Toshitaro Nakagawa

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.

Multiple Point Mutation of N-ras and K-ras Oncogenes in Myelodysplastic Syndrome and Acute Myelogenous Leukemia

We analyzed activating mutations of N-ras and K-ras by the polymerase chain reaction and oligonucleotide hybridization in hematological disorders. Activating mutations of these codons were detected in 4 of 20 cases of myelodysplastic syndrome (MDS) and 15 of 77 cases of acute myelogenous leukemia (AML). Our of 19 cases of MDS and AML who carried active mutations, 7 cases were found to have two or more distinct mutations in activating codons of N-ras and K-ras. Ras mutation was found preferentially in progressive disease such as refractory anemia with excess of blasts (RAEB) of RAEB in transformation (RAEB-t). A relatively high incidence of ras mutation was found in M5 AML (40%). No ras mutations were found in other hematological disorders, such as acute lymphoblastic leukemia and chronic myelogenous-leukemia. The most frequent amino acid substitution was that of an aspartate for glycine at codon 12 of N-ras resulting from G to A mutation (11/35). The survival of AML patients who carried ras mutations showed no significant differences from those without ras mutations calculated by Kaplan-Meier. Seven cases of MDS and 7 cases of AML patients could be investigated at various points during their clinical course. Among these 14 cases, we found 2 interesting cases of MDS. The first case lost multiple clones carrying ras mutations during disease progression, the second case acquired mutation of the ras gene during disease progression. These results suggested that multiple point mutations of ras genes may not be initiating events but may contribute to a clonal evolution of MDS and AML.

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.

Successful treatment of angioimmunoblastic lymphadenopathy with dysproteinemia with cyclosporin A

Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is a lymphoma‐like disease. Although the histologic appearance of this disease is benign, it is clinically malignant, with chemotherapy being ineffective and the prognosis being poor. In this article, the authors report the results of treating two patients with AILD with cyclosporin A. One patient had cervical lymphadenopathy and fever, and AILD was refractory to both chemotherapy and a‐interferon. However, treatment with cyclosporin A achieved complete remission. The other patient had generalized lymphadenopathy, and cyclosporin A was used as the treatment of first choice. Complete remission also was achieved in this patient, who still is in remission. These two cases demonstrate that cyclosporin A can be effective for treating AILD.

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.

Glucocorticoids induce apoptosis in acute myeloid leukemia cell lines with a t(8;21) chromosome translocation

The t(8;21) chromosome translocation frequently occurs in the AML, acute myeloid leukemia, M2 sub-type. This translocation juxtaposes the AML1 gene on chromosome 21 with the MTG8(ETO) gene on chromosome 8, resulting in the expression of the AML1-MTG8(ETO) fusion transcript. The fusion product is thought to play a critical role in the abnormal proliferation and differentiation of myeloid leukemia cells. We investigated the effects of various differentiation inducers of myeloid leukemia cells on the growth and differentiation of Kasumi-1 and SKNO-1 cells, AML cell lines with t(8;21). These cells resisted differentiation into mature granulocytes and macrophages in response to various inducers of myelomonocytic differentiation, such as dimethyl sulfoxide, retinoic acid, butyrate, 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1alpha,25-dihydroxyvitamin D3. On the other hand, dexamethasone can induce apoptosis in these cells at low concentrations, whereas other myelomonocytic leukemia cell lines tested were resistant to glucocorticoid-induced apoptosis. The levels of glucocorticoid receptor gene expression were high in Kasumi-1 and SKNO-1 cells. Expression of the AML1-MTG8(ETO), bcl-2, and c-myc genes was unchanged following exposure to dexamethasone. Glucocorticoids might induce the apoptosis of some types of AML cells, just like that of some lymphoid leukemia cells.

14q11 abnormality and trisomy 8q are not common in Japanese T-cell prolymphocytic leukemia.

We studied ten cases of Japanese T-cell prolymphocytic leukemia (T-PLL) collected over the last 9 years. Median age was 61 years with a male predominance (M:F, 8:2). The main disease features were splenomegaly, lymphadenopathy, hepatomegaly, skin lesions and serous effusions. The clinical course was progressive with a median survival of 10 months. Immunophenotyping showed that the prolymphocytes had a post-thymic phenotype (TdT-, CD1a-, CD2+, CD3+, CD5+, CD7+) with a predominant CD4+ immunophenotype. Cytogenetic analysis showed no consistent abnormalities. 14q abnormality and trisomy 8q, which are frequently seen in T-PLL of Western countries, were found in only two and zero cases, respectively. We conclude that the clinical and biological characteristics of T-PLL in Japan are almost the same as those in Western countries. However, the cytogenetic findings of T-PLL in Japan might be different.

Establishment of a leukaemic cell line from a patient with acquisition of chromosomal abnormalities during disease progression in myelodysplastic syndrome

Summary. A cell line designated SKM‐1 was newly established from leukaemic cells of a 76‐year‐old Japanese male patient with monoblastic leukaemia following myelodysplastic syndrome (MDS). The cells were obtained from peripheral blood of the patient when he lost multiple point mutations of ras genes with acquisition of chromosomal abnormalities during disease progression in MDS. The cells grew as a single floating cell, and have been continuously growing with the morphological characteristics of immature monoblasts by serial passages during the past 42 months with a doubling time of about 48 h. By cytochemical analysis. the cloned cells were positive for butyrate esterase, but negative for the Epstein‐Barr virus associated nuclear antigen. Phenotypic analysis revealed the expression of myelomonocyte specific antigens such as CD4, CD13, CD33 and HLA‐DR. Cells from the primary peripheral blood and those from SO passages of the SKM‐1 cell line both possessed no activated ras genes but showed karyotype abnormalities with 46.XY, del(9)(q13;q22), der(17) t(17:?)(p13:?). The SKM‐1 cells have two mutations in p53 gene and overexpress the pS3 products. This cell line may contribute to a better understanding of molecular mechanisms in the progression from MDS to myelogenous leukaemia.