Katsuhisa Kogawa

Aberrant Crypt Foci of the Colon as Precursors of Adenoma and Cancer

Background Aberrant crypt foci of the colon are possible precursors of adenoma and cancer, but these lesions have been studied mainly in surgical specimens from patients who already had colon cancer. Methods Using magnifying endoscopy, we studied the prevalence, number, size, and dysplastic features of aberrant crypt foci and their distribution according to age in 171 normal subjects, 131 patients with adenoma, and 48 patients with colorectal cancer. We also prospectively examined the prevalence of aberrant crypt foci in 11 subjects (4 normal subjects, 6 with adenoma, and 1 with cancer) before and after the administration of 100 mg of sulindac three times a day for 8 to 12 months and compared the results with those in 9 untreated subjects (4 normal subjects and 5 with adenoma). All 20 subjects had aberrant crypt foci at base line. Results We identified 3155 aberrant crypt foci, 161 of which were dysplastic; the prevalence and number increased with age. There were significant (P<0.001) correlations between...

Effect of Helicobacter pylori eradication on platelet recovery in Japanese patients with chronic idiopathic thrombocytopenic purpura and secondary autoimmune thrombocytopenic purpura

Summary. The prevalence of Helicobacter pylori infection and the effect of its eradication on platelet count in 48 Japanese patients with autoimmune thrombocytopenic purpura (AITP), including 40 chronic idiopathic thrombocytopenic purpura (ITP) and eight secondary AITP, were investigated. H. pylori infection was found in 25 ITP patients (62·5%) and in two secondary AITP (25%). H.pylori eradication was obtained in 19 of 19 infected ITP patients (100%), who were not in remission (platelets < 100 × 109/l) at the time of infection assessment. During follow‐up (median 14·8 months), 12 of 19 H. pylori‐eradicated patients (63·2%) showed a significant increase in platelet count accompanied by a significant decrease of platelet‐associated immunoglobulin G (IgG). This response was maintained in all responding patients throughout the follow‐up period. However, two infected patients with secondary AITP did not show platelet increase after eradication. The assessment of H. pylori infection and its eradication should be attempted in ITP as this approach could be an effective strategy, at least for some of these patients.

Induction of Heat Shock Protein 47 Synthesis by TGF-β and IL-1β Via Enhancement of the Heat Shock Element Binding Activity of Heat Shock Transcription Factor 1

With most immunological reactions, tissue fibrosis, collagen overproduction caused by immune cytokines, is inevitably associated. Among the various immune cytokines, heat shock protein 47 (HSP47) is a procollagen-specific molecular chaperon and is essential for secretion of procollagen from cells. Induction of HSP47 by TGF-β has been previously reported in rat skeletal myoblasts and mouse osteoblasts, but not in human diploid fibroblasts. As for IL-1β, its effect on HSP47 has not been elucidated. In the present study, using human embryonic lung fibroblast cells, we first disclosed that both TGF-β and IL-1β induced HSP47 synthesis. We then revealed that the binding of the heat shock element (HSE) by heat shock transcription factor 1 (HSF1) was enhanced by both cytokines. We further demonstrated that trimer formation of HSF1, which is essential for its binding to HSE, was induced by these cytokines. The enhancement of HSP47 synthesis and their trimer formation of HSF1 were augmented by using a combination of both cytokines. Collectively, TGF- β and IL-1β were found to induce trimer formation of HSF1 which in turn bound to HSE of HSP47, resulting in the enhancement of HSP47 expression. Thus, HSP47 could well be a good candidate for molecular targeting in controlling tissue fibrosis, given that both principal fibrinogenetic cytokines (TGF-β, IL-1β) are commonly involved in its induction through HSF1 trimerization.

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)

Treatment of murine collagen-induced arthritis by ex vivo extracellular superoxide dismutase gene transfer

UNLABELLED OBJECTIVE; Superoxide dismutase (SOD) is a potent antiinflammatory enzyme that has received growing attention for its therapeutic potential. This study was undertaken to examine the efficacy of extracellular SOD (EC-SOD) gene therapy in murine collagen-induced arthritis. METHODS Embryonic DBA/1 mouse fibroblasts were infected with a recombinant retrovirus expressing human EC-SOD. DBA/1 mice that had been treated with type II collagen were administered subcutaneous injections of 2 x 10(7) EC-SOD-expressing fibroblasts on day 29, when symptoms of arthritis were already present. The severity of arthritis in individual mice was evaluated in a double-blind manner; each paw was assigned a separate clinical score, and hind paw thickness was measured with a caliper. Mice were killed on day 50 for histologic examination of the joints. RESULTS High serum concentrations of EC-SOD were maintained for at least 7 days. Mice treated with the transgene exhibited significant suppression of clinical symptoms such as disabling joint swelling, deformity, and hind paw thickness, compared with the untreated group (mean +/- SD maximum clinical score in the untreated and the transgene-treated groups 2.71 +/- 1.08 and 1.35 +/- 1.22, respectively; P < 0.01, and hind paw thickness 3.04 +/- 0.18 mm and 2.56 +/- 0.12 mm, respectively; P < 0.05). Histologic abnormalities, including destruction of cartilage and bone, infiltration of mononuclear cells, and proliferation of synovial cells, were also markedly improved in the EC-SOD-treated mice compared with the control group (histopathologic score 7.50 +/- 1.13 and 4.13 +/- 1.88 in the untreated and transgene-treated groups, respectively; P < 0.05). CONCLUSION These results indicate that EC-SOD gene transfer may be an effective form of therapy for rheumatoid arthritis.

Establishment of high- and low-invasion clones derived for a human tongue squamous-cell carcinoma cell line SAS

Distant-organ metastasis and regional lymph node metastasis are still the major cause of mortality of oral-cavity squamous-cell cancer (SCC). However, only a few studies have been undertaken to elucidate the mechanism of invasion and metastasis of oral SCC. In this study, we attempted to establish human oral SCC clones with different invasiveness, defined by endothelial cell monolayer assay, which can be used for the study of invasion and metastasis of oral SCC. We established five clones from the human oral SCC cell line SAS by a limiting-dilution method. Two distinct clones, SAS-L1 with very low invasive potential and SAS-H1 with very high invasive potential, were picked out by rat lung endothelial cell monolayer assay. The number of SAS-H1 that penetrated the rat lung endothelial cell monolayer was six fold higher than the number of SAS-L1. There were no differences of metalloproteinase production and cell adhesiveness to Matrigel of SAS-L1 and SAS-H1. However, SAS-H1 exhibited a higher migration ability than SAS-L1. This pair of clones would be a useful experimental model to help in the study of the invasiveness of human oral SCC.

Suppression of intracellular Cu-Zn SOD results in enhanced motility and metastasis of Meth A sarcoma cells

We have previously described an inverse relationship between Cu‐Zn superoxide dismutase (SOD) activity and invasiveness of a clone of human tongue cancer cells. In these cells, suppression of Cu‐Zn SOD activity by transfection with anti‐sense cDNA enhanced motility in vitro. The present studies were undertaken to determine whether the inverse relationship between intracellular Cu‐Zn SOD activity and motility is a general property of other tumor cells and whether this enzyme indeed defines in vivo metastatic potential. Murine Meth A sarcoma‐derived ML‐01 cells, which have low metastatic activity, were transfected with anti‐sense Cu‐Zn SOD cDNA. Two clones with very different SOD activities—ML‐AS2, with the most suppressed, and ML‐AS5, with the least suppressed activity—were analyzed for their motility and metastatic capability. Compared to the mock‐transfectant ML‐neo, the metastatic potential and motility of the ML‐AS2 and ML‐AS5 were increased 4.5‐ and 2.1‐fold, respectively. Superoxide treatment enhanced the motility of the AS clones but not that of the ML‐neo cells. Our results clearly show that there is an inverse relationship between the intracellular level of Cu‐Zn SOD, cell motility and in vivo metastatic potential. Int. J. Cancer 73:187–192, 1997.

A proof of glutathione S-transferase-π-related multidrug resistance by transfer of antisense gene to cancer cells and sense gene to bone marrow stem cell

In order to directly prove the involvement of GST-pi in drug resistance, its antisense gene was transduced into human colorectal cancer cell line which has been shown to express high level of GST-pi and the sensitivity of this cell line to anticancer drugs were assessed. The transfectant showed higher sensitivity to adriamycin (3.3-fold), Cisplatnum (2.3-fold), Melphalan (2.2-fold), Etoposode (2.2-fold) than the parental cell, while the sensitivity to vincristine, mitomicin C, 5-fluorouracil was unchanged by transfection. When the transfectant and parental cells were innoculated in nude mice and treated with adriamycin, a significant suppression of tumor growth was observed with the transfectant as compared to the parental cell. On the basis of this observation, we then transduced sense GST-pi gene into human bone marrow stem cells (CD34+ cells) to protect them from toxicity of anticancer drug. The gene transduced CD34+ cells formed more CFU-GM than nontransduced CD34+ cell in the presence of adriamycin (30 ng/ml). Thus, the autotransplantation of GST-pi gene transduced cell into cancer patients to protect the bone marrow from subsequent highdose chemotherapy is considered to be a new strategy for cancer gene therapy.

GST-π gene-transduced hematopoietic progenitor cell transplantation overcomes the bone marrow toxicity of cyclophosphamide in mice

Autologous transplantation of bone marrow cells (BMCs) transduced with the multidrug resistance 1 (MDR1) gene or dihydrofolate reductase (DHFR) gene has already been applied in clinical chemoprotection trials. However, anticancer drugs frequently used in high-dose chemotherapy (HDC), such as alkylating agents, are not relevant to MDR1 or DHFR gene products. In this context, we have previously reported that glutathione S-transferase-pi (GST-pi) gene-transduced human CD34(+) cells showed resistance in vitro against 4-hydroperoxicyclophosphamide, an active form of cyclophosphamide (CY). In the present study, a subsequent attempt was made in a murine model to evaluate the effectiveness of transplantation of GST-pi-transduced BMCs to protect bone marrow against high-dose CY. The gene transfection was carried out retrovirally, employing a recombinant fibronectin fragment. Transfection efficiency into CFU-GM was 30%. After the transplantation, recipient mice (GST-pi mice) received three sequential courses of high-dose CY. As the chemotherapy courses advanced, both shortening of recovery period from WBC nadir and shallowing of WBC nadir were observed. In contrast to the fact that three of seven control mice died, possibly due to chemotoxicity, all seven GST-pi mice were alive after the third course, at which point the vector GST-pi gene was detected in 50% of CFU-GM derived from their BMCs and peripheral blood mononuclear cells. When BMCs obtained from these seven mice were retransplanted into secondary recipient mice, 20% of CFU-GM from BMCs showed positive signals for vector GST-pi DNA after 6 months. These data indicate that the GST-pi gene can confer resistance to bone marrow against CY by being transduced into long-term repopulating cells.

Mechanisms for increment of platelet associated IgG and platelet surface IgG and their implications in immune thrombocytopenia associated with chronic viral liver disease

In addition to hypersplenism, immunological destruction of platelets by elevated platelet associated IgG (PAIgG) and platelet surface IgG (PSIgG) has been proposed as a causative factor for thrombocytopenia in chronic liver disease (CLD), although the implication of PAIgG may be debatable since recent investigations on idiopathic thrombocytopenic purpura disclosed the fact that PAIgG largely relates to the intra-platelet IgG in alpha-granules and not to PSIgG. Further, with regard to the elevated PSIgG of CLD, characterization as to whether it mainly represents anti-platelet glycoprotein (GP) antibodies or IgG contained in the immune complex has not been elucidated. Thirty-seven patients with chronic viral liver disease (CVLD); 31 hepatitis C and 6 hepatitis B were included in this study. First we monitored the changes in levels of PAIgG, alpha-granule IgG, PSIgG and mean platelet volume (MPV) during the course of partial splenic arterial embolization (PSE). The elevated level of PAIgG decreased after PSE, paralleling that of alpha-granule IgG, while PSIgG showed no change; MPV decreased reciprocally with the increase of platelet count. These results indicate that the increment of PAIgG in CVLD may be caused by accelerated destruction of platelets; this generally evokes hyperproduction of large-sized thrombocytes, which have an increased capability to uptake circulating IgG. To characterize PSIgG, we then tested CVLD patients for antiplatelet GP antibodies and found only a 5.4% positivity. It was also found that circulating immune complex levels in CVLD patients were clearly elevated, correlating with the levels of PSIgG. Thus, it was surmised that immune complexes bound to the platelet surface, and not platelet specific GP antibodies, may be playing a crucial role in platelet destruction of CVLD, possibly through phagocytosis by macrophages.