Tamio Koizumi

Establishment of a myeloid leukaemic cell line (SKNO-1) from a patient with t(8;21) who acquired monosomy 17 during disease progression

A novel cell line SKNO‐1 was established from the bone marrow cells of a 22‐year‐old male suffering from acute myeloblastic leukaemia (AML) M2 with t(8;21) whose disease became resistant to chemotherapy after acquisition of 17 monosomy. SKNO‐1 has been maintained for more than 36 months as a granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) dependent line.

Effects of Corticosteroid and 1,24R-Dihydroxy-Vitamin D3 Administration on Lymphoproliferation and Autoimmune Disease in MRL/MP-lpr/lpr Mice

The pharmacological effects of prolonged administration of a corticosteroid, betamethasone, and active vitamin D3 [1,24R-(OH)2D3] on lymphoproliferation and autoimmune disease of MRL/MP-lpr/lpr (MRL/1) mice were examined. Relatively high doses of betamethasone (0.25 mg/kg/day) prevented lymphoproliferation, reduced serum levels of anti-dsDNA, anti-ssDNA, and anti-poly (ADP-ribose) antibodies, and brought about clinical improvement, such as reduced proteinuria and diminution of skin lesions. It is noteworthy that not only did prevention of lymphoproliferation occur, but also recovery of the Lyt-2+ T cell subset in the thymus and the spleen was observed. The administration of 1,24R-(OH)2D3 (0.1 microgram/kg/day) similarly prevented proteinuria, and produced recovery of a Lyt-2+ subset in the thymus.

Quantitation of minimal residual disease in t(8;21)-positive acute myelogenous leukemia patients using real-time quantitative RT-PCR.

t(8;21) is one of the common chromosomal translocations in acute myelogenous leukemia (AML). Using a recently developed real‐time quantitative polymerase chain reaction (PCR) system, we analyzed the minimal residual disease (MRD) in bone marrow samples from seven AML patients with t(8;21) at different time points during the clinical courses of their disease. Four of these patients received chemotherapy and allogenic bone marrow transplantation (allo‐BMT), and the other three were treated with chemotherapy alone. Two of the patients that received allo‐BMT suffered a relapse. In these patients, the levels of AML1‐MTG8 mRNA expression were shown to quantitatively increase. After re‐induction chemotherapy and donor lymphocyte infusion therapy, AML went into remission and the expression levels decreased. In the other two patients receiving allo‐BMT, the disease went into remission and the level of AML1‐MTG8 mRNA expression remained under the detectable range. The other three patients received several courses of chemotherapy, without allo‐BMT, and all of them clinically reached the hematological and cytogenetic remission state. However, there were low but detectable levels of MRD in their bone marrow samples. These results suggest that the real‐time quantitative PCR assay is very useful for the monitoring of MRD and detecting an early relapse. This assay may also be useful in determining the quantitative difference in myelo‐ablative activity between the chemotherapy alone and chemotherapy in conjunction with allo‐BMT. Am. J. Hematol. 64:101–106, 2000.

Expression of α and β genes of human chorionic gonadotropin in lung cancer

To confirm the ectopic production of human chorionic gonadotropin (hCG) in lung cancer, we attempted to detect the presence of mRNA transcripts of the α and β genes for hCG in lung cancer tissues obtained from surgical operations. Although we were able to show the presence of hCGβ mRNA transcripts in lung cancer tissue by Northern blot, the sensitivity of the assay was too low for a precise analysis of hCGB mRNA transcripts in most lung cancers. Using reverse transcription PCR (RT‐PCR) and Southern blot analysis, however, various amounts of mRNA transcripts of hCGβ genes 3, 5, 7 and 8 were demonstrated in 9 of the 14 lung cancer tissues examined, while no mRNA transcripts were detectable in 12 normal lung tissues from the same patients. Our results are consistent with a clear difference in serum and urinary hCGβ levels observed between normal subjects and lung cancer patients. The expression of the hCGα gene, however, was detected in normal lung tissues more frequently than in lung cancer tissues using RT‐PCR Southern blot. Our results strongly suggests the production of hCGβ as being part of the phenotype of malignantly transformed lung cells and further strengthen its superior specificity over intact hCG or hCGα as a tumor marker for lung cancers. Int. J. Cancer 71:539‐544, 1997.

Hepatitis C virus structural proteins induce liver cell injury in transgenic mice

To develop an animal model of hepatitis C virus (HCV) infection, transgenic mice carrying part of the HCV cDNA (C980) encoding HCV‐core and envelope proteins under control of the mouse class I major histocompatibility complex gene (H‐2K) regulatory region were produced. HCV‐C980 RNA and HCV‐core protein were present in livers from line H36 as determined by RNase protection assay and immunostaining, respectively. More than 40 animals from line H36 were examined histologically. Most of these H36 mice after 10 months of age developed spontaneous focal infiltration of lymphocytes, hepatocyte necrosis, degeneration, and altered foci with mitotic hepatocytes. These pathological lesions were absent in livers from the age‐matched control littermates. Liver cells from these H36 mice were sensitive to damage induced by intravenous administration of an anti‐Fas antibody. It is suggested that HCV‐C980 proteins by themselves may be one causative agent of liver cell injury in subjects with HCV infection. J. Med. Virol. 59:281–289, 1999.

Levels of soluble FasL and FasL gene expression during the development of graft‐versus‐host disease in DLT‐treated patients

Three patients with different clinical symptoms of graft‐versus‐host disease (GVHD) who had received donor lymphocyte transfusion (DLT) for the treatment of relapsed leukaemia after an allogeneic bone marrow transplantation (BMT) from HLA‐matched sibling donors were analysed for the presence of soluble FasL (sFasL) in the sera and for the expression of the Fas ligand (FasL) gene in the peripheral blood mononuclear cells (PBMNC). Two patients who demonstrated liver damage with increased levels of serum bilirubin showed significantly increased levels of serum sFasL. The increase in the sFasL level was observed prior to the increase in the bilirubin during the clinical courses of both patients. The high dose of methyl predonisolone administered to one of these patients greatly reduced the levels of sFasL in the serum. The bilirubin levels were also reduced thereafter. The third patient (without liver damage) did not show any increase in the serum sFasL level.

Immunohistochemical expression of gastric mucin and p53 in minimal deviation adenocarcinoma of the uterine cervix.

Comparative immunostaining with antibodies against gastric mucin and p53 was performed on 6 cases of minimal deviation adenocarcinoma (MDA) of the uterine cervix. The MDAs consisted of predominant areas of glands lined by extremely well-differentiated tall columnar epithelial cells with little cytological atypia and minor foci of less well-differentiated glandular epithelium with a minor degree of nuclear atypia. Immunostaining for gastric mucin with a monoclonal antibody HIK1083 revealed that all the tumors areas of typical MDA were partly immunoreactive for HIK1083, but the coexisting less well-differentiated glands were essentially negative. Four MDAs focally contained cells with p53 positive nuclei that were located exclusively in the less well-differentiated cells that lacked gastric mucin. In the pelvic lymph nodes in 2 cases, most of the metastatic tumor was less differentiated and a positive reaction for HIK1083 was observed only in small foci of typical MDA. No significant overexpression of p53 was observed in the metastases. Immunohistochemical expression of gastric mucin and p53 may be related to the histological differentiation of MDA, and detection of p53 overexpression may help to identify critical steps in the local progression of MDA.

1,25-dihydroxyvitamin D3 regulates proliferation of activated T-lymphocyte subsets

The biologically active vitamin D metabolite, 1,25-(OH) 2D3 suppressed phytohaemagglutinin (PHA)-induced lymphocyte proliferation dose-dependently (0.1 nM-100 nM), and decreased the OKT4+/OKT8+ ratio and transferrin-receptor-positive (OKT9+) cells. A possible parallelism between expression of 1,25-(OH) 2D3 receptors and interleukin 2 (IL2)-receptors recognized by anti-Tac antibody was not confirmed in this study. However, the addition of exogenous IL2 abolished the inhibitory effects of 1,25-(OH) 2D3 on PHA-stimulated T-cell proliferation, and the decrease of OKT4+ and OKT9+ T-cell in this population. Among various vitamin D3 analogues examined, 1,25-(OH) 2D3 was the most potent anti-proliferative effect, followed in order by 1,24S-(OH) 2D3, 1 alpha OH D3, 25 OH D3 and 24,25-(OH) 2D3.

Effect of 1,25-dihydroxyvitamin D3 on cytokine-induced thymocyte proliferation☆

To clarify the mechanism of the inhibitory effect of 1,25(OH)2D3 on lymphocyte proliferation, the effect of 1,25(OH)2D3 on murine thymocyte proliferation induced by interleukin 1 (IL-1), or 2 (IL-2) was examined. Physiological concentrations of 1,25(OH)2D3 inhibited thymocyte proliferation induced by IL-1 and IL-2 in similar fashion suggesting an inhibition of the response to IL-2 by this hormone. In addition, cortisone-resistant thymocytes (including a majority of medullary thymocytes), which proliferate more vigorously in response to IL-1 than do untreated thymocytes, were more sensitive to 1,25(OH)2D3 inhibition. Therefore, the inhibition of IL-2 production of the mature medullary thymocyte by this hormone was also suggested.

Induction of apoptosis and manganese superoxide dismutase gene by photodynamic therapy in cervical carcinoma cell lines

AbstractBackground. Photodynamic therapy (PDT) is a cancer treatment modality in which systemic administration of a tumor-localizing photosensitizer is followed by irradiation of the tumor with visible light. Although PDT is undergoing clinical trials for various cancers, the mechanisms of its action are not fully understood. To investigate the mechanism of cell death by PDT, we performed in-vitro PDT, using Photofrin II as a photosensitizer, in two human cervical carcinoma cell lines, HeLa and CaSki. Methods. Cells were incubated with Photofrin II for 24 h, followed by illumination, using a YAG-OPO laser. Cell survivability after PDT was evaluated by an MTT assay. Cytotoxicity was assayed by measuring the release of lactate dehydrogenase (LDH) into the supernatant. DNA of the PDT-treated cells was electrophoresed in an agarose gel to determine fragmentation. In situ detection of apoptosis in the PDT-treated cells was performed by identification of the 3′-OH ends of DNA. In addition, induction of manganese superoxide dismutase mRNA (MnSOD) was analyzed in the PDT-treated cells. Results. The CaSki cells were more sensitive to this PDT treatment than were the HeLa cells. DNA fragmentation was observed with less than 5 μg/ml of Photofrin II in both cell lines, whereas PDT-induced cell membrane destruction, determined by LDH release, was observed only at 10 μg/ml. The MnSOD mRNA was induced in the HeLa cells in the early hours after PDT with a non-lethal dose of Photofrin II, but was reduced with a high dose, whereas the CaSki cells did not show any induction of the MnSOD gene by PDT. Conclusion. The present results suggest that PDT induces cell death by a mechanism involving membrane destruction and apoptosis. Differences in cell susceptibilities to PDT may depend upon a protective mechanism, such as MnSOD gene induction.