Identification of negative regulatory regions within the first exon and intron of the BCL6 gene.

Chromosomal translocations involving BCL6 gene are frequent in human B-cell lymphomas. Chromosomal breaks preferentially occur within a 3-kb region containing the first exon and intron. Recent reports have revealed that internal deletions or point mutations also are common in this region, suggesting that structural alteration of this region may be a crucial event in the development of lymphomas. In this study, we identified two regions in the BCL6 gene that negatively regulate BCL6 expression. One region, ES, is located within the first exon between nucleotides +472 and +543, and a second region, IS, is located between +783 and + 918 of the first intron. A consensus nucleotide sequence for the binding of the BCL6 protein itself was found within the ES region. An electrophoretic mobility shift assay and a co-transfection experiment using a BCL6 expression vector showed that transcription of the BCL6 gene was negatively regulated by the BCL6 gene product. The IS region which is included in the regions commonly deleted in B-cell lymphomas had a silencer activity. Structural alterations of these two regions may play roles in the deregulated expression of the BCL6 gene in B-cell lymphomas.

The proto-oncogene Bc16 inhibits apoptotic cell death in differentiation-induced mouse myogenic cells.

The Bc16 gene is located at chromosomal band 3q27, a breakpoint for translocation that frequently occurs in B cell lymphomas. Bc16 has been found to be preferentially expressed in germinal center B cells, and expression of this gene has been shown to be essential for germinal center formation in vivo. The physiological function of Bc16 and its role in lymphomagenesis, however, are not yet known. Since significant expression of Bc16 has been demonstrated in skeletal muscle, we have utilized a differentiation-inducible mouse myogenic cell line, C2C12, to elucidate the function of the Bc16 gene product. Expression of Bc16 mRNA was very low in growing myocytes, but was increased in differentiating myocytes cultured in serum-starved medium. Incubation of these cells with cytokines or chemicals that are known to block differentiation suppressed this increased Bc16 message abundance, indicating that Bc16 induction is related to the process of terminal differentiation in muscle cells. While a fraction of myocytes is known to undergo apoptosis after serum-starvation to induce differentiation, adenovirus-mediated overexpression of Bc16 enhanced the viability of the differentiating cells by preventing the apoptosis. High levels of Bc16 antisense mRNA expression induced substantial apoptosis during the differentiation of C2C12 cells, but this was effectively prevented by infection with adenovirus that expressed Bc16 sense mRNA. These results indicate that Bc16 acts to prevent apoptotic cell death in differentiating myocytes. The deregulation of expression of this antiapoptotic gene may also contribute to the development of B cell lymphomas.

1alpha,25-dihydroxyvitamin D(3) and its potent synthetic analogs downregulate tissue factor and upregulate thrombomodulin expression in monocytic cells, counteracting the effects of tumor necrosis factor and oxidized LDL.

BACKGROUND: We have recently found that a hormonally active form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], exerts anticoagulant effects by upregulating the expression of an anticoagulant glycoprotein, thrombomodulin (TM), and downregulating the expression of a critical coagulation factor, tissue factor (TF), in monocytic cells including human peripheral monocytes. In this study, we investigated the counteracting effects of 1,25(OH)(2)D(3) and its potent analogs on TF induction and TM downregulation by tumor necrosis factor and oxidized LDL in monocytic cells and the modulatory effects of potent analogs on TF and TM expression. METHODS AND RESULTS: Effects of 1,25(OH)(2)D(3) and its potent synthetic analogs (22R)-22-methyl-20-epi-1,25(OH)(2)D(3) (KY3) and 22-oxacalcitriol on TF and TM antigen levels, cell surface activities, and mRNA levels in monocytic cells were examined. 1, 25(OH)(2)D(3) and its potent analogs showed anticoagulant effects in monocytic cells by downregulating TF and upregulating TM expression, counteracting the effects of tumor necrosis factor and oxidized LDL. KY3 was most potent in its regulatory effect on TF and TM expression. CONCLUSIONS: Because KY3 has the highest affinity for vitamin D receptor, our findings suggest that TF and TM regulation by 1, 25(OH)(2)D(3) analogs is also mediated by vitamin D receptor. The 1, 25(OH)(2)D(3) analogs KY3 and 22-oxacalcitriol may have the potential to serve as an agent for preventing and treating atherosclerotic and other cytokine-mediated thrombotic diseases and as a tool for studying the molecular mechanisms of TF and TM regulation.

A steroidogenic cell line with differentiation potential from mouse granulosa cells, transfected with Ad4BP and SV40 large T antigen genes.

Several steroidogenic cell lines of granulosa cells (GC) have been used to elucidate differentiation mechanisms of GC during folliculogenesis. These cell lines, however, are of limited usefulness since they have lost some of their differentiation potential. The transcription factor adrenal-4 binding protein (Ad4BP), also known as steroidogenic factor-1 or NR5A1, is essential for the expression of all P-450 steroidogenic enzymes. By transfection with the Ad4BP gene together with SV40 DNA, we have generated several steroidogenic cell lines. One selective clone, named 4B2, retained its steroidogenic potential and was therefore analyzed in depth. This cell line responded to 8-Br-cAMP by displaying differentiation characteristics similar to those occurring in the differentiation process of primary cultured GC, including enhanced progesterone secretion, a cell shape change from a fibroblastic to epithelioid conformation, elongated mitochondria, increased gap junction formation and inhibition of cell proliferation. Prostaglandin E2 (PGE2), an intraovarian regulator of GC, stimulated cAMP production, and this eicosanoid, like 8-Br-cAMP, induced differentiation properties with the exception of cell conformation in 4B2 cells. These results suggest that expression of Ad4BP may provide the basis for a repertoire of cAMP-sensitive differentiation properties, including morphological alterations and growth inhibition. Thus, the 4B2 cell line may serve as a tool for elucidation of differentiation mechanisms that are under the control of Ad4BP.

Expression of inducible nitric oxide synthase (NOS) in bone marrow cells of myelodysplastic syndromes.

Nitric oxide (NO) is a biological mediator which is synthesized from L-arginine by a family of nitric oxide synthases (NOS). We have studied the expression of the inducible NOS (iNOS) by bone marrow cells from the patients with myelodysplastic syndromes (MDS) at the mRNA level by RT-PCR assay and at the protein level by immunohistochemical staining using a specific anti-iNOS monoclonal antibody. The iNOS message was present in 92% of bone marrow tissues from MDS patients (11 out of 12) by an examination using RT-PCR. Basically, iNOS message was negative or very weak in control (1/9) and AML (0/7) cases. This was supported by immunohistochemical findings that the iNOS was positive in most of the bone marrow samples from MDS patients (9 out of 12), while bone marrow cells of control (O out of 12) and AML (O out of 5) cases were basically negative. Double immunostaining for CD68 antigen, which is a marker for macrophage lineage cells, and iNOS was performed on MDS bone marrow sections. iNOS was dominantly localized to bone marrow macrophages, although a part of myeloid cells were also positively stained with anti-iNOS antibody in a part of cases. These results indicated that there is some in vivo induction of iNOS expression for local NO production that might be involved in the dysregulation of hematopoiesis in bone marrow of MDS.

Localization of Fas and Fas ligand in bone marrow cells demonstrating myelodysplasia.

Frequent apoptosis in the bone marrow of patients with myelodysplastic syndromes (MDS) was demonstrated on frozen sections using the terminal deoxytransferase (TdT)-mediated dUTP nick end labeling (TUNEL) method. The overall mean percentage of TUNEL-positive cells was about 17% in the bone marrow of MDS, while bone marrow from control cases exhibited a mean of 3.4% (P < 0.001). To elucidate the mechanism of apoptosis in bone marrow cells of MDS, the expression of Fas antigen and Fas ligand (FasL) was examined by RT-PCR and immunohistochemistry. All MDS cases showed expression of Fas mRNA (12/12) and most exhibited an expression of FasL mRNA (10/12) by RT-PCR. Basically, control cases did not show positive signals for Fas and FasL mRNA, however, a very weak band was detected in three cases (3/10) for Fas and in one case (1/10) for FasL mRNA by RT-PCR. Immunohistochemical examination revealed positive staining for Fas (11/12) and FasL (12/12) in the bone marrow of MDS, while all the bone marrow samples from control cases were negative for anti-Fas (0/15) and for anti-FasL (0/15) antibody. Double staining clarified that TUNEL-positive apoptotic cells expressed Fas antigen on the cell surface, although not all Fas-positive cells were TUNEL positive. The Fas-positive cells of MDS bone marrow included hematopoietic cells expressing CD34 antigen, neutrophil elastase, a marker for myeloid series of cells, or glycophorin A, a marker for erythroid cells. However, CD68-positive cells which were macrophage lineage cells, did not express Fas antigen strongly. In contrast, positive staining for FasL was detected in hematopoietic cells and CD68-positive cells in the bone marrow of MDS. These results suggest that the Fas-FasL system plays an important role in inducing apoptosis in the bone marrow of MDS and works in an autocrine (hematopoietic cell-hematopoietic cell interaction) and/or paracrine (hematopoietic cell-stromal cell interaction) manner.

Overexpression of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma by bone marrow cells from patients with myelodysplastic syndromes.

To clarify whether regulatory cytokines inhibit hematopoiesis in patients with myelodysplastic syndromes (MDS), malignancies characterized by the formation of cytopenias despite the presence of cellular bone marrow, expression of TNF-alpha and IFN-gamma by bone marrow cells was investigated using specific reverse transcriptase-polymerase chain reaction assays. An enhanced expression of the mRNA for TNF-alpha was observed in most of the samples from MDS patients (11/14, 79%), whereas no enhancement was observed in bone marrow samples from AML (0/6), CML (0/2) or control cases (0/8). The expression of IFN-gamma was also enhanced in some of MDS cases (5/12, 42%) while AML (0/5), CML (0/2) and control cases (0/6) showed very low levels of IFN-gamma mRNA expression. Immunohistochemical examination confirmed the scattered presence of TNF-alpha or IFN-gamma producing cells in the bone marrow of MDS patients. The majority of these cells were CD68-positive macrophage lineage cells. These results suggested that disruption of hematopoiesis in MDS might be caused by enhanced production of inhibitory regulatory cytokines especially TNF-alpha and occasionally IFN-gamma by bone marrow macrophages.

Expression of the proliferating cell nuclear antigen in bone marrow cells from patients with myelodysplastic syndromes and aplastic anemia.

To determine the proliferative activity of the hematopoietic cells under nonneoplastic and/or neoplastic conditions, the expression of a cell cycle-related antigen, the proliferating cell nuclear antigen (PCNA), was examined in the bone marrow trephines of 79 individuals, 12 of whom had no hematologic disorder, 32 of whom had a diagnosis of myelodysplastic syndromes (MDSs), 20 of whom suffered from aplastic anemia, and 15 of whom had a diagnosis of acute myeloid leukemia. Most of the patients with MDS had more than 15% PCNA-positive cells (23.5% +/- 1.5%) while patients with no hematologic disorder showed fewer than 15% PCNA-positive cells (11.7% +/- 0.7%). The overall ratio of the PCNA-positive cell fraction in the bone marrow was considered of prognostic value for predicting transition into overt leukemia from MDS. Aplastic anemia cases usually exhibited hypocellular bone marrow and an infrequent labeling with the anti-PCNA antibody (3.3% +/- 0.5%). However, a few aplastic anemia cases showed hypercellular bone marrow and a significantly high PCNA-positive cell ratio (32.0% +/- 4.4%). In the bone marrow of acute myeloid leukemia patients more than 20% of total nucleated cells were positive for PCNA (30.0% +/- 2.2%). The results suggest that the expression of PCNA is associated with the regulation of bone marrow cell proliferation and the bone marrow cellularity, and that these findings would serve as an early indicator of evolution of overt leukemia in MDS and also would be useful in distinguishing MDS cases from aplastic anemia cases when the bone marrow is hypocellular or normocellular.

Immunolocalization of platelet-derived growth factor, transforming growth factor-beta, and fibronectin in acute megakaryoblastic leukemia manifesting tumor formation.

Acute megakaryoblastic leukemia (AMKL) manifesting myelofibrosis and tumor formation in the liver with marked increase of reticulin is described. The megakaryoblastic nature of the leukemic cells of the bone marrow and the hepatic tumor nodule was established by positive immunohistochemical stains for CD41a and CD41b on frozen tissue sections. Immunolocalization of the platelet-derived growth factor (PDGF) protein and transforming growth factor (TGF)-beta protein also was demonstrated in the leukemic cells of the bone marrow and the hepatic tumor. Further, the deposition of fibronectin that has been known as the ligand of CD41a molecule and collagen types I and IV were recognized in the extracellular matrix of the bone marrow and the hepatic tumor. These results suggest that specific expression of growth factor proteins by the leukemic cells may selectively regulate the fibrosis of the bone marrow as well as the tumor formation of AMKL. The expression of adhesion molecules and growth factor proteins by the leukemic cells and the deposition of extracellular matrix are discussed in relation to the myelofibrosis as well as the tumor-forming nature of AMKL.

A variant Burkitt-type translocation (2p-;8q+) in a patient with diffuse large cell lymphoma.

A case of diffuse large cell lymphoma with t(2p-;8q+) is reported. Immunologically the lymphoma cells were shown to be of B-cell origin and positive for surface gamma and kappa chains, B4, CALLA, and Ia1 markers. Karyotypically three major clones were detected: 47,XX, + 12,t(2;8)(p11-13;q24) (52%); 47,XX, + 12 (26%); and 46,XX,t(2;8)(p11-13;q24) (15%). A t(2p-;8q +) has been exclusively reported in cases of Burkitt's lymphoma or Burkitt-type acute lymphocytic leukemia. The present case is the first one with t(2p-;8q +) observed in non-Burkitt-type lymphoid malignancy of the B-cell lineage. The t(2p-;8q +) may play a primary role in the early stage of transformation of B cells, and trisomy 12 may provide them secondarily with an advantage for tumor progression. The phenotypic pictures provided by 8q24 rearrangements seem to be heterogeneous, as previously suggested.

Fibromuscular dysplasia in the extremities.

Four patients had fibromuscular dysplasia of the extremities involving the brachial, deep femoral, popliteal, anterior and posterior tibial and peroneal arteries. One of them demonstrated a string-of-beads appearance in the superficial veins of the arm and this was pathologically confirmed as fibromuscular dysplasia. We believe this is the first report of this disease affecting the peripheral arteries and the superficial veins of the extremities. The sympathetic system seems to be related to the development of fibromuscular dysplasia.

Determination of a common clonal origin of gastric and pulmonary mucosa-associated lymphoid tissue lymphomas presenting five years apart.

Mucosa-associated lymphoid tissue (MALT) lymphoma is often mis-diagnosed as a benign tumor. Dissemination to other sites occurs in MALT lymphoma. We report a 60-year-old man with gastric and pulmonary tumors of MALT lymphoma which occurred 5 years apart. Initially, the gastric tumor had been diagnosed as reactive lymphoreticular hyperplasia. To determine whether the two tumors arose from the same malignant clone, we amplified and sequenced the complementarity-determining region 3 of the immunoglobulin heavy chain gene using the polymerase chain reaction (PCR). The sequences were identical except for 11-nucleotide difference, suggesting identical clonality.

Primary chondroid chordoma arising from the base of the temporal bone. A 10-year post-operative follow-up.

A 36-year-old male with a primary chondroid chordoma is presented. This tumour arose from the base of the temporal bone and extended to the mastoid cavity. It involved the facial nerve and was adherent to the internal jugular vein and internal carotid artery. The tumour was excised and the patient has been carefully followed up for 10 years. He has shown no evidence of local recurrence, intracranial extension of the residual tumour and distant metastasis.

[HCG-positive cells in seminoma of the testis].

In a retrospective study of 38 patients with pure seminoma, serum and urine levels of human chorionic gonadotropin (HCG) were measured and the cellular origin of HCG-like substance was searched using the technique of indirect immunoperoxidase on step sections of the tumors. Eight of the patients had elevated HCG in serum or urine, and 5 had HCG-positive cells in the sections of tumor specimens. With this technique, two types of HCG-positive cells were identified, syncytiotrophoblastic giant cells (STGC) and mononuclear cells otherwise indistinguishable from seminoma cells. Patients in the present series responded well to conventional radiation therapy or cytotoxic chemotherapy and had a favorable outcome regardless of the presence of STGC or slightly elevated HCG levels.

[Plasma cell leukemia with amyloid deposition and osteogenetic change at the site of an extramedullary plasmacytoma].

A 49-year-old man was admitted with swelling in the left lower extremity, and a mass in the left lower abdomen. Laboratory findings showed an increased WBC of 15,000/microliter with 41% plasma cells, and immunoglobulin (Ig) A of 2,557mg/dl with a monoclonal component. A roentgenogram and computed tomograph of the abdomen revealed that a 5 x 10 cm mass with calcification located in the iliopsoas muscle. Plasma cell leukemia with extramedullary plasmacytoma was diagnosed, and the patient was treated with high-dose dexamethasone (40 mg/day for 4 days), resulting in a good response with the disappearance of plasma cells in peripheral blood and a marked decrease in serum Ig A. However, the patient's condition deteriorated in spite of various treatments, and he died of heart failure 5 months after admission. With informed consent from relatives, a necropsy was performed and infiltration of plasma cells in the mass in the iliopsoas muscle was noted. We reported this case because plasma cell leukemia with amyloid deposition and osteogenesis at the site of extramedullary plasmacytoma is very rare.