Establishment of a new human acute monocytic leukemia cell line TZ-1 with t(1;11)(p32;q23) and fusion gene MLL-EPS15.

Human leukemia cell lines are of great value in investigating basic and applied aspects of cell biology and clinical medicine. There have been 37 leukemia cell lines carrying 11q23 translocation and MLL rearrangements; however, cell lines harboring with t(1;11)(p32;q23) have not been established. We report here for the first time a new acute monocytic leukemia (AMoL) cell line with t(1;11)(p32;q23), designated TZ-1, and herein describe its biological characteristics. Mononuclear cells isolated from the ascites from a patient with AMoL (French-American-British classification; acute myeloid leukemia M5a) were isolated and passaged by liquid culture medium for a year. TZ-1 cells revealed typical monocytic features in morphology and had a t(1;11)(p32;q23) translocation. The immunoprofiling as determined by flow cytometry showed that TZ-1 cells are positive for myeloid and monocytic markers with lymphoid-associated markers. Fluorescence in situ hybridization and reverse transcription-polymerase chain reaction analyses revealed MLL-EPS15 fusion transcript and protein. Taken together, these results suggest that TZ-1 is a new monocytic leukemia cell line with t(1;11) translocation and fusion gene MLL-EPS15. The established cell line, TZ-1, could provide a valuable model in the analysis of the pathogenesis of MLL-EPS15-positive leukemia and in the development of new agents for this type of leukemia.

Establishment of a retinoic acid-resistant human acute promyelocytic leukaemia (APL) model in human granulocyte-macrophage colony-stimulating factor (hGM-CSF) transgenic severe combined immunodeficiency (SCID) mice.

To understand the mechanisms and identify novel approaches to overcoming retinoic acid (RA) resistance in acute promyelocytic leukaemia (APL), we established the first human RA-resistant APL model in severe combined immunodeficiency (SCID) mice. UF-1 cells, an RA-resistant APL cell line established in our laboratory, were transplanted into human granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing SCID (hGMTg SCID) mice and inoculated cells formed subcutaneous tumours in all hGMTg SCID mice, but not in the non-transgenic control SCID mice. Single-cell suspensions (UF-1/GMTg SCID cells) were similar in morphological, immunological, cytogenetic and molecular genetic features to parental UF-1 cells. All-trans RA did not change the morphological features of cells or their expression of CD11b. RA did not alter the growth curve of cells as determined by MTT assay, suggesting that UF-1/GMTg SCID cells are resistant to RA. These results demonstrate that this is the first RA-resistant APL animal model that may be useful for investigating the biology of this myeloid leukaemia in vivo, as well as for evaluating novel therapeutic approaches including patients with RA-resistant APL.

Restoration of retinoid sensitivity by MDR1 ribozymes in retinoic acid-resistant myeloid leukemic cells.

Complete remission is achieved in a high proportion of patients with acute promyelocytic leukemia (APL) after all-trans retinoic acid (RA) treatment, but most patients relapse and develop RA-resistant APL. We have previously reported that both RA-resistant HL-60 (HL-60R) and APL cells express P-glycoprotein and MDR1 transcripts; and these cells differentiate to mature granulocytes after culture with RA and P-glycoprotein antagonist. Ribozymes have been shown to be able to intercept a target RNA by catalytic activity. To address the role of MDR1 in overcoming RA-resistance in APL cells, we investigated the biologic effects of ribozymes against the MDR1 transcript in HL-60R cells. These ribozymes efficiently cleaved MDR1 mRNA at a specific site in vitro. The 196 MDR1 ribozyme was cloned into an expression vector, and stably transfected (HL-60R/196Rz) cells were obtained. Expression of MDR1 transcripts was decreased in HL-60R/196Rz cells compared with parental HL-60R and empty vector-transfected (HL-60R/neo) cells. Interestingly, RA inhibited cellular proliferation and induced differentiation of HL-60R/196Rz cells in a dose-dependent manner, suggesting reversal of drug resistance in HL-60R cells by the MDR1 ribozyme. These data are direct evidence that P-glycoprotein/MDR1 is responsible in part for acquired resistance to RA in myeloid leukemic cells. The MDR1 ribozyme may be a useful tool for investigating the biology of retinoid resistance and may have therapeutic potential for patients with RA-resistant APL.

Retinoid resistance in leukemic cells.

Recent studies have shown that a high proportion of patients with acute promyelocytic leukemia (APL) achieve complete remission after treatment with all-trans retinoic acid (RA). Nevertheless, despite an initial good response, most patients who received continuous treatment with all-trans RA relapsed and develop RA-resistant disease. The detailed mechanisms for this development of RA resistance by APL cells are still unclear. Several possible mechanisms have been considered to explain in vitro resistance to RA. One obvious explanation is the generation of new mutations in the retinoid receptors. However, UF-1 cells (the first permanent APL cell line with RA-resistant features) had no point mutations in the ligand-binding domain of the RAR-alpha gene. Another potential mechanism for clinical RA resistance is the pharmacologic alteration in the metabolism of all-trans RA. Continuous treatment with all-trans RA in APL is associated with a progressive reduction of the plasma concentrations of RA. Induction of cytochrome P-450, cellular RA-binding protein (CRABP) and P-glycoprotein resulted in lower plasma and cellular levels of active retinoids. Thus, acquired resistance to RA may be explained at least in part by drug metabolism in leukemic cells.

Circulating endogenous thrombopoietin, interleukin-3, interleukin-6 and interleukin-11 levels in patients undergoing allogeneic bone marrow transplantation.

To elucidate the physiologic role of thrombopoietin (TPO) for hematologic reconstitution following allogeneic bone marrow transplantation (BMT), serum TPO levels as well as interleukin-3 (IL-3), IL-6 and IL-11 were serially measured in 55 samples from 3 patients who underwent allogeneic BMT using an enzyme-linked immunosorbent assay (ELISA). The TPO level was higher in the serum taken during marrow aplasia than in the pretransplant serum. The serum TPO levels and platelet counts showed a strong inverse relationship in all patients examined. We also sequentially measured endogenous serum TPO levels before and within 36 h after platelet transfusions. Endogenous serum TPO levels were inversely correlated with platelet mass following platelet transfusions. Serum levels of IL-3 had no apparent correlation with platelet counts and serum levels of IL-11 remained below the detection levels (31.3 pg/ml) in all samples. Serum levels of IL-6 were high during myeloaplasia and more upregulated in the febrile period. These findings support the view that TPO is the central regulator for megakaryopoiesis in vivo and the rationale for its clinical use after allogeneic BMT.

Serial quantification of minimal residual disease of t(8;21) acute myelogenous leukaemia with RT-competitive PCR assay.

The chromosomal translocation (8;21)(q22;q22) in the AML M2 subtype according to the FAB classification, results in the production of a novel fusion gene AML1/ETO. The chimaeric AML1/ETO transcript is useful for the detection of minimal residual disease (MRD). Recently, several studies on the detection of AML1/ETO transcripts in t(8;21) AML have been reported. However, the clinical significance of a small number of AML1/ETO transcripts by a reverse transcription-polymerase chain reaction (RT-PCR) remains to be elucidated. We have developed a novel quantitative RT-competitive PCR assay and evaluated the clinical usefulness of this method by the monitoring of MRD in eight patients with t(8;21) AML. In four patients in first continuous complete remission (CR) the value of MRD was always < 0.1 fg of the competitor dose throughout their courses, whereas in four relapsed patients there was an increase in the value of MRD to > 0.1 fg of the competitor dose before cytogenetic relapse. We conclude that the detection of the presence of cells with AML1/ETO fusion transcripts by our RT-competitive PCR assay may be useful to monitor disease progression and to predict subsequent relapse.

Establishment and characterization of a novel acute promyelocytic leukemia cell line (UF-1) with retinoic acid-resistant features.

All-trans retinoic acid (RA) induces complete remission in a high proportion of patients with acute promyelocytic leukemia (APL). Nevertheless, most of these patients develop RA resistance and relapse. The mechanisms of RA resistance by APL cells are still unclear. To understand the characteristics of human leukemia, human leukemic cell lines are useful tools for study. APL cells have a strikingly low proliferation potential in vitro; thus, only one APL cell line has been established. We developed a novel APL cell line (UF-1) from a patient clinically resistant to all-trans RA. Cell surface markers in the UF-1 cells were positive for CD7, CD13, CD33, and CD38. Cytogenetic analyses revealed additional abnormalities, 46XX, add(1)(q44), add(6)(q12), add(7)(q36), t(15;17) (q21;q21). Molecular analyses showed a PML/RAR alpha fusion transcript. Sequence analysis of the RAR alpha gene in RA-resistant HL-60 cells disclosed a point mutation in codon 411 (C to T substitution), whereas UF-1 cells showed the normal sequence. All-trans RA did not change morphological features of the cell, NBT reduction activity, or their expression of CD11b antigens as determined by FACS analysis except at 10(-6) mol/L. RA also did not alter the growth curve of the cells as determined by the MTT assay. These findings suggest that the UF-1 cell is the first permanent cell line with spontaneous RA-resistant APL cells. This RA-resistant APL cell line may be a useful model for molecular studies on the block of leukemic cell differentiation and as a means to investigate the mechanisms of RA resistance.

[Erythroblastic transformation of chronic myelogenous leukemia associated with an additional chromosome abnormality translocation (6;9].

A 57-year-old female was diagnosed being in the blastic phase of chronic myelogenous leukemia (CML) on her second admission in May 1993. The patient was previously treated with vincristine and prednisolone in the accelerated phase of CML in December 1991 without improvement. Other chemotherapeutic agents such as BHAC-DMP (enocitabine, daunorubicin, mercaptopurine, prednisolone), interferon, mercaptopurine and ranimustine were also administered. After the second chronic phase was achieved, she was treated with busulfan as an outpatient. On her second admission, the diagnosis of erythroblastic transformation was made, and cytogenetic study revealed t(9;22) (q34;q11) with the additional chromosomal abnormalities, t(6;9) (p23;q34). This karyotype rearrangement has been reported neither in Ph-positive CML nor in blastic crisis.

Serious adverse effects induced by simultaneous administration of two nonsteroidal anti-inflammatory drugs.

We have described the case of a 58-year-old woman in whom acute hemolytic anemia, renal failure, and granulocytopenia developed after intake of mefenamic acid and diclofenac. Results of direct and indirect Coombs' tests were negative, but the lymphocyte transformation test was positive for these two nonsteroidal antiinflammatory drugs (NSAIDs). We suggest that the immune complex mechanism could have induced these adverse reactions. Although these drugs are very popular, simultaneous administration should be avoided because serious adverse effects can potentially occur.

[Development of Stevens-Johnson syndrome following sulindac administration in a patient with mixed connective tissue disease].

Nineteen-year-old woman with mixed connective tissue disease developed Stevens-Johnson syndrome following treatment of arthritis using sulindac. Involvements of infectious and malignant diseases have been ruled out and sulindac has strongly been suspected as a causative agent for Stevens-Johnson syndrome. Ten out of 13 cases with Stevens-Johnson syndrome associated with non-steroidal anti-inflammatory drugs, sulindac has been administrated. Four cases also presented with severe liver disease. Patients who developed Stevens-Johnson syndrome following sulindac administration did not have apparent common clinical or laboratory findings which might be implicated for development of this severe side effects. Among the various non-steroidal anti-inflammatory drugs, safety of sulindac has widely been appreciated. However, occurrence of severe adverse events as reported here indicated that sulindac should be administrated as carefully as other non-steroidal anti-inflammatory drugs.

Delineation of the gastric muscularis mucosae and assessment of depth of invasion of early gastric cancer using a 20-megahertz endoscopic ultrasound probe.

Using a 20 MHz endoscopic ultrasound system, delineation of the gastric muscularis mucosae and estimation of the depth of malignant invasion was attempted by in vivo scanning during the process of routine endoscopic observation or in vitro scanning of excised sections of 34 early gastric cancers in 32 patients. The muscularis mucosae was visualized as a single hypoechoic layer in 16 of 32 lesions (50%) scanned in vitro. Comparison of lesions in which delineation of the muscularis mucosae was or was not possible revealed no significant differences with respect to either the thickness of the lamina propria and muscularis mucosae or with respect to the degree of inflammatory cell infiltration of the lamina propria or the conditions of the boundary between the lamina propria and the muscularis mucosae. This indicates that improvement of the operability characteristics of the ultrasonic apparatus will be needed to achieve improved delineation of the muscularis mucosae. The accuracy of invasion depth estimation of early gastric cancer was 67% (16 of 24 lesions scanned in vivo) and 73% (8 of 11 lesions) in cases in vivo where the muscularis mucosae and the tumor were delineated on the same screen. The principal factors causing erroneous staging were the presence of dilated benign glandular ducts, ulcer scars, and attenuation of the ultrasound waves.