressed region around the basal part of the elevated lesion. Because these findings differ significantly from previous reports of elevated SRCC, this report provides further insight into the nature of SRCC.