Nobuyoshi Tsuruoka

Iron deprivation‐induced apoptosis in HL‐60 cells

Iron deprivation of HL‐60 cells with deferoxamine B mesylate (DFO) induced apoptosis. DNA fragmentation became apparent with 10−6 M DFO after 48 h treatment. The apoptosis peak according to the DNA histogram on flow cytometory and typical nuclear collapse and were observed microscopically after 48 h treatment with 10−4 M DFO. Cells treated with 10−4 M DFO for as little as 24 h were shown to be committed to apoptosis, as chromatin condensation progressed gradually thereafter.

Frequent loss of heterozygosity in the region of the D7S523 locus in advanced ovarian cancer

Loss of heterozygosity (LOH) of the long arm of chromosome 7 occurs frequently in many types of primary cancers. We analyzed 22 primary ovarian cancers for LOH of chromosome arm 7q using a set of 16 microsatellite markers in order to determine the location of a putative tumor suppressor gene (TSG). Eleven samples (50%) showed LOH at least at one locus on chromosome arm 7q. We identified the smallest commonly deleted region to be at 7q31.1, which includes D7S523. LOH of chromosome arm 7q was more frequent in advanced stages (III–IV) (7/9, 78%) than in early stages (I–II) (4/13, 31%) of ovarian cancer (P<0.05). These data suggest that alteration of a TSG at 7q31.1 gene plays an important role in advanced ovarian cancer. Genes Chromosom. Cancer 19:1–5, 1997.

Spontaneous cytokine overproduction by peripheral blood mononuclear cells from patients with myelodysplastic syndromes and aplastic anemia

We studied spontaneous cytokine production by peripheral blood mononuclear cells (PBMC) obtained from 14 patients with aplastic anemia (AA) and 28 various myelodysplastic syndromes (MDS). The levels of interleukin-6, interleukin-1 beta, and tumor necrosis factor-alpha in cultured PBMC were measured by ELISA. The average levels of these cytokines were higher in AA or in refractory anemia (RA) than in RA with excess of blasts (RAEB) or in RAEB in transformation (RAEB-T). Marked cytokine overproduction was observed in RA as well as in AA. High cytokine levels were observed in hypocellularity and low blast cell counts in the bone marrow. These results may suggest that the increase of cytokines may be a reactive response in hypocellular bone marrow.

Immunodeficiency and IL‐6 production by peripheral blood monocytes in multicentric Castleman's disease

Summary. To study the pathogenesis of multicentric Castlemans disease (MCD), IL‐6 producing cells and immune function were investigated in four MCD patients. The expression of IL‐6 mRNA in one MCD lymph node was analysed by in situ hybridization. IL‐6 mRNA expressing cells were scattered in the interfollicular areas and did not resemble plasma cells. Spontaneous IL‐6 production was detected in the culture supernatants of peripheral blood mononuclear cells (PBMNC) from four patients. The IL‐6 producing cells among the PBMNC were found to be monocytes by both in situ hybridization and immunohistochemistry. We evaluated immune function in four MCD patients. These studies show: (1) a negative PPD skin test in 3/4 patients. (2) decreased IL‐2 production in 3/4 patients, (3) decreased T cell colony formation in 3/4 patients, (4) decreased NK activity and NK cell number in 2/4 patients, (5) increased soluble IL‐2 receptor in 4/4 patients, and (6) decreased CD4/CD8 ratio in 3/4 patients. These results show that MCD resembles, in several ways, acquired immunodeficiency syndrome (AIDS).

Allelotyping of acute myelogenous leukemia: loss of heterozygosity at 7q31.1 (D7S486) and q33-34 (D7S498, D7S505).

Loss of a whole chromosome 7(-7) or a deletion of the long arm of chromosome 7 del(7q) occurs frequently in many types of primary cancers including cases of acute myelogenous leukemia (AML). We analyzed for loss of heterozygosity (LOH) of chromosome arm 7q in 26 AML cases using a set of 15 microsatellite markers in order to begin to determine the location of putative tumor suppressor genes (TSG) important to this disease. Seven samples (27%) showed LOH at one or more loci on chromosome 7q. We identified the smallest commonly deleted regions to be at 7q31.1 (D7S486) and 7q33-34 (D7S498, D7S505) suggesting that alterations of a TSG in each region have an important role in de novo AML.

Structural integrity of the cyclin-dependent kinase inhibitor genes, p15, p16 and p18 in myeloid leukaemias.

Summary The cyclin‐dependent kinase inhibitors known as p15, p16 and p18 have been suggested as candidates for tumour suppressor genes. We examined these genes for their alterations in 46 myeloid leukaemias and 15 myeloid leukaemia cell lines, p16 mRNA expression was studied in 41 myeloid leukaemias. The p15 and p16 genes were either deleted or mutated in myeloid leukaemia lines at a high frequency [6/15 (40%) for p15; 8/15 (53%) for p16] but alterations in primary myeloid leukaemias are much less frequent [2/46 (4%) for p15; 3/46 (6%) for p16]. Alterations of p18 were not found in any of the samples. 13 primary myeloid leukaemia samples had negligible levels of p16 mRNA. In summary, the deletions of p15 and p16 genes identified in the myeloid leukaemia cell lines probably occurred during their in vitro immortalization. Alterations of the pl6 or pl5 gene only occurred in primary acute myeloid leukaemia samples that were of mixed myeloid/ lymphoid lineage (CD19/CD20‐positive acute myeloid leukaemia [AML], CD2/CD19‐positive AML, and lymphoid blastic crisis of chronic myeloid leukaemia). Further studies are required to determine if the absence of mRNA expression results from inactivation of the p16 gene.

Molecular analysis of the Cip1/Waf1 (p21) gene in diverse types of human tumors.

We have screened for mutations in the Cip1/Waf1 gene using Southern blot analysis and the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method in diverse human tumors. Seven of 102 (7%) human tumor samples were identified to have point mutations within the coding region of the Cip1/Waf1 gene. Two of the seven mutated cases showed gene rearrangements. These results suggest that the frequency of genetic alterations in the Cip1/Waf1 gene is relatively low in comparison with several known tumor suppressor genes.

Enhancement by bufalin of retinoic acid-induced differentiation of acute promyelocytic leukemia cells in primary culture

Bufalin, a cardiotonic steroid isolated from the Chinese toad venom preparation Chansu, has differentiation-inducing activity in several myeloid leukemia cell lines. We examined the effect of bufalin on differentiation of leukemic cells from acute myeloid leukemia (AML) patients in primary culture. Bufalin significantly stimulated functional and morphologic differentiation of leukemia cells in four of 20 cases, suggesting that bufalin alone is only a modest inducer of differentiation of AML cells in primary culture. In contrast, acute promyelocytic leukemia (APL) cells showed synergistic differentiation after treatment with all-trans retinoic acid (RA) and bufalin. In some cases, bufalin restored RA sensitivity to previously resistant APL cells. The effective concentration of bufalin for differentiation-inducing activity in APL cells was lower than for its cardiac action. Combined treatment with bufalin and RA may be more effective than RA alone in differentiation therapy of APL.

Granulocyte-colony Stimulating Factor and Retinoic Acid Cooperatively Induce Granulocytic Differentiation of Acute Promyelocytic Leukemia Cells in vitro

The interaction of granulocyte‐colony stimulating factor (G‐CSF) and retinoic acid (RA) in proliferation and differentiation of acute promyelocytic leukemia (APL) cells was examined. G‐CSF stimulated proliferation of APL cells at concentrations of 0.1 to 50 ng/ml in a dose dependent manner. More than 10−8M RA induced granulocytic differentiation of APL cells. Although G‐CSF induced lysozyme activities in APL cells, it alone did not induce terminal differentiation of APL cells. G‐CSF significantly enhanced the RA‐induced granulocytic differentiation of APL cells in vitro. Enhancement by G‐CSF was not due to the prolongation of survival of RA‐induced differentiated cells, but the differentiation‐inducing effects of G‐CSF might be evident only in the presence of RA. Since G‐CSF has a potential to induce the granulocytic differentiation of myeloid leukemia cells, G‐CSF in combination with RA may be applicable in differentiation induction therapy for some types of myeloid leukemia.

DNA damage induces p21 protein expression by inhibiting ubiquitination in ML-1 cells

We previously reported that deferoxamine, an iron chelating agent, induced p53 and cell accumulation in the G1 phase of ML-1 cells in the same way as the DNA damaging agent, etoposide. Etoposide treatment increased expression of the p21 gene, a cyclin kinase inhibitor, at both the mRNA and protein levels. However, deferoxamine treatment only increased the p21 mRNA level without the appearance of a detectable protein product. A substrate for cyclin kinase, pRB, was unphosphorylated by etoposide treatment, but remained unaffected by deferoxamine, indicating that p21 was functional after etoposide, but not after deferoxamine treatment. Therefore, in the present study, we investigated the involvement of the ubiquitin proteasome pathway in post-transcriptional regulation of p21. By the addition of lactacystin, a proteasome inhibitor, to deferoxamine treatment, the level of unubiquitinated p21 protein product was similar to that induced by etoposide treatment, and the ubiquitinated p21 bands became apparent. After etoposide treatment, the level of ubiquitinated p21 was diminished and a high level of unubiquitinated p21 expression was observed. We concluded that (1) efficient expression of p21 protein requires inhibition of the ubiquitin-proteasome pathway, and (2) DNA damage inhibits the ubiquitination of p21.