Sumio Sakamaki

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...

Interaction between leukemic-cell VLA-4 and stromal fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia

Bone-marrow minimal residual disease (MRD) causes relapse after chemotherapy in patients with acute myelogenous leukemia (AML). We postulate that the drug resistance is induced by the attachment of very late antigen (VLA)-4 on leukemic cells to fibronectin on bone-marrow stromal cells. We found that VLA-4-positive cells acquired resistance to anoikis (loss of anchorage) or drug-induced apoptosis through the phosphatidylinositol-3-kinase (PI-3K)/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA-4 and fibronectin. This resistance was negated by VLA-4-specific antibodies. In a mouse model of MRD, we achieved a 100% survival rate by combining VLA-4-specific antibodies and cytosine arabinoside (AraC), whereas AraC alone prolonged survival only slightly. In addition, overall survival at 5 years was 100% for 10 VLA-4-negative patients and 44.4% for 15 VLA-4-positive patients. Thus, the interaction between VLA-4 on leukemic cells and fibronectin on stromal cells may be crucial in bone marrow MRD and AML prognosis.

Primary intestinal lymphangiectasia successfully treated with octreotide

Abstract: A 21-year-old man with diarrhea and edema was admitted to our hospital and diagnosed with protein-losing enteropathy caused by primary intestinal lymphangiectasia. He was placed, in turn, on a low-fat diet, an elemental diet, and, subsequently, fasting therapy with total parenteral nutrition (TPN) support. However, his symptoms were not relieved, but, rather were exacerbated. On the 45th day of hospitalization, octreotide therapy was initiated. After 2 weeks of treatment, his clinical symptoms, as well as hypoproteinemia and hypoalbuminemia, gradually became alleviated. The improvement was confirmed in terms of scintigraphy, endoscopy, and histology of the duodenum. The patient remained healthy until 6 months after the commencement of octreotide treatment, when he discontinued octreotide at his own discretion, at which point the symptoms recurred. Resumption of the drug; however, again brought about remission, which has continued until the present, March 2000. Thus, octreotide therapy is one modality which may be useful for refractory primary intestinal lymphangiectasia.

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)

Cytomegalovirus DNA in the sera of patients with cytomegalovirus pneumonia

Summary We attempted to detect cytomegalovirus DNA (CMV‐DNA) in the sera of four leukaemia patients who underwent an allogeneic bone marrow transplant (BMT), in six leukaemia patients who suffered from pneumonia and in 16 healthy subjects, using the polymerase chain reaction (PCR). Three of the four BMT patients subsequently developed CMV pneumonia. In two cases, CMV‐DNA was detected in the sera at about the time the pneumonia occurred, and the amount of DNA increased with disease progression. The serum of the third patient became positive for CMV‐DNA before he developed pneumonia. The fourth patient did not develop CMV pneumonia, but his urine became persistently positive for CMV‐DNA soon after the BMT, whereas the serum was negative. A relationship was found between the occurrence of pneumonia and the serum level of CMV‐DNA. CMV‐DNA was also detected in three of six pneumonia patients whose anti‐CMV IgM antibodies were elevated in the circulation. Sera from the 16 normal subjects were negative for CMV‐DNA, regardless of their being seropositive or seronegative for CMV.

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.

Aberrant Crypt Foci as Precursors of the Dysplasia-Carcinoma Sequence in Patients with Ulcerative Colitis

Purpose: Long-standing ulcerative colitis (UC) predisposes patients to the development of colorectal cancer, but surveillance of colitis-associated cancer by detecting the precancerous lesion dysplasia is often difficult because of its rare occurrence and normal-looking appearance. In sporadic colorectal cancer, aberrant crypt foci (ACF) have been reported by many investigators to be precursor lesions of the adenoma-carcinoma sequence. In the present study, we analyzed the genetic background of ACF to determine whether they could be precursors for dysplasia, and we examined the usefulness of endoscopic examination of ACF as a surrogate marker for surveillance of colitis-associated cancer. Experimental Design: ACF were examined in 28 UC patients (19 patients with UC alone and 9 patients with UC and dysplasia; 2 of those patients with dysplasia also had cancer) using magnifying endoscopy. K-ras, APC, and p53 mutations were analyzed by two-step PCR RFLP, in vitro–synthesized protein assay, and single-strand conformation polymorphism, respectively. Methylation of p16 was analyzed by methylation-specific PCR. Results: ACF that appeared distinct endoscopically and histologically were identified in 27 out of 28 UC patients. They were negative for K-ras, APC, and p53 mutations but were frequently positive for p16 methylation (8 of 11; 73%). In dysplasia, K-ras and APC mutations were negative but p53 mutation (3 of 5; 60%) and p16 methylation (3 of 5; 60%) were positive. There was a significant stepwise increase in the number of ACF from patients with UC alone to patients with dysplasia and to patients with cancer. Univariate and multivariate analyses showed significant correlations between ACF and dysplasia. Conclusions: We have disclosed an ACF-dysplasia-cancer sequence in colitis-associated carcinogenesis similar to the ACF-adenoma-carcinoma sequence in sporadic colon carcinogenesis. This study suggests the use of ACF instead of dysplasia for the surveillance of colitis cancer and warrants further evaluation of ACF as a surveillance marker in large-scale studies.

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.

Cytokine mRNA expression of bone marrow stromal cells from patients with aplastic anaemia and myelodysplastic syndrome

Summary We studied mRNA expression of the cytokine granulocyte‐colony stimulating factor (G‐CSF), interleukin‐1β (IL‐1β), IL‐6, IL‐8 and stem cell factor of stromal cells derived from bone marrows of nine normal volunteers, eight patients with aplastic anaemia (AA) and seven patients with myelodysplastic syndrome (MDS). The proportion of endothelial cells. macrophages. fibroblast‐like cells and adipocytes in stromal cells showed no differences between normal volunteers and the patients. Levels of cytokine inKNA expression were determined by reverse transcription‐polymerase chain reaction. Spontaneous expression occurred and this was augmented by IPS stimulation in cells of all the normal volunteers and in most patients.

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.