Adrenomedullin inhibits angiotensin II-induced expression of tissue factor and plasminogen activator inhibitor-1 in cultured rat aortic endothelial cells.

Adrenomedullin (AM) is a potent vasodilating peptide having a variety of pharmacological properties mainly in respect to vascular pathophysiology. We have previously demonstrated that angiotensin II (Ang II) or natriuretic peptides have influence on the expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) in vascular endothelial cells. The aim of this study was to elucidate the effects of AM on TF and PAI-1 mRNA and protein expression in endothelial cells. As a result, AM inhibited Ang II-induced TF and PAI-1 mRNA expression in a dose- and time-dependent manner. Because the expression of TF and PAI-1 mRNA induced by Ang II was attenuated by the increase of intracellular concentrations of cAMP by forskolin and 8-bromo-cAMP and because AM increased the intracellular level of cAMP in rat aortic endothelial cells, it was indicated that the inhibitory effect of AM on the expressions of TF and PAI-1 was mainly mediated by the cAMP-dependent signal transduction. Furthermore, the inhibitory effect of AM on TF and PAI-1 expression was partly attenuated by an NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester. In conclusion, AM is shown to contribute to the regulation of blood coagulation and fibrinolysis by vascular endothelial cells mainly via the cAMP pathway.

[Transverse lesion of the spinal cord due to mucormycosis in an AML patient].

A 54 year old man complained exertional dyspnea and palpitation since November 1989. As he was diagnosed with marked anemia, leukocytosis and thrombocytopenia by his work place doctor, he was admitted to our hospital. Acute myelogenous leukemia was diagnosed based on laboratory findings. BHAC-DMP, BHAC-MEP and A triple V therapies were only partially effective. Fine nodular shadows in all lung fields and a semicircular mass in the right lower lobe next to the thoracic vertebra were evident on the chest X-P since the end of March 1990. He was treated with antibiotics and amphotericin B but the abnormal lung shadows did not disappear. He had sudden onset of paraplegia and loss of all sensation below Th6 on May 1. Aparavertebral mass in the right lower lobe was detected by CT and MRI, for which radiotherapy was performed but without improvement. He died of respiratory failure on May 12. Autopsy showed that the semicircular paravertebral mass continued to the main pulmonary vein and epidural area of the thoracic cord (Th6-8). Microscopically, mucormycosis was found. Necrosis due to mucor embolism was found in the thoracic cord. It is usually difficult to diagnose mucormycosis in immunocompromised patients while they are alive. It is important to suspect mucormycosis if any infarction symptoms or infections resistant to antibiotics develop in immunocompromised patients.

Megakaryocytic differentiation of a leukemic cell line, MC3, by phorbol ester: induction of glycoprotein IIb/IIIa and effects on expression of IL-6, IL-6 receptor, mpl and GATA genes.

We investigated megakaryocytic differentiation in a newly-established Ph1-positive leukemic cell line, MC3, which showed tri-lineage immunophenotypes (myeloid antigens2+, CD19(1+) and CD41a1+) and was positive for CD34 and CD38. TPA induced MC3 cells to differentiate to an early stage of megakaryocyte lineage exhibiting an increase in the expression of platelet glycoproteins (GP) IIb/IIIa (CD41a), and an increase in cell size and nuclear ploidy. TPA treatment also enhanced the expression of GPIIb mRNA, and induced the expression of interleukin-6 (IL-6) and its receptor mRNAs, while it did not induce transcripts of the genes IL-11 and mpl ligand, and further decreased the transcript of the mpl gene. Consistent with these findings, MC3 cells treated with TPA showed an increased expression of GATA-1, but not GATA-3 transcripts, whereas those without TPA treatment expressed only the GATA-2 transcript. These results provide an insight into the study for the regulatory mechanism of megakaryocytopoiesis and leukemic cell differentiation.

Analysis of the p53 gene mutations in acute myelogenous leukemia: the p53 gene mutations associated with a deletion of chromosome 17.

In order to determine the relevance of the p53 tumor suppressor gene mutations in acute myelogenous leukemia (AML), we analyzed the p53 gene in genomic DNA of 18 unselected cases of AML by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing. We detected three cases (16.7%) with the p53 gene mutations showing only the mutant alleles; the high incidence in cases with loss of a whole chromosome 17 (two of three) contrasted with the low incidence in cases without abnormalities of chromosome 17 (one of 15). These cases containing the mutations of the p53 gene showed a poor prognosis. Although we analyzed a rather small series of patients, these findings suggest that the p53 gene mutations might be involved in the progression and prognosis of at least some cases of AML.

Establishment and characterization of a new Ph1-positive chronic myeloid leukemia cell line MC3 with trilineage phenotype and an altered p53 gene.

A new Ph1-positive leukemic cell line (MC3) expressing the P210bcr/abl oncoprotein was established from a patient with CML in blast crisis. The MC3 cells showed the trilineage phenotype of myeloid, lymphoid (CD19) and megakaryocytoid lineages, and had a proliferative response to rhIL-1 and rhIL-3 in the serum-free culture. These results and the expression of CD34 indicated that the MC3 cells have characteristics of hematopoietic progenitor cells. Recently, it has been documented that alterations of the p53 gene in leukemic cells are frequently detected during the blast crisis of CML. The MC3 cells contained the altered p53 gene. In addition, the original leukemic cells showed the point-mutational activation of the N-ras gene and an additional chromosomal abnormality inv(3q), but the MC3 cells contained no such abnormalities, indicating that not all of the original leukemic cells had these abnormalities. Thus, the MC3 cell line may provide several insights into investigations of the blast crisis in CML as well as hematopoietic progenitor cells.

bcr-abl hybrid messenger RNA in a patient with Philadelphia-negative essential thrombocythemia.

We describe a case of Philadelphia-negative essential thrombocythemia in whom bcr-abl hybrid messenger RNA was detected. The patient suffered from frequent splenic infarctions and myelofibrosis. Interestingly, a transformation to acute leukemia which was commonly seen in patients with bcr-abl-positive chronic myelogenous leukemia did not occur until he died from heart failure due to severe anemia 8 years after the diagnosis. The heterogeneity of bcr-abl-positive thrombocythemia is emphasized.

In vivo antitumor activity of herbimycin A, a tyrosine kinase inhibitor, targeted against BCR/ABL oncoprotein in mice bearing BCR/ABL-transfected cells.

Herbimycin A, a benzoquinoid ansamycin antibiotic, has been shown to reverse the oncogenic phenotype of p60v-src transformed cells because of the inhibition of src protein tyrosine kinase. We previously demonstrated that herbimycin A displayed antitumor activity on the in vitro growth of Philadelphia chromosome-positive leukemia cells and BCR/ABL-transfected murine hematopoietic FDC-P2 cells through the inhibition of BCR/ABL protein tyrosine kinase. In this study, the transformed FDC-P2 cells were demonstrated to be tumorigenic in syngeneic DBA/2 mice. The intraperitoneal (i.p.) injection of the transformed tumor cells into DBA/2 mice induced infiltrations of abdominal organs, and then all of the mice died within time periods proportional to the cell numbers of inoculation. In mice that received an i.p. inoculation with greater than 1 x 10(5) cells, in vivo administration of herbimycin A by i.p. injection inhibited tumor formation and significantly prolonged survival time, and further, in mice inoculated with 1 x 10(4) cells, herbimycin A completely suppressed the in vivo growth of transformant FDC-P2 cells and brought about a complete remission. The present study revealed the in vivo efficacy of herbimycin A in mice bearing BCR/ABL-transfected cells.

Chronic myeloid leukemia presenting ALL-type BCR/ABL transcript.

We investigated the breakpoints of the bcr gene in 46 Ph1-positive CML cases by Southern blot analysis of bcr rearrangement, and in 17 CML cases by a combination of Southern blot analysis and RT-PCR. By Southern blot, the breakpoint was not identified on M-bcr in three CML cases, of which one case showed the P210-type bcr/abl transcript and two cases showed the ALL-type (P190-type) bcr/abl transcript with or without P210 transcript. Later two cases showed unique hematological profiles such as thrombocytosis, mild myelofibrosis, and relative resistance to alkylating agents. Therefore, the present study suggests that expression of the P190-type transcript may affect clinical and hematological findings in CML.

Establishment and characterization of a new Ph1-positive ALL cell line (ALL/MIK) presenting bcr gene rearrangement on bcr-2 and ALL-type bcr/abl transcript: suggestion of in vitro differentiation to monocytoid lineage.

A new Ph1-positive acute lymphoblastic leukemia cell line, designated as ALL/MIK, has been developed from a patient with Ph1-positive acute leukemia. The ALL/MIK cells showed an immunophenotype of common ALL with rearranged JH and Jk genes. The ALL/MIK cells showed no M-bcr rearrangement using Southern blot analysis with either 3' or 5' M-bcr probes, but had the bcr gene rearrangement on bcr-2 within the first intron of the bcr gene. Consistent with this result, the reverse transcriptase-dependent polymerase chain reaction (RT-PCR) assay revealed that the ALL/MIK cells contained the transcript derived fusion of the first exon of bcr gene and the second exon of abl gene. Although the ALL/MIK cells were defined as early pre-B cells by immunophenotypical and genotypical analyses, they were capable of differentiating into monocytoid lineage by when cultured with TPA. Furthermore, another Ph1-positive ALL cell line, (TOM-1), was investigated for its ability to differentiate to monocytoid lineage. TOM-1 was also induced to monocytoid lineage by TPA. Thus, the present study suggested that the leukemic transformation in some Ph1-positive ALL may occur at the level of multipotential hematopoietic cells capable of differentiating towards lymphoid and myelo-monocytoid lineage.

BCR/ABL oncoprotein-targeted antitumor activity of antisense oligodeoxynucleotides complementary to bcr/abl mRNA and herbimycin A, an antagonist of protein tyrosine kinase: inhibitory effects on in vitro growth of Ph1-positive leukemia cells and BCR/ABL oncoprotein-associated transformed cells.

We investigated whether antisense oligodeoxynucleotides complementary to bcr/abl mRNA or protein kinase antagonists display antitumor activity on Ph1-positive leukemia cell lines. bcr/abl antisense oligomers showed inhibitory effects on the in vitro growth of Ph1-positive leukemia cell lines in liquid culture, and further displayed an inhibitory effect on transformed murine hematopoietic cells using transfection with a retroviral vector expressing P210bcr/abl oncoprotein. However, in vitro treatment with a bcr/abl antisense oligomer did not completely abolish the expression of bcr/abl mRNA and did not display the desired "killing effect" on Ph1-positive leukemia cells. On the other hand, investigation of the effect on Ph1-positive leukemia cells by various types of protein kinase antagonists revealed that herbimycin A, a protein tyrosine kinase antagonist, displays preferential and remarkable suppression of the growth of Ph1-positive leukemia cells and P210bcr/abl associated transformed cells by virtue of suppressing bcr/abl protein tyrosine kinase activity. These results may provide important future insights in developing a new category of antitumor therapy by targeting oncogene products.

[The cellular and molecular-biological studies on Philadelphia chromosome-positive acute lymphocytic leukemia].

Philadelphia chromosome (Ph1) is detected in more than 95% of chronic myelogenous leukemia (CML) and approximately 20% of adult acute lymphocytic leukemia (ALL). In order to discriminate Ph1-positive ALL from Ph1-positive CML as a clinical entity, I studied on biological and genetic characteristics of Ph1-positive ALL cells. Two cases out of 11 Ph1-positive ALL showed hybrid leukemia phenotypes; in one hybrid case simultaneous proliferation of lymphoid and myeloid blast cells was observed and contained rearranged alleles of heavy chain genes, thus indicating that both blast cells might originate from a common precursor. Two Ph1-positive ALL cell lines (TOM-1 and ALL-MIK) were established from two patients and were investigated for their differentiation potential in vitro. Both cell lines showed the potency to differentiate into monocytic lineage cells, thus suggesting that these Ph1-positive ALL cells might reside at the stage of multipotent progenitor cell along hematopoietic cell differentiation. As to Ph1-chromosome, 4 out of 9 Ph1-positive ALL cases showed rearrangements within the classical sequence (M-bcr), similar to those in CML cases. Two out of 5 cases without rearrangement of M-bcr showed breakpoints in the first intron of the BCR gene. In the rest of 3 cases, BCR-ABL rearrangement was not detected by Southern analysis. However, a leukemic cell line established from one of these patients (TOM-1) were contained P190bcr-abl mRNA as analyzed through RT-PCR. Thus, breakpoints of the BCR gene in Ph1-positive ALL cases were heterogenous, in contrast to those of CML. Then, I investigated whether or not the activation of transforming genes other than BCR-ABL might be involved in pathogenesis of Ph1-positive ALL. Three out of 15 Ph1-negative ALL cases showed the mutations of RAS gene by the PCR. However, no activated oncogene was detected in Ph1-positive ALL cases by both DNA transfection assay and PCR.

[Rearrangement and expression of bcr-abl genes in CML and ALL].

We have carried out the molecular and cell-biological analysis on Ph1-positive leukemias in this study. Five out of nine Ph1-positive ALL cases showed molecular rearrangement within the classical bcr sequence (or M-bcr), similar as those in 47 CML cases. We examined 4 cases of Ph1-positive ALL presenting no rearrangement of M-bcr and found that, in 2 of 4 cases, one showed the breakpoint in a 5 kb segment of the bcr gene first intron (bcr-2) and the other in bcr-1, 16 kb upstream of bcr-2. Ph1-positive ALL frequently showed biphenotypical or biclonal phenotypes of myeloid and lymphoid lineages. Furthermore, we demonstrated the ability of two Ph1-positive ALL cell lines to differentiate into monocytic lineage in vitro, thus suggesting the possibility that these Ph1-positive ALL cells might reside on the stage of multipotent stem cell along the hematopoietic cell differentiation. Two out of 31 CML cases showed the mutations of the ras genes by the polymerase chain reaction; one case in the crisis phase and the other in the chronic phase. However, no mutations of the fms genes was detected. Two cases in the crisis phase of 24 CML patients (11 cases in the chronic phase and 13 cases in the crisis phase) contained rearrangements of the p53 gene by Southern analysis. Furthermore, the transcriptional alteration was found in 2 CML-BC and 2 CML-BC derived cell lines' samples, suggesting a important role of the p53 gene in the transformation of CML into the crisis phase.

[Therapeutic effect of ranimustine(MCNU) on myeloproliferative disorder and chronic myelomonocytic leukemia].

Seventeen patients with myeloproliferative disorders and one patient with chronic myelomonocytic leukemia (CMMoL) were treated with ranimustine++ (MCNU), and the efficacy was evaluated. MCNU was given intravenously by drip infusion at an usual dose of 100 approximately 150 mg with intervals arranged according to the counts of peripheral blood cells. A complete remission was achieved in all 10 patients with chronic myelogenous leukemia (CML) in chronic phase. In three of patients with polycythemia vera (PV) the excellent effects were obtained, and the other 2 cases showed moderate effect. An excellent effect was obtained in both 2 patients with essential thrombocythemia (ET). A patient with CMMoL revealed partial remission. The overall efficacy rate was 100%. The cases with CML needed more long term and much more dose of the drug in order to get remission compared with PV and ET. After remission in both PV and ET, well controlled states were maintained for a relatively long period with no additional administration. In CMMoL, MCNU combined with 6-mercaptopurine also showed remarkable anti-tumor effects. It suggests that MCNU may be one of the useful drugs for the treatment of CMMoL. The side effects observed with MCNU were a slight degree of nausea and vomiting (28%), however they showed no trouble on carrying out the therapy.

[A case of adrenal myelolipoma with colonic cancer and gallstones].

A 47-year-old female, referred to Kushiro Rohsai Hospital because of upper abdominal pain on January 10, 1986, was found to have a gallbladder stone on X-ray examination of her abdomen. On admission, the occult blood found in her stool was strongly positive and a barium enema and a colonoscopy showed an elevated lesion in the caecum. Subsequently, a diagnosis of a colonic cancer was made after a biopsy. Sonography and CT scans revealed a gallbladder stone and a right suprarenal mass 4 cm in diameter. At surgery, this gallbladder stone was found, as was a Borrman II type tumor in the caecum. The surface of an adrenal mass was hard and smooth and a cut of this surface showed a yellowish gray color. Microscopic examination revealed adrenal cortical tissue beneath the capsule with a mixture of hematopoietic and adipose tissue. These findings were compatible with myelolipoma. Postoperatively, a bone marrow aspiration was performed, but no definite abnormality was detected.

[Bone marrow necrosis in thymic T cell lymphoma].

A 29 year-old male patient was admitted because of exertional dyspnea, due to the pleural effusion on chest X-P on June 18 of 1988. On admission, enlargement of left supraclavicular and mediastinal lymph nodes were found. Cytological studies of pleural exudate cells showed the proliferation of atypical lymphoid cells with a few eosinophiles. Surface markers of the tumor cells were just positive for both CD5 and CD38, thus indicated that they resided as early thymocyte along T cell differentiation. Moreover, according to Southern blotting analysis, these DNA's showed rearrangement of TCR-beta genes. Based on these results, he was diagnosed to be suffered from thymic T cell lymphoma. Bone marrow biopsy on left posterior iliac bone showed tumor necrosis owing to extensive tumor which were diffusely infiltrated among collagen fibers with reticulin fibrosis. He was treated with aggressive combination chemotherapy but achieved no remission and died of cerebral vascular bleeding on Jan. 7, 1989.

[Tumor lysis syndrome at the induction therapy of the first remission in two cases of T-ALL].

The anti-tumor therapy followed by tumor lysis syndrome may cause the metabolic disorders including hyperkalemia, hyperphosphatemia and hyperuricemia. It should be known that it occurs frequently in lymphoproliferative diseases, especially in Burkitt's lymphoma. Two cases of T-ALL accompanied by this syndrome, from which the patients were recovered, at the induction therapy of the first complete remission are reported here. Case 1. A 28-year-old man received VP therapy under the diagnosis of T-ALL with massive hepatosplenomegaly and bilateral enlarged kidneys. During the therapy, metabolic disorders with both renal failure and ventricular tachycardia happened. They were resolved by certain series of treatments. The patient was brought to a complete remission with normal size of liver, spleen and kidneys. Case 2. A boy aged 15 having received the intrasubarachnoidal infusion of MTX and 1-Ad-VP therapy under the diagnosis of T-ALL accompanied by this syndrome which was improved by an appropriate treatment, and the patient was lead to the remission. The risk factors of this syndrome, such as 1-high drug sensitivity of the tumor; 2-renal dysfunction; 3-rapid cytokinetics of the tumor cell; 4-bigger size of the tumor, as well as the preventive treatment of this syndrome are reviewed.

[A case of adrenal metastasis of hepatocellular carcinoma effectively treated with local ethanol injection].

We experienced a case of hepatocellular carcinoma with adrenal metastasis that was effectively treated with ultrasonographically guided percutaneous ethanol injection. Effectiveness was proved by various imaging procedures and decreased serum alpha-fetoprotein. No complications during and after the treatment were deserved.