Toshiki Natazuka

Transport mechanism of anthracycline derivatives in human leukemia cell lines: uptake and efflux of pirarubicin in HL60 and pirarubicin-resistant HL60 cells

Abstract We studied the transport mechanism of pirarubicin (THP) in HL60 and its THP-resistant (HL60/THP) cells, which showed no expression of mdr1 mRNA on Northern blot analysis. Under physiological conditions, the uptake of THP by both types of cell was time- and temperature-dependent. The amount of drug transport in the resistant cells was significantly less than that in the parent cells within 3 min of incubation. THP uptake was significantly higher in the presence than in the absence of 4 mM 2,4-dinitrophenol (DNP) in glucose-free Hanks’ balanced salt solution in both HL60 and HL60/THP cells and the increases were approximately equal. In the presence of DNP, the uptake of THP by both types of cell was concentration-dependent, and there were no significant differences in the apparent kinetic constants (Michaelis constant (Km), maximum velocity (Vmax) and Vmax/Km) for THP uptake between HL60 and HL60/THP cells. Additionally, THP transport was competitively inhibited by its analogue doxorubicin. The efflux of THP from HL60/THP cells was significantly greater than that from HL60 cells, and the release from both types of cell was completely inhibited by decreasing the incubation temperature to 0°C and by treatment with DNP in glucose-free medium. In contrast, the P-glycoprotein inhibitors verapamil and cyclosporin A did not inhibit THP efflux. However, genistein, which is a specific inhibitor of multidrug resistance-associated protein (MRP), increased the THP remaining in the resistant cells, and the value was approximately equal to that of the control group in the sensitive cells. These results suggest that THP is taken up into HL60 and HL60/THP cells via a common carrier by facilitated diffusion, and then pumped out in an energy-dependent manner. Furthermore, the accelerated efflux of THP by a specific mechanism, probably involving MRP, other than the expression of P-glycoprotein, resulted in decreased drug accumulation in the resistant cells, and was responsible, at least in part, for the development of resistance in HL60/THP cells.

Association between non-insulin dependent diabetes mellitus and non-Hodgkin's lymphoma

Nodal and extranodal lymphomas are considered to be distinct entities on the basis of histological distribution, response to chemotherapy, and prognosis.1 The risk of non-Hodgkins lymphoma is significantly increased in patients in an immunosuppressive state,2 and diabetes mellitus impairs the immune response to bacterial infections.3 We therefore retrospectively investigated the prevalence of diabetes mellitus in patients with nodal and extranodal lymphomas to determine whether the risk of lymphoma is increased in patients with diabetes. From 1986 to 1993, 160 patients with nodal and extranodal lymphoma, excluding those who were positive for human T cell leukaemia/lymphoma virus type I antibody, were treated at Kobe University School of Medicine Hospital. No patients were positive for HIV-1 antibody. We excluded 27 patients (21 with nodal lymphoma, six with extranodal lymphoma) because adequate …

Proliferative reaction of myelogenous leukemia cells with cytokines G-CSF, GM-CSF, M-CSF, SCF and TPO

In recent years, many cytokines have been defined and some of them used clinically. In hematological malignancies, cytokines, including granulocyte colony-stimulating factor (G-CSF), have been widely used for leukopenia after chemotherapy. However, in acute myelogenous leukemia (AML), some leukemic cells may be induced to proliferate by these cytokines and they must be used with care. In this study, we have investigated cell reactivity and proliferation with G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CMF), macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF) and thrombopoietin (TPO) in cases of AML. We have also investigated the reactivity of some myeloid leukemia cell lines to TPO. G-CSF, GM-CSF, M-CSF, SCF and TPO caused proliferation of leukemic cells in 25%, 58.3%, 8.3%, 21.1% and 0% of cases, respectively. Because of this result, the use of G-CSF in AML should be regarded as potentially hazardous. TPO did not cause proliferation of leukemic cells in any case of AML, or in cell lines except MO7E, which is a megakaryocytic cell line. This result suggests that TPO might cause proliferation of some megakaryocytic leukemia cells. We cannot conclude that TPO does not cause proliferation of other AML cells, as the number of cases was small and it has been reported elsewhere that leukemia cells may proliferate when exposed to TPO in 50% of AML cases. Reactivity of AM L cells to TPO is an important factor when deciding the indications of TPO in AML and myelodysplastic syndrome.

Combination Assay of Urinary β-Core Fragment of Human Chorionic Gonadotropin with Serum Tumor Markers in Gynecologic Cancers

Ectopic production of the immunoreactive β‐subunit of human chorionic gonadotropin (IR‐hCGβ) by gynecologic malignancies has been well recognized, but IR‐hCGβ has not yet been established as a clinically useful tumor marker, except for germ cell tumors. We measured the concentrations of IR‐hCGβ‐related molecules, intact hCG, free hCGβ, and β‐CF, in the sera and urine of patients with various gynecologic cancers (cervical, endometrial, and ovarian cancers) to assess their clinical usefulness as a tumor marker in comparison with serum tumor markers such as CEA, SCC, CA125, and CA19‐9. The highest incidence of IR‐hCGβ was obtained in tbe assay for β‐CF in the urine, with positive rates of 47.7% (94 of 197) for cervical, 37.8% (14 of 37) for endometrial, and 84.4% (38 of 45) for ovarian cancers with a cut‐off value of 0.2 ng/mg of creatinine. In cervical cancer, there was no significant correlation between the concentrations of urinary β‐CF and serum SCC, and 57.9% (114 of 197) of the patients were detected by the combination assay of these tumor markers. Serial determination in 22 cervical cancer patients with elevated urinary β‐CF level prior to therapy showed that its level decreased after successful treatment, but 4 of 5 patients with persistent or recurrent disease had elevated levels of urinary β‐CF. All of the ovarian cancer patients examined were detected by the combination assay of urinary β‐CF and serum CA125. The levels of urinary β‐CF showed little correlation with those of the serum tumor markers, indicating the usefulness of the combination assay of urinary β‐CF with serum tumor markers for detecting cervical and ovarian cancers.

Rare point mutation at codon 301 and 969 of FMS/M-CSF receptor in acute myelomonocytic and monocytic leukemia

We have investigated whether point mutations occurred at codon 301 or 969 of FMS (M-CSF receptor) in 19 patients with acute myelomonocytic (M4) and monocytic leukemia (M5). Nineteen peripheral blood and bone marrow blood samples collected from M4 and M5 patients were examined by using polymerase chain reaction and hybridization to allele specific oligonucleotide probes. Mutations at codon 301 and 969 of FMS were not detected in any samples. FMS gene mutations at codon 301 and 969 were rarely involved in M4 and M5 patients in Japan.

Feasibility of high-dose chemotherapy without stem cell support as a first-line treatment for non-Hodgkin's aggressive lymphoma: a pilot study.

A regimen which incorporates cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP) is the standard treatment for patients with non-Hodgkins lymphoma (NHL), but it has not been effective in patients with aggressive NHL who are at high risk. The aim of the present trial was to investigate the feasibility of high-dose chemotherapy (HDC) without stem cell support as a first-line treatment. The primary endpoint was a complete remission rate. The second endpoint was survival. Fourteen patients with aggressive NHL entered the study and were treated according to the K93 protocol (3 cycles of CHOP, high-dose etoposide and ifosfamide, and high-dose methotrexate) Eleven patients (79%) achieved complete remission (CR) and two (14%) achieved partial remission (PR). Overall survival (OS) after five years was 79%. The actuarial five year disease free survival (DFS) for the eleven cases of CR was 75%. During chemotherapy, grade IV hematologic toxicity was observed in all patients and grade IV non-hematologic toxicity in only one patient, who experienced oral ulcers. Peripheral blood stem cell (PBSC) apheresis was performed in eight cases. One harvesting was enough to provide an adequate number of CD34+ cells for the subsequent PBSC transplantation (PBSCT). In conclusion our study confirmed the efficacy of the K93 protocol in obtaining a good response (CR + PR) rate and a very good DFS rate in most cases of aggressive NHL, with acceptable toxicity. This regimen may improve the outcome in cases of aggressive NHL without stem cell support. It seems worthwhile to conduct a randomized controlled study comparing the K93 protocol with the standard CHOP regimen.

Plasma cell leukemia with myelofibrosis

SummaryWe describe a case of plasma cell leukemia associated with myelofibrosis. A 60-year-old woman was admitted due to lumbago and monoclonal hypergammaglobulinemia. Peripheral blood showed about 40% of plasma-cell-like cells. A bone marrow aspiration was dry tap. The patient was diagnosed as having plasma cell leukemia with myelofibrosis by bone marrow biopsy. Plasma cell leukemia as well as myelofibrosis improved with combination chemotherapy using vincristine, pirarubicin, and dexamethasone. However, when plasma cell leukemia became resistant to chemotherapies, myelofibrosis also reappeared. This case strongly suggests the pathogenetic relationship between plasma cell leukemia and myelofibrosis.

Therapeutic Trial of Hematological Disorders with Intermittent Administration of High-Dose 1-Alpha-Hydroxyvitamin D3

Aplastic anemia and myelodysplastic syndrome were successfully treated with an intermittent administration of high-dose 1 alpha-hydroxy-vitamin D3, an active analogue of 1,25-dihydroxyvitamin D3. This effect was considered to be through the differentiation-inducing and immunomodulatory actions of 1,25-dihydroxyvitamin D3. The only adverse effect was hypercalciuria which was controllable by decreasing the dose.

Effect of combination chemotherapy with cefoperazone on non-hodgkin's lymphoma

Abstract A patient with non-Hodgkins lymphoma stage III, relapsing after a partial response, was refractory to 3 cytostatic combinations, but responded to one of these given together with cefoperazone.