Biomedical subjects

H Ohtsubo

Publications and source records attributed to H Ohtsubo.

At least 37 records · Page 2Linked to original sources

PubMed1999

NF-kappaB involvement in the activation of primary adult T-cell leukemia cells and its clinical implications.

The HTLV-I provirus-encoded Tax protein induces NF-kappaB in Tax-transfected Jurkat T cells or HTLVL-I- infected T cells in vitro. Tax induction of NF-kappaB is presumed to be involved in proliferation and activation of primary leukemia cells in vivo. Recent studies have demonstrated that NF-kappaB activities in human T cells are mediated by at least four c-Rel-related DNA binding proteins - p50, p55, p75 and p85. We examined the significance of NF-kappaB induction in primary adult T cell leukemia cells and the induction kinetics of each of the four NF-kappaB species. Marked NF-kappaB activity was detected using an electrophoretic mobility shift assay (EMSA) in the primary cells of patients with acute disease, but little activity was noted in the cells of chronic patients. NF-kappaB activity was enhanced in a time-dependent manner in acute type cells cultured with mitogen-free medium; there was no induction of activity in chronic type cells. UV crosslinking demonstrated all four species of NFkappaB complex - high levels of p50 and lower levels of p55 and p75, in acute type cells; chronic type cells showed only the p50. As a control, normal resting T cells similarly showed only p50; control cells showed little change in activity when cultured without mitogenic stimulation, analogous to chronic type ATL. Northern blotting revealed enhancement of c-rel (encoding p85) and KBFI (encoding p50 and p55) expression in acute type cells during culture, while there was no significant enhancement of mRNAs in chronic type ATL cells or unstimulated normal T cells. Northern blotting also revealed that Tax is upregulated at the mRNA level in acute- but not chronic-type cells during culture. Expression of c-rel and KBF1 mRNAs in acute type cells appeared to be related to Tax mRNA expression. These results suggest that Tax is capable of inducing nuclear expression of all four NF-kappaB species in primary ATL cells of acute type patients, with marked effects on p55, p75, and p85. Tax induction of NF-kappaB species is regulated, at least in part, at a pretranslational level involving increases in c-rel and KBF1 mRNA.

Acute Disease↗

PubMed1999

Identification and phylogenetic analysis of gypsy-type retrotransposons in the plant kingdom.

PCR was performed with degenerate primers which hybridized to the homologous sequences in the reverse transcriptase (rt) genes of gypsy-type retrotransposons from rice (RIRE3, RIRE8 and RIRE2), using total DNA samples from various plants (monocots, dicots, pine, ginkgo, horsetail, liverwort and algae) as templates. Cloning and sequencing showed that the amplified fragments had various degrees of homology to the rt sequences of rice retrotransposons. Phylogenetic analysis showed that these retrotransposon homologues and some additional gypsy-type retrotransposons previously identified from plants could be classified into two families, A and B. In each family, the retrotransposons were further classifiable into several subfamilies. Interestingly, retrotransposons from a single or related plant species were clustered in each subfamily. This indicates that sequence divergence during vertical transmission has been a major influence on the evolution of gypsy-type retrotransposons in plants. The retrotransposons isolated from one plant species could often be classified into the two families. This indicates that the gypsy-type retrotransposons of a family evolved independently within a species without affecting the evolution of retrotransposons of the other family. Retrotransposons in each subfamily are characterized by the lengths of LTR, by the nucleotide sequences in the terminal regions of LTRs, and by the PBS (primer binding site) sequence complementary to the 3' sequence of a particular tRNA species.

DNA Primers↗

PubMed1999

RIRE2, a novel gypsy-type retrotransposon from rice.

The 441-bp DNA segment in a PCR-amplified fragment from Oryza sativa cv. IR36 was found to have a sequence with features characteristic of LTRs of retroelements, which was named RIRE2 (Rice retroelement #2) and further analyzed. Cloning and sequencing analyses of the DNA segments connected to LTR-like sequence showed that RIRE2 has a long internal region almost 10 kb long that is flanked by LTR-like sequences. This internal region carries a primer binding site (PBS) and polypurine tract (PPT) which are necessary for cDNA synthesis of retroelements. The PBS sequence is complementary to the 3' end region of tRNA(Arg). The internal region has an rt gene homologous to that of gypsy-type retrotransposons, evidence that RIRE2 is indeed a retrotransposon related to gypsy from Drosophila. RIRE2 has an extra sequence more than 4 kb long in the region downstream of gag-pol. Phylogenetic analysis of the putative amino-acid sequences of the rt gene as well as the int gene showed that RIRE2 is related to a group of gypsy-type retrotransposons of a large size that include Grande1-4 of teosinte, Tat4-1 and Athila1-1 of Arabidopsis thaliana, and Cyclops-2 of pea, but distantly related to any other group of gypsy-type retrotransposons, including RIRE3 and RIRE8 of rice. RIRE2 and Grande1-4 had the highest homology in the gag-pol region, but the nucleotide sequences of the LTR regions differed. Both elements had significant homology in the middle area of the extra regions downstream of gag-pol, in which they had an open reading frame encoding a protein with no known function on the opposite strand from that coding for gag-pol.

Amino Acid Sequence↗

PubMed1999

Epstein-Barr virus involvement in T-cell malignancy: significance in adult T-cell leukemia.

Epstein-Barr virus (EBV) was first reported as the causative virus of Burkitt's lymphoma in 1964. Since then, EBV has also been associated with infectious mononucleosis, AIDS and transplant-related B cell lymphomas, and nasopharyngeal cancer. The virus has further been linked with T cell lymphomas, Hodgkin disease, and NK leukemia or LGL leukemia, establishing a concept of a wide spectrum of EBV associated malignant disorders. EBV DNA encodes several proteins such as EBNA1-6, LMP 1, 2 and others. Recent studies have demonstrated that EBNA2, EBNA5, EBNA3A, EBNA 3C are essential for transformation, and that any gene product is not sufficient to transform cells by itself. Further there are different mechanisms of virus-associated transformation or carcinogenesis among EBV-associated malignant disorders. On the other hand, human T lymphotropic virus type I (HTLV-I) is known as a causative virus of adult T cell leukemia (ATL). However, precise molecular mechanisms of leukemogenesis in ATL still remains unclear. Some additional factors to HTLV-I infection are supposed to be involved in complete leukemogenesis. We demonstrated that HTLV-I infected T cells and primary ATL cells express EBV receptor/CD21 on the cell surface. Therefore, it is possible that EBV infection is one of the factors. We further investigated this possibility in 6 HTLV-I infected T cell lines and primary ATL cells from 18 patients with ATL. However, no EBV genome was detected in both T cell lines and primary ATL cells. EBV involved T-cell lymphoma has unique clinical manifestations as compared to non-EBV involved T-cell lymphoma. Therefore, it is still possible that a small group of ATL patients with unique clinical manifestations is associated with EBV.

Adult↗

PubMed1999

Long-term maintenance combination chemotherapy with OPEC/MPEC (vincristine or methotrexate, prednisolone, etoposide and cyclophosphamide) or with daily oral etoposide and prednisolone can improve survival and quality of life in adult T-cell leukemia/lymphoma.

Acute leukemia and lymphoma varieties of adult T-cell leukemia/lymphoma (ATL) usually carry a poor prognosis. While etoposide is generally useful for treating ATL, especially as a daily oral maintenance regimen, etoposide has not proven effective in severe types of ATL efficient in some patients. Of 87 ATL patients whom we have treated, 51 had acute leukemia, 22 lymphoma and 14 progressive chronic leukemia. Seventy-nine patients were treated with a long term maintenance combination protocol, OPEC/MPEC (weekly doses of vincristine, 0.7 mg/m2 or methotrexate, 14 mg/m2; prednisolone, 20 mg/m2; etoposide, 70 mg/m2 and cyclophosphamide, 200 mg/m2). The other 8 patients, 3 with acute leukemia, 2 with lymphoma and 3 with progressive chronic leukemia, were treated with daily oral administration of 25 mg of etoposide and 10 mg of prednisolone (DOEP). The dose administered was modified in individual cases to maintain the granulocyte count and reduce the number of ATL cells. Considering both protocols, a complete response and a partial response were achieved in 31.0% and 58.6% patients, respectively. Median survival times (MST) of all patients and, acute leukemia, lymphoma and progressive chronic leukemia types were 7.5, 6.7, 9.6 and 12.4 months, respectively. Respective MST of patients treated with OPEC/MPEC or DOEP protocols were 7.1 and 18.0 months. Relatively normal WBC counts, lower lactate dehydrogenase concentration and normal calcium concentration, limited numbers of anatomic sites involved, good performance status and good response to chemotherapy were significantly associated with long survival time. Drug toxicity was not apparent, and about half of patients were treated in an outpatient setting.

Administration, Oral↗

PubMed1999

[Primary myelofibrosis with fatal mesenteric arterial thromboembolism caused by antiphospholipid syndrome].

A 60-year-old woman was admitted to our hospital in February 1993 due to dizziness, dyspnea, abdominal pain, and high susceptibility to bleeding. Physical examination revealed livedo reticularis of the foot, but did not detect hepatosplenomegaly. Examination of the peripheral blood detected pancytopenia, leukoerythroblastosis, and tear-drop erythrocytes. Primary myelofibrosis (PMF) was diagnosed on the basis of bone marrow biopsy findings. Antiphospholipid syndrome (APS) was confirmed by positive response to anti-cardiolipin antibody and recurrent splenic infarction. Because of factor XIII deficiency, the patient experienced severe gingival bleeding after tooth extraction. Her condition was complicated by mesenteric arterial thromboembolism and she died of sepsis 5 years after onset. Although the incidence of immunopathy in PMF patients is high, few studies to date have focused on APS patients presenting with a variety of severe embolic symptoms. Our patient required careful monitoring due to bleeding tendency and thromboemboli.

Antiphospholipid Syndrome↗

PubMed1998

IL-2-induced growth of CD8+ T cell prolymphocytic leukemia cells mediated by NF-kappaB induction and IL-2 receptor alpha expression.

The binding of interleukin-2 (IL-2) to its receptor on normal T cells induces nuclear expression of nuclear factor kappaB (NF-kappaB), activation of the IL-2 receptor (IL-2R) alpha chain gene, and cell proliferation. In the present study, the role of IL-2R signaling in the growth of CD8+ T cell prolymphocytic leukemia (T-PLL) cells has been investigated. Flow cytometry revealed that primary leukemia cells from a patient with CD8+ T-PLL expressed IL-2Ralpha and beta chains, and the cells showed a proliferative response and an increase in IL-2Ralpha expression on culture with exogeneous IL-2. Northern blot analysis failed to detect IL-2 mRNA, suggesting that IL-2 may act in a paracrine manner in vivo. Electrophoretic mobility-shift assays revealed that recombinant IL-2 increased NF-kappaB binding activity in nuclear extracts of the leukemia cells, and Northern blot analysis showed that IL-2 increased the abundance of mRNAs encoding the NF-kappaB components c-Rel and KBF1 in these cells. IL-2 binding analysis demonstrated that IL-2 markedly increased the number of low affinity IL-2Rs on the leukemia cells, without an effect on the number of high-affinity IL-2Rs. These results show that IL-2 is capable of inducing the nuclear expression of NF-kappaB in primary CD8+ T-PLL cells, and that this effect is mediated, at least in part, at a pretranslational level.

Blotting, Northern↗

PubMed1998

Neurotropic melanoma invading the median nerve.

We report a case of acral lentiginous melanoma of the right thumbnail bed which demonstrated characteristic intraneural invasion and extension along the median nerve. Four years after amputation of the involved thumb, a melanotic tumor recurred on the right thenar. Radiation therapy was given. The tumor invaded the median nerve, however, causing progressive pain and paralysis of the right hand. Despite right arm amputation, the tumor extensively metastasized, and the patient died three years later. Histopathologically, the tumors were characterized by extensive proliferation of spindle-shaped cells forming neuroid fascicles especially prominent in the metastatic region. Tumor cells were positive immunohistochemically with S-100 protein antisera.

Amputation, Surgical↗

PubMed1997

Frequent expression of interleukin-9 mRNA and infrequent involvement of interleukin-9 in proliferation of primary adult T-cell leukemia cells and HTLV-I infected T-cell lines.

To examine the possibility that interleukin-9 (IL-9) may be involved in oncogenesis and the proliferation of adult T-cell leukemia (ATL) cells, we examined the expression of IL-9 mRNA and growth response to IL-9 in five human T-lymphotropic virus type-I (HTLV-I) infected T-cell lines and in primary leukemia cells in peripheral blood from eight patients with ATL (four acute ATL and four chronic). Four out of five cell lines expressed IL-9 mRNA not correlated with Tax expression. Primary ATL cells from all patients also expressed IL-9 mRNA not correlated with the clinical forms. Recombinant IL-9 showed growth enhancing activity in only one out of five cell lines and one out of eight patients' primary leukemic cells. These results suggest the infrequent involvement of IL-9 in the proliferation of ATL cells, both primary tumor cells and HTLV-I infected T-cell lines.

Adult↗

PubMed1997

Granulocyte-colony stimulating factor induces differentiation and apoptosis of CD2, CD7 positive hybrid leukemia cells in vivo and ex vivo.

We report a case of a 70-year-old man with a hybrid leukemia treated successfully with granulocyte-colony stimulating factor (G-CSF) combined with a cytocine arabinoside regimen through the induction of differentiation of leukemic cells into monocytoid cells resulting in apoptosis. The leukemic cells demonstrated a TCR-gamma rearrangement, and expressed CD2, CD7, CD33 and G-CSF receptors but not CD11b on the cell surface nor non-specific esterase in cytoplasm. Several days following the administration of G-CSF, the cells with monocytoid characteristics such as CD11b and cytoplasmic non-specific esterase appeared in the peripheral blood replacing the blastic cells. The cells were shown to be derived from the same clone of the leukemic cell because of the identical TCR-gamma gene rearrangement. The short-term culture of leukemic cells with G-CSF induced the differentiation into a monocyte lineage, resulting in apoptosis. Although there is no denying the possibility that cytosine arabinoside is partly responsible, our results strongly suggest that G-CSF plays the main role in differentiation of leukemic cells into a monocyte lineage inducing apoptosis in vivo in this patient.

Aged↗

PubMed1997

Repetitive sequences: cause for variation in genome size and chromosome morphology in the genus Oryza.

Large variation in genome size as determined by the nuclear DNA content and the mitotic chromosome size among diploid rice species is revealed using flow cytometry and image analyses. Both the total chromosomal length (r = 0.939) and the total chromosomal area (r = 0.927) correlated well with the nuclear DNA content. Among all the species examined, Oryza australiensis (E genome) and O. brachyantha (F genome), respectively, were the largest and smallest in genome size. O. sativa (A genome) involving all the cultivated species showed the intermediate genome size between them. The distribution patterns of genome-specific repetitive DNA sequences were physically determined using fluorescence in situ hybridization (FISH). O. brachyantha had limited sites of the repetitive DNA sequences specific to the F genome. O. australiensis showed overall amplification of genome-specific DNA sequences throughout the chromosomes. The amplification of the repetitive DNA sequences causes the variation in the chromosome morphology and thus the genome size among diploid species in the genus Oryza.

Base Sequence↗

PubMed1997

Granulocyte-colony stimulating factor-induced proliferation of primary adult T-cell leukaemia cells.

Granulocyte-colony stimulating factor (G-CSF) is known to induce proliferation and differentiation of granulocyte progenitors, and is widely used to treat neutropenia induced by intensive chemotherapy for malignant lymphoma or adult T-cell leukaemia/lymphoma (ATL). G-CSF is thought not to stimulate malignant lymphoid cells. In the present study we examined the ability of G-CSF to induce in vitro growth of primary ATL cells from 14 patients (nine acute-type, two chronic-type and three lymphoma-type), and we analysed the in vivo counts of ATL cells in patients who received G-CSF for neutropenia. FACS analysis using phycoerythrin-labelled recombinant G-CSF demonstrated that ATL cells from 11/14 patients express some G-CSF receptor (G-CSFR), with a range between 5.4% and 87.3%. Cells expressing G-CSFR also expressed CD4. Reverse polymerase chain reaction (PCR) analysis demonstrated expression of G-CSFR messenger RNA in G-CSFR expressing cells. Leukaemic cells derived from seven (four acute-type, one chronic-type and two lymphoma-type) of the 14 patients proliferated in vitro in response to G-CSF, as measured by [3H]thymidine incorporation; maximum responses were at G-CSF concentrations of 10-100 ng/ml. Nine of 14 patients receiving rG-CSF for neutropenia were analysed retrospectively for ATL cell numbers. Four patients whose primary tumour cells proliferated in response to rG-CSF in vitro showed a significant increase in ATL cell count after administration of rG-CSF (P = 0.038), whereas five patients whose leukaemic cells did not proliferate in vitro showed no significant increase in ATL cell count. G-CSF can stimulate proliferation of ATL cells which may complicate therapy for this disease.

Aged↗

PubMed1997

RIRE1, a retrotransposon from wild rice Oryza australiensis.

RIRE1 is a retrotransposon present in wild rice Oryza australiensis in an extraordinary number of copies, and only a portion of the LTR sequence has been determined previously. Here, we isolated and sequenced DNA segments of various portions of RIRE1, revealing that the sequences of LTR and the internal region were 1523 and 5277 bp in length, respectively. The internal region shows homology with the pol region in copia, a Drosophila retrotransposon, indicating that RIRE1 is a copia-like retrotransposon. The internal region of RIRE1 contained an open reading frame coding for genes, gag, pro, int, rt and rh, like copia and retroelements related to it. A clone screened from a library of the O. australiensis genomic DNA contained solo LTR, which was flanked by direct repeats of a 5-bp sequence. This suggests that RIRE1 generates a duplication of the target sequence of 5 bp upon retroposition. We observed that many RIRE1 members were nested by another RIRE1 member. This indicates that these RIRE1 members have received another RIRE1 to make an extraordinary number of copies in the O. australiensis genome without giving a deleterious effect on the growth of rice cells.

Amino Acid Sequence↗

PubMed1997

Human T lymphotropic virus-type I Tax induction of CD21/Epstein-Barr virus receptor expression on T cells and its significance in leukemogenesis of adult T cell leukemia.

CD21, which is expressed on B cells, is also expressed on human T lymphotropic virus-type I (HTLV-I)-infected T cell lines. CD21 also serves as a receptor of Epstein-Barr virus (EBV). We evaluated the mechanism of CD21 induction on HTLV-I-infected T cells and its clinical significance in the leukemogenesis of adult T cell leukemia (ATL). CD21 induction was detected at very low levels in T cell lines (Jurkat and CEM cells), and in non- or low-Tax-producing HTLV-I-infected T cell lines (Oh13T, S1T, and Su9T01 cells). In contrast, marked induction of CD21 was detected in high-Tax-producing HTLV-I-infected T cell lines (K3T, F6T, and MT-2). A Jurkat T cell clone stably transfected with tax-expressing cDNA expressed a significant amount of CD21 on the cell surface. These results strongly suggest that HTLV-I Tax induces CD21 on T cells. On two-color analysis, CD21 expression was detected in CD4+ T cells of the primary ATL cells from a subset of patients, suggesting that EBV infection may be associated with the leukemogenesis of ATL, at least in part. However, no genome of EBV was detected in the genomic DNA of six HTLV-I-infected T cell lines or the primary ATL cells separated from all patients, indicating the irrelevance of EBV infection to ATL leukemogenesis.

DNA, Viral↗

PubMed1996

Relation of autonomous and interleukin-2-responsive growth of leukemic cells to survival in adult T-cell leukemia.

We examined autonomous and interleukin-2 (IL-2)-responsive growth activities of leukemic cells derived from peripheral blood, as well as several clinical manifestations, including serum lactate dehydrogenase (LDH) level, of 35 patients with adult T-cell leukemia (ATL) to determine whether these properties were related to prognosis. Growth activities were measured by [3H]-thymidine incorporation of the cells after 24 hours' culture with or without exogenous IL-2. Both autonomous and IL-2-responsive growth activities were higher in the patients than in healthy controls and were significantly correlated with each other (P < .0001, r = .956). Both higher growth activities were significantly associated with shorter survival times (P = .0042, r = .472 and P = .0117, r = .421, respectively). An increased serum LDH value was also significantly associated with shorter survival times (P = .0011, r = .530), but corrected calcium level, sex, white blood cell count, or age were not. These results strongly suggest that both growth activities of primary tumor cells, in addition to the serum LDH value, are prognostic determinants in ATL. We propose a new prognostic classification combining LDH values and autonomous growth activity into three groups: (1) high growth activity and high LDH; (2) high growth activity and low LDH, or low growth activity and high LDH; and (3) low growth activity and low LDH, which showed a significant relationship to survival time (P = .0014; the median survival time for each group was 39, 94, and 340 days, respectively).

Adult↗

PubMed1996

Identification of Tnr3, a suppressor-mutator/enhancer-like transposable element from rice.

We isolated members of the retroposon family p-SINE1 in rice and found that one member contained an insertion.Aa 3-bp sequence at the insertion site within p-SINE1 appeared duplicated. The insertion sequence, 1539 bp in length, carried imperfect inverted repeats of about 13 bp at its termini which begin with 5'-CACTA---3'; these repeats are similar to those found in members of the En/Spm transposable element family. These results indicate that the insertion sequence is a transposable element belonging to the En/Spm family and is thus named Tnr3 (transposable element in rice no. 3). In fact, Tnr carried long subterminal regions containing direct and inverted repeats of short DNA sequences of 15 bp, another characteristic of the En/Spm family. The subterminal repeat sequences in Tnr3 are, however, of two kinds, although they share homology with each other. Tnr3 and its relatives were present in multiple copies in rice. considering the length of Tnr3, it cannot represent an autonomous type element, but is a non-autonomous element probably derived by deletion from an autonomous transposon.

Base Sequence↗

PubMed1996

Interleukin-2-mediated growth of leukemic cells in lymph nodes of patients with adult T-cell leukemia/lymphoma.

We investigated the effect of interleukin-2 (IL-2) on tumor growth of primary adult T-cell leukemia/lymphoma (ATL) cells in biopsied lymph node cells obtained from 14 patients (seven [corrected] with acute-type disease, one with chronic-type disease and six [corrected] with lymphoma-type disease). Biological activity of IL-2 in culture supernatants of the cells was detected in six out of 12 cases. The IL-2 mRNA in the lymph node cells was detected in four out of nine patients by northern blotting. However, it was detected in all nine patients examined by reverse polymerase chain reaction (PCR) method. Lymph node cells from 12 out of 14 patients showed a high or moderate proliferative response to IL-2; the remaining two patients showed a slight response. These results suggest that malignant growth of primary tumor cells in lymph nodes may be associated with the IL-2-IL-2 receptor system in patients with ATL more frequently than had been previously thought.

Adult↗

PubMed1996

Expression of cadherin-catenin complexes in human leukemia cell lines.

Cadherins are Ca2(+)-dependent cell-cell adhesion molecules, and are involved in the formation and maintenance of the histo-architecture. Using cultured human leukemia cell lines (adult T cell leukemia and thymus-derived lymphoma cell lines), we obtained evidence that cadherins and catenins are expressed in these cell lines but not in normal leukocytes. Immunoblot analysis of cells using a pan-cadherin serum, directed against the conserved carboxyl-terminus of cadherins, revealed a major band of 130 kDa and a minor one of 135 kDa. The 130 kDa cadherin was also recognized by anti-N-cadherin antibodies. A human N-cadherin cDNA probe hybridized to a 4.3 kb mRNA isolated from cells immunologically positive for N-cadherin. Sequencing of the cDNA fragments isolated from the cells revealed a N-cadherin sequence. Cell surface expression of N-cadherin was confirmed by indirect immunofluorescence staining of the cells. Immunoblot and Northern blot analyses also revealed the presence of alpha-catenin, beta-catenin, and gamma-catenin (plakoglobin) in these cell lines. Immunoprecipitation with anti-N-cadherin antibodies and subsequent immunoblot analysis with anti-catenin antibodies revealed that N-cadherin is associated with alpha- and beta-catenins, a prerequisite for cadherins to be functional. These results suggest an important role of the cadherin-catenin complexes in the behavior of the leukemia cells.

Base Sequence↗