Methylation analysis of asparagine synthetase gene in acute lymphoblastic leukemia cells.
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Aspartate-Ammonia Ligase↗
Biomedical subjects
N Kawamata
Publications and source records attributed to N Kawamata.
Dlk1 in normal and abnormal hematopoiesis.
Dlk1 (Pref-1) is a transmembrane and secreted protein, which is a member of the epidermal growth factor-like family, homologous to Notch/Delta/Serrate. We have found by real-time RT-PCR that Dlk1 mRNA levels were high in CD34(+) cells in 10 of 12 MDS samples compared with CD34(+) cells from 11 normals. Also, Dlk1 mRNA was elevated in mononuclear, low density bone marrow cells from 11/38 MDS patients, 5/11 AML M6 and 2/4 AML M7 samples. Furthermore, 5/6 erythroleukemia and 2/2 megakaryocytic leukemia cell lines highly expressed Dlk1 mRNA. Levels of Dlk1 mRNA markedly increased during megakaryocytic differentiation of both CMK megakaryoblasts as well as normal CD34(+) hematopoietic stem cells. High serum levels of Dlk1 occurred in RA (4/10) and essential thrombocythemia (2/10) patients. Functional studies showed that forced expression of Dlk1 enhanced proliferation of K562 cells growing in 1% fetal bovine serum. Analysis of hematopoiesis of Dlk1 knockout mice suggested that Dlk1 contributed to granulocyte, megakaryocyte and B-cell clonogenic growth and was needed for generation of splenic B-cells. In summary, Dlk1 is overexpressed in selected samples of MDS (especially RA and RAEB) and AML (particularly M6, M7), and it appears to be associated with normal development of megakaryocytes and B cells.
Animals↗
Molecular analysis of tumor suppressor genes, Rb, p53, p16INK4A, p15INK4B and p14ARF in natural killer cell neoplasms.
Natural killer (NK) cell neoplasms, which are derived from mature or precursor NK cells, are rare diseases and are observed predominantly in Asian countries. We analyzed the status of the Rb, p53, p15INK4B, p16INK4A and p14ARF genes in these diseases by Southern blot, polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and western blot analysis. We used 31 NK cell neoplasms, including four cell lines derived from NK cell neoplasms, 3 myeloid / NK cell precursor acute leukemias, 4 blastic NK cell lymphoma / leukemias, 4 aggressive NK cell leukemia / lymphomas, 4 nasal NK cell lymphomas, and 12 chronic NK lymphocytosis. We found gene amplification of the p53 gene in one nasal NK cell lymphoma, and point mutations of the p53 gene in one blastic NK cell lymphoma / leukemia and one chronic NK lymphocytosis. In addition, homozygous deletions of p15, p16 and p14 genes in 5 out of 31 samples were detected; 3 were from nasal NK cell lymphoma and 2 from blastic NK cell lymphoma / leukemia. Also hemizygous deletion of the Rb gene in one blastic NK cell lymphoma was detected. Rb proteins were highly expressed in one cell line as well as two myeloid / NK cell precursor acute leukemias. In other cell lines, complete loss and an aberrant migration pattern of Rb protein expression were observed. Comparative genomic hybridization suggested that the homozygous deletions of the p15, p16 and p14 were subtle chromosomal deletions and could not be identified by standard karyotyping in some cases. Although the number of cases we analyzed was not large, alterations identified in the Rb, p53, p16, p15 and p14 genes are of significance and might be associated with tumorigenesis in NK cell neoplasms.
Blotting, Southern↗
A novel mutation that leads to a congenital factor XI deficiency in a Japanese family.
We have identified a novel mutation leading to a congenital deficiency of the coagulation factor XI (FXI) in a Japanese family. A propositus was a 42-year-old female patient without bleeding tendency. Coagulant activity and the antigen level of FXI in her plasma were below the detectable range. The nucleotide sequences of the FXI gene of this patient were determined by a direct sequence method established in this study. A novel nonsense mutation (CAA; Gly263 --> TAA; stop) was identified in exon 8 of the FXI gene. Her parents are first cousins, and a polymerase chain reaction-restriction-fragment length polymorphism analysis revealed that her parents were heterozygous at this nucleotide position. This patient inherited mutant alleles from her parents and is homozygous at this nucleotide position. The nonsense mutation in the FXI gene is responsible for her deficiency of FXI.
Adult↗
[Autoimmune hemolytic anemia].
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Anemia, Hemolytic, Autoimmune↗
P-glycoprotein expression on normal and abnormally expanded natural killer cells and inhibition of P-glycoprotein function by cyclosporin A and its analogue, PSC833.
P-glycoprotein (P-gp), a transmembrane efflux pump encoded by the MDR1 gene, has been found to be expressed in many normal bone marrow and peripheral blood cells. Among normal leukocytes, CD3(-)CD16(+) or CD3(-)CD56(+) lymphocytes, ie, natural killer (NK) cells, express relatively high levels of P-gp, but little is known about P-gp in abnormally expanded NK cells. In this study, we examined the expression and activity of P-gp on NK cells derived from three normal donors, six patients with indolent NK cell-lineage granular lymphocyte-proliferative disorder (NK-GLPD), three patients with aggressive NK cell tumors (one NK cell leukemia and two nasal NK cell lymphoma), and two NK cell lines. By flow cytometric analysis using the monoclonal antibody (MoAb) MRK16 and rhodamine 123 dye (Rh123), P-gp expression and the efflux of Rh123 were found in all NK samples except one NK cell line. The Rh123 efflux of NK cells was inhibited by cyclosporin A (CsA) and its analogue PSC 833, but the aggressive NK tumor cells were less inhibited than were the other NK cells. The percent inhibition of efflux in the normal NK cells, indolent NK-GLPD cells and aggressive NK cell tumors was 81.8% +/- 0. 9%, 93.4% +/- 3.1% and 36.9% +/- 11.7%, respectively, by 1 micromol/L CsA, and 80.2% +/- 3.6%, 91.7% +/- 2.6% and 32.7% +/- 10. 1%, respectively, by 1 micromol/L PSC833. In reverse transcription-polymerase chain reaction (RT-PCR) analysis, the low inhibitory effect of P-gp modulators in aggressive NK cell tumors did not correlate to the expression level of MDR1 gene, multidrug resistance-associated protein gene, or human canalicular multispecific organic anion transporter gene. This phenomenon could be related to the presence of other transporters or to unknown cellular or membrane changes. Some patients with NK cell tumors have been reported to show a highly aggressive clinical course and to be refractory to chemotherapy, and this could be related to the expression of P-gp on NK cells. Our results suggest that, although the inhibitors for P-gp have been used in combination with chemotherapy in some hematologic tumors, these inhibitors may be less effective against aggressive NK cell tumors.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
[Primary splenic lymphoma with hypoplastic bone marrow].
A 44-year-old man was admitted because of persistent fever and pancytopenia. Because his bone marrow was hypoplastic and the karyotype of his marrow cells was normal, he was given a diagnosis of aplastic anemia, and treated with glucocorticoids and granulocyte colony-stimulating factor. Splenomegaly was later found and a splenectomy performed: pathological findings on resected tissue specimens disclosed non-Hodgkin's lymphoma, B-cell diffuse large. The patient was transferred to our hospital, where a bone marrow biopsy revealed lymphoma cells infiltrating his hypoplastic marrow. Complex chromosomal abnormalities were detected in marrow cells, but no lymphadenopathy was observed. A diagnosis of primary splenic lymphoma with infiltration of lymphoma cells into bone marrow was made, and chemotherapy was accordingly started. After multiple cycles of chemotherapy, the patient's marrow recovered to a normal state and his karyotype abnormalities disappeared. Six months later, pancytopenia reappeared and lymphoma cells were again detected in the patient's bone marrow. We reasoned that the hypoplastic state of his bone marrow was associated with the lymphoma, and that cytokines, including interferon-gamma, may have been responsible for this association.
Adult↗
[Clinical effects of combination therapy with cefozopran and tobramycin for severe infections in patients with hematologic diseases].
We studied clinical effect of a combination therapy with cefozopran (CZOP) and tobramycin (TOB) for infections in 80 patients with hematologic diseases in 15 institutes. Combined doses with CZOP 2 g and TOB 60-90 mg twice a day had been given intravenously. Of the 80 patients, 61 patients (42 with acute leukemia, 10 with malignant lymphoma, 3 with aplastic anemia, 2 with chronic myeloid leukemia, 2 with multiple myeloma, and 2 with myelodysplastic syndrome) were evaluable. Those consisted of 6 patients with septicemia, 49 with suspected septicemia, 3 with pneumonia, and 3 with other infections. Clinical efficacy by the treatment was excellent in 24, good in 17, fair in 9, and poor in 11 patients, and the overall efficacy rate including excellent and good was 67.2%. Microbiologically, 5 of the 6 patients with septicemia (1 coagulase negative Staphylococcus, 2 S. pneumoniae, 1 S. oralis, and 1 E. coli) were responded. The efficacy rate in patients with severe granulocytopenia showing 100/microliter or lesser neutrophil counts during the drug administration was 57.1% (12/21). Side effects and abnormal changes of clinical laboratory findings were observed in 5 patients, and 16 patients, respectively, but most of them were mild. The findings above suggested that the combination therapy with CZOP and TOB is useful as an empiric therapy for severe infections in patients with hematologic diseases.
Adult↗
Detection of chimaeric transcripts of the immunoglobulin heavy chain and BCL6 genes by reverse-transcriptase polymerase chain reaction in B-cell non-Hodgkin's lymphomas.
T(3;14)(q27;q32) is frequently detected in B-cell non-Hodgkin's lymphomas, especially the diffuse large cell type and the follicular type. The BCL6 gene encoding a putative transcriptional factor which resides on 3q27 rearranges to the immunoglobulin heavy chain (IgH) gene on 14q32 in this chromosomal translocation. The upstream regulatory region of the BCL6 gene is replaced by the IgH gene. Deregulation of the BCL6 gene may contribute to tumourigenesis of these diseases. The rearrangement between the IgH and BCL6 genes generates chimaeric transcripts in which the joining (J) region of the IgH gene fuses to exon 3 of the BCL6 gene. We established a method to detect these chimaeric transcripts by reverse transcriptase polymerase chain reaction (RT-PCR) using the consensus sequence of the J region and the sequence of exon 3 of the BCL6 gene as primers. Using the semi-nested RT-PCR method and a cell line carrying t(3;14)(q27;q32), we detected one lymphoma cell among 10,000 background cells. We detected these chimaeric transcripts in two out of 13 clinical samples by this method. This method can detect t(3;14)(q27;q32) easily, whereas this alteration is frequently overlooked by routine karyotype analysis. Since this technique is sensitive enough to detect a small number of lymphoma cells with this genetic abnormality, it could be employed to detect contaminating lymphoma cells in bone marrow and peripheral blood and minimal residual diseases.
Base Sequence↗
[Mantle cell lymphoma with marked lymphocytosis at the presentation and with discordant morphology between the lymph node and peripheral blood].
A 72-year-old man was referred to our hospital because of lymphadenopathy, splenomegaly, and leukocytosis. His WBC count was 54,300/microliter, with 89.6% atypical lymphocytes two to three times the diameter of red blood cells, cleaved nuclei, and one or two nucleoli. A lymph node specimen revealed a vaguely nodular pattern, and the diagnosis of mantle cell lymphoma (MCL) was made. The lymphoma cells appeared smaller and more mature than the leukemic cells. The phenotype of the peripheral blood and the lymph node cells was CD5+ CD10- CD19+ CD20+ and the same rearranged JH bands were detected, suggesting that their lymphocytes were of the same origin. In addition, the phenotype of the leukemic cells was CD23+ CD38+ CD43- CD44+ FMC-7+ micro+ chi+. Cytogenetic analysis revealed complex anomalies but not t(11; 14). Cyclin D1 protein was not detected. Because the lymphocyte morphology of the peripheral blood and lymph nodes was discordant, we speculated that variant large cells had proliferated mainly in the peripheral blood. The patient achieved a partial response after 6 courses of CHOP regimen, and was then placed on a COP regimen. He seemed to have MCL, but the following findings were unusual: marked lymphocytosis at initial presentation, discordant morphology, CD5+ CD10- CD23+ CD43- phenotype with neither t(11; 14) or cyclin D1 over-expression.
Aged↗
Molecular analysis of the secretory phospholipase A2 gene, a candidate of Mom1 gene, in neuroblastomas.
Mice with hereditary intestinal polyposis have mutations of the APC gene which causes formation of multiple polyps. At least one other gene influences the susceptibility for development of polyps in mice, and the locus was named Mom1. The causative gene for the Mom1 locus has recently been cloned and was found to be identical to the secretory type II phospholipase A2 (PLA2S-II) gene. Although the mechanism of contribution of PLA2S-II to formation of polyps is unclear, abnormalities of the PLA2S-II gene contribute to cellular transformation in mice. We speculated that this gene could contribute to tumorigenesis in human neoplasms. The human homologue of this gene maps to 1p35-36.1. Chromosomal deletions involving this region are frequently observed in neuroblastomas. We analyzed 19 neuroblastomas to detect point mutations of the PLA2S-II gene by PCR-single strand conformational polymorphism (SSCP). A polymorphism was detected at codon 32; no point mutations were found in the coding region of the gene. Moreover, in cases that were heterozygous at codon 32, three samples had hemizygous deletion of the gene. Taken together, PLA2S-II is frequently hemizygously deleted, but no point mutations are observed in neuroblastomas.
Abdominal Neoplasms↗
[Primary pulmonary non-Hodgkin's lymphoma in a patient with dermatomyositis].
A 47-year-old man with dermatomyositis and interstitial pneumonia had been treated with prednisolone since May, 1992, and with azathioprine since April, 1993. During the sixth month of this treatment, primary pulmonary non-Hodgkin's lymphoma (T-cell, diffuse, pleomorphic) developed. Chemotherapy (vincristine and adriamycin) was begun but there was no response. An invasive lesion of the brain was seen on a CT image. Despite cranial radiotherapy, the patient died of respiratory suppression due to progressive brain disease on December 14, 1993. Primary pulmonary non-Hodgkin's lymphoma develops only rarely in patients with dermatomyositis. In this case, oncogenesis may have been related to the use of immunosuppressants.
Azathioprine↗
Two Burkitt-type lymphoma/leukemia-derived cell lines presenting 3q27 translocations and immunoglobulin/BCL6 chimeric transcripts.
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Adult↗
p27/Kip1 mutation found in breast cancer.
The p27/Kip1 protein belongs to the recently identified family of proteins called cyclin-dependent kinase inhibitors. These proteins play an important role as negative regulators of cell cycle-dependent kinase activity during progression of the cell cycle. Since cyclin-dependent kinase inhibitors can inhibit cell proliferation, they may have a role as tumor suppressor genes. To determine whether p27 alterations may be involved in tumorigenesis, we examined its mutational status in 36 primary breast carcinomas and 9 breast cancer cell lines using PCR-single-strand conformational polymorphism, direct DNA sequencing, and Southern blot analysis. Southern blot analysis showed no homozygous deletions of the p27 gene in either the clinical samples or cell lines. Two point mutations were found in primary tumors. One represents a previously undescribed polymorphism at codon 142; another is a nonsense mutation at codon 104. The latter mutation was absent in the normal matched control sample, and, in addition, it was accompanied with the loss of heterozygosity (LOH) of a microsatellite marker in the vicinity of the p27 gene on chromosome 12p13. These data indicate that p27 mutations are a rare event in breast cancer, but may play an important role in the development of a minority of these cancers. Furthermore, LOH analysis of the 12p13 locus revealed that an additional four of six matched DNA samples had LOH at 12p13 but did not have an alteration of the p27 gene, suggesting that another tumor suppressor gene is located on the short arm of human chromosome 12 which may be frequently involved in the pathogenesis of breast cancers.
Base Sequence↗
Molecular analysis of the cyclin-dependent kinase inhibitor family: p16(CDKN2/MTS1/INK4A), p18(INK4C) and p27(Kip1) genes in neuroblastomas.
BACKGROUND: Chromosomal abnormalities involving band 1p32, especially deletions, are frequent in neuroblastomas, indicating that a tumor suppressor gene(s) is localized at this region. The p18 gene, one of the cyclin-dependent kinase inhibitor (CDKI) genes, maps to this chromosomal region. Complexes of cyclin and cyclin-dependent kinase (CDK) play important roles in the cell cycle. CDKIs inhibit the kinase activities of these complexes and block transitions of the cell cycle. Some of the CDKI genes may be tumor suppressor genes. For example, the CDKI genes p16 and p15 are frequently deleted in various malignancies and are thought to contribute to cellular transformations. METHODS: To elucidate the importance of CDKI genes, including the p18 as well as the p16 and p27 genes in tumorigenesis of neuroblastoma, 25 neuroblastomas were analyzed for deletions by Southern blot analysis and for point mutations by polymerase chain reaction-single strand conformational polymorphism. RESULTS: No deletions, rearrangements, nor mutations were detected in these genes, however, polymorphisms reported previously were detected. CONCLUSIONS: Abnormalities, including deletions and point mutations of the p16, p18, and p27 genes, were not observed in this series of neuroblastomas. Other mechanisms to inactivate these genes, such as transcriptional or translational defects, must be analyzed. CDKI genes rarely contributed to tumorigenesis in neuroblastomas.
Base Sequence↗
Point mutations of the Mxil gene are rare in prostate cancers.
Overexpression of the Myc genes promote cellular transformation. Max protein exerts a pivotal function with the Myc family, and Mxi1/Mad proteins play a positive and negative activity, respectively, on transcription. The function of Mxi1 suggests that it might be a tumor suppressor protein. The Mxi1 gene map to 10p24-25 and deletions of this locus are frequently observed in prostate cancers. The N-terminal helical motif, helix-loop-helix (HLH) and leucine zipper (ZIP) regions of the Mxi1 gene are functionally important. We analyzed most of the coding region of the Mxi1 gene, including these three important regions in 32 prostate cancers and three cell lines by PCR-single strand conformational polymorphism (SSCP). To enrich neoplastic cells, the microdissection was performed on clinical samples. We detected a silent mutation in the HLH region, but no point mutations reflecting the functional change of Mxi1 were found in human prostate cancers. Point mutations of the Mxi1 gene, if they occur, must be minor events in primary prostate cancers.
Basic Helix-Loop-Helix Proteins↗
Alterations of the p15, p16,and p18 genes in osteosarcoma.
Activation of cyclin-dependent kinases (CDKs) by interaction with cyclins regulates progression through cell cycle checkpoints. This process is counterbalanced by CDK inhibitors (CDKIs), which can inhibit progression through the cell cycle. Because CDKI expression acts to inhibit cellular proliferation, CDKIs may have a role as tumor suppressors. One class of CDKIs, characterized by the presence of ankyrin repeats, has at least four members (p15INK4B), p16INK4, p18, and p19). Two of these, p15INK4B, p16INK4, have been mapped to chromosome 9p21, a region of frequent loss in a wide variety of cancers. Alterations of p16INK4 have been detected in various tumors and cell lines. We analyzed p15INK4B, p16INK4, and p18 alterations in 52 osteosarcomas (including 11 explants), and 23 other various sarcomas. Single-stranded conformation polymorphism analysis [polymerase chain reaction (PCR-SSCP)] of the coding regions of these CDKI genes detected a missense mutation of p16INK4 exon 1 in one soft tissue sarcoma. Southern blotting detected complete deletion of p15INK4B and p16INK4 genes in osteosarcomas from 2 patients and a soft tissue sarcoma from another individual. Loss of heterozygosity (LOH) at chromosome 9p21 was observed with a microsatellite probe closely linked to the INK4 genes in the latter case. Deletions of both p15INK4B and p16INK4 genes were detected in five of eight osteosarcoma cell lines. By contrast, no alterations of p18 were detected in any sample. Together these data suggest that alterations of the p15INK4B and p16INK4 genes, but not p18, may occur in approximately 5% of sarcomas. However, deletions of the p15INK4B and P16INK4 genes are frequent in osteosarcoma cell lines and probably have a role in tumor cell growth in culture. Notably, all seven detectable deletions involved both p15INK4B and p16INK4 genes, suggesting that both contribute individual tumor suppressor activity.
Base Sequence↗
mdm-2 oncogene expression in non-Hodgkin's lymphomas.
The mdm-2 protein is a 90-kD protein that forms a complex with the p53 protein, enabling cells from some human neoplasms to overcome the growth-suppressing activity of p53. Most non-Hodgkin's lymphomas lack p53 mutations, and the mechanism of inactivation of tumor suppressive function remains obscure. To assess the role of mdm-2 in lymphomagenesis, 22 cases were evaluated for mdm-2 gene amplification or rearrangement in Southern blots. Localization of the mdm-2 protein was performed on cryostat sections and compared with expression of the p53 gene product. No case exhibited mdm-2 gene amplification or rearrangement, but overexpression of nuclear mdm-2 gene protein product was found in three of six diffuse large cell (B-cell immunoblastic) lymphomas (30-70% of the tumor cells stained). The mdm-2 protein was absent from low- and intermediate-grade lymphomas with the exception of a few cells (5% or less) in four cases. The mdm-2-positive cases stained negative for p53. Southern blot analysis showed that samples overexpressing mdm-2 did not have amplification or rearrangement of the gene. In summary, amplification of the mdm-2 gene does not appear to play a prominent role in the pathogenesis of non-Hodgkin's lymphomas, although overexpression of the protein gene product occurs, particularly in high-grade neoplasms.
Blotting, Southern↗