Yoshikazu Kawanishi

Apoptosis/differentiation-inducing effects of Vitamin K2 on HL-60 cells : dichotomous nature of Vitamin K2 in leukemia cells

We originally reported that vitamin K2 (VK2) analogs, including menaquinone 4 (MK4) but not vitamin K1, effectively induce apoptosis in various types of primary cultured leukemia cells and leukemia cell lines in vitro. It has also been reported by others that VK2 showed the differentiation-inducing activity in leukemia cell lines. To investigate the discrepancy between apoptosis- and differentiation-inductions of leukemia cells by VK2 treatment, we used bcl-2 gene transfected HL-60 cells (HL-60-bcl-2) which resulted in five-fold over-expression of BCL-2 protein, and then compared the effects of MK4 to the control HL-60-neo cells. Seventy-two hours of exposure to various concentrations of MK4 resulted in growth inhibition of these cells in a dose-dependent manner (0.1–50 μM), however, HL-60-bcl-2 was less sensitive against MK4. MK4 potently induced apoptosis of HL-60-neo cells along with the depolarization of mitochondrial membrane potential and caspase-3 activation. Notably, HL-60-bcl-2 was almost completely resistant to apoptosis induction in response to MK4, although cell growth inhibition was still observed. In spite of the abrogation of apoptosis induction, about 90% of HL-60-bcl-2 cells were arrested in the G0/G1 phase within 48 h of exposure to 10 μM of MK4 accompanied by up-modulation of p27KIP1 expression. Concomitantly, HL-60-bcl-2 cells underwent monocytic differentiation. These data suggest that VK2 also shows the differentiation inducing effects on leukemia cells which are resistant against VK2-inducing apoptosis. The dichotomous nature of VK2 against leukemia cells appears to have clinical benefits for the treatment of patients with leukemias and myelodysplastic syndromes.

Vitamin K2 selectively induces apoptosis of blastic cells in myelodysplastic syndrome: flow cytometric detection of apoptotic cells using APO2.7 monoclonal antibody

We have previously reported that vitamin K2 (VK2) but not VK1 has a potent apoptosis-inducing effect on freshly isolated leukemia cells from patients with various types of leukemia. By multi-color flow cytometric analysis using monoclonal antibody (mAb), APO2.7, which detects mitochondrial 7A6 antigen specifically expressed by cells undergoing apoptosis, we further investigated the apoptosis-inducing effect of VK2 on minor populations of leukemic blast cells in bone marrow from patients with myelodysplastic syndrome (MDS) and overt myeloid leukemia (post-MDS AML). Limiting dilution of CD95 (anti-Fas) mAb-treated apoptotic Jurkat cells with nonapoptotic CTB-1 cells revealed that APO2.7-positive Jurkat cells were consistently detectable by flow cytometry when present at levels of at least 5% in the CTB-1 suspension. In patient samples the gating area for leukemic clone was determined using cell surface antigen-specific mAbs conjugated with either fluorescein isothionate (FITC) or phycoerythrin (PE) and subsequently the cells stained with phycoerythrin cyanine (PE-Cy5)-conjugated APO2.7 mAb were assessed within the gating area of the leukemic clone for monitoring apoptosis. Treatment of the bone marrow mononuclear cells with 3–10 μM of VK2 (menaquinone-3, -4 and -5) in vitro potently induced apoptosis of the leukemic blast cells as compared with the untreated control cells in all 15 MDS patients tested. This effect was more prominent on blastic cells than that on mature myeloid cells such as CD34−/CD33++ gated cells. In addition, VK2 performed much less effectively on CD3-positive lymphoid cells. In contrast to VK2, VK1 did not show apoptosis-inducing activity. These data suggest that VK2 may be used for treatment of patients with MDS in blastic transformation.

Comparative multi-color flow cytometric analysis of cell surface antigens in bone marrow hematopoietic progenitors between refractory anemia and aplastic anemia.

Refractory anemia (RA) in myelodysplastic syndrome (MDS) without prominent dysplasia closely resemble the mild type of aplastic anemia (AA) in their hematological features. This sometimes makes it difficult to distinguish clearly between the two diseases. Using the multi-color flow cytometric technique, we compared cell surface antigen expression patterns on bone marrow hematopoietic progenitor cells which were isolated as a CD34 positive- CD45 dull positive with low side scatter intensity (CD34(+)CD45(dull+)SSC(low)) population in flow cytogram between RA (n=12) and AA (n=11). The antigens analyzed in CD34(+)CD45(dull+)SSC(low) mononuclear cells were: CD38 and CD71 for cell growth-related antigens, CD 33 and CD13 for myeloid and monocytoid lineage-associated antigens, CD7 and CD19 for lymphoid lineage, and CD14 for a monocytic lineage specific antigen. The percentages of CD34(+)CD45(dull+)SSC(low) cells in bone marrow non-erythroid mononuclear cells, and the expression frequencies of CD38, CD71, CD33 and CD13 antigens in CD34(+)CD45(dull+)SSC(low) progenitors were all significantly decreased in AA compared to normal bone marrows (n=7) (P<0.005). In contrast, in RA bone marrows the percentages of CD34(+)CD45(dull+)SSC(low) cells showed wide distribution and the cell surface antigen expression patterns varied among each case: some cases showed low frequencies of CD38 and CD71 expression as well as AA, whereas the others showed high expression frequency of specific antigen(s) which may reflect the clonal expansion of an abnormal clone in bone marrow. An MDS patient who had progressed from RA to RAEB showed further projecting pattern of expression of CD38 and CD33 in CD34(+)CD45(dull+)SSC(low) population in accordance with the disease progression. These data suggest that analysis of cell surface antigen expression patterns of CD34(+)CD45(dull+)SSC(low) progenitor cells by multi-color flow cytometry appears to be a useful method for qualitative and quantitative assessment of marrow progenitor states in AA and RA, therefore this method could be helpful for early detection of clonal evolution in MDS.

Vitamin K2 induces apoptosis of a novel cell line established from a patient with myelodysplastic syndrome in blastic transformation

We have previously reported that vitamin K2 (VK2) has a potent apoptosis inducing activity toward various types of primary cultured leukemia cells including acute myelogenous leukemia arising from myelodysplastic syndromes (MDS). We established a novel cell line, designated MDS-KZ, from a patient with MDS in blastic transformation, and further investigated the effects of VK2 using this novel cell line. MDS-KZ shows complex chromosomal anomaly including −4, 5q−, −7, 13q+, 20q−, consistent with that seen in the original patient. Culture of MDS-KZ cells in RPMI1640 medium containing 10% FBS lead to steady but very slow proliferation with a doubling time of 14 days. However, the cellular growth rate was significantly accelerated in the presence of various growth factors such as granulocyte colony-stimulating factor, stem cell factor, granulocyte–macrophage colony-stimulating factor, interleukin-3, and thrombopoietin. Most of the cultured cells show the morphological features of myeloblasts. They are positive for CD7, CD33, CD34, CD45, CD117, and HLA-DR. However, about 10% of the cells are more mature metamyelocytes and neutrophils with various dysplastic characteristics such as pseudo-Pelger nuclear anomaly and hypersegmentation, suggesting a potential for differentiation in this cell line. As previously reported for cultured primary leukemia cells, exposure to VK2, but not to VK1, resulted in induction of apoptosis of MDS-KZ cells in a dose-dependent manner (IC50: 5 μM). In addition, VK2 treatment induced down-regulation of BCL-2 and up-regulation of BAX protein expression with concomitant activation of caspase-3 (CPP32). A tetrapeptide functioning as antagonist of caspase-3, Ac-DEVD-H, suppressed the VK2-induced inhibition of cell growth, suggesting that caspase-3 is, at least in part, involved in VK2-induced apoptosis. These observations suggest that the MDS-KZ cell line can serve as a model for the study of the molecular mechanisms of VK2-induced apoptosis.

Membranous glomerulonephritis with nephrotic syndrome associated with chronic lymphocytic leukemia.

A 66-year-old woman was admitted to our hospital for evaluation of edema of the extremities. Laboratory findings suggested that she had nephrotic syndrome and chronic lymphocytic leukemia (CLL). Renal biopsy (with PAM staining) showed a spike formation in the capillary wall. Immunofluorescent staining revealed deposition of immunoglobulin G (IgG) and the third component of complement in the glomerular basement membrane. Electron microscopy showed fibrillary deposits in the subepithelium. These findings indicated membranous glomerulonephritis (MGN). In addition, focal segmental sclerosis and interstitial lymphocytic infiltration were observed in the renal biopsy specimen. In CLL patients nephrotic syndrome occurs rarely. Even if the complication occurs, MGN is not frequent. Both diseases are suspected to occur in association with each other, and immunologic abnormality contributes to their coexistence. Although administration of prednisolone and endoxan improved leukocytosis, proteinuria was not sufficiently improved with combination therapy.

Thrombocytopenia induced by imatinib mesylate (Glivec) in patients with chronic myelogenous leukemia: is 400 mg daily of imatinib mesylate an optimal starting dose for Japanese patients?

Imatinib mesylate (Glivec) is a potent and selective inhibitor of the tyrosine kinase activity of BCR-ABL. Because the mechanism of action of imatinib mesylate is inhibition of BCR-ABL tyrosine kinase, it seems likely that to achieve maximum therapeutic benefit for chronic myelogenous leukemia (CML), imatinib mesylate must be administered at a dose that maximally inhibits BCR-ABL kinase activity or, alternatively, that inhibits sufficient BCR-ABL kinase activity to induce apoptosis. According to in vitro study results, 1 M levels of imatinib mesylate are optimal for inducing cell death, and 1 M trough levels are achieved in patients using imatinib mesylate at a daily dose of more than 300 mg [1]. In a phase I/II study in which the dose-response curve in chronic-phase CML patients was examined, the therapeutic effect was reported to plateau at a dose at or slightly above 300 mg. In addition, there was no convincing evidence of dose-limiting toxicity in patients receiving a maximum dose of 1000 mg [2]. These data were used to set the standard therapeutic dose of imatinib mesylate for chronicphase patients at 400 mg daily, and thereafter, the therapeutic benefits for CML patients of imatinib mesylate at this dose were demonstrated in clinical trials [2,3]. This standard therapeutic dose has been used in administering imatinib mesylate to Japanese patients as well as patients in the United States and Europe. At our institute, however, a series of CML patients treated with 400 mg of imatinib mesylate showed thrombocytopenia, and imatinib mesylate adminisThrombocytopenia Induced by Imatinib Mesylate (Glivec) in Patients with Chronic Myelogenous Leukemia: Is 400 mg Daily of Imatinib Mesylate an Optimal Starting Dose for Japanese Patients?

Combination of granulocyte colony-stimulating factor and low-dose cytosine arabinoside further enhances myeloid differentiation in leukemia cells in vitro.

We examined the differentiation-inducing effect on freshly isolated myeloid leukemia cells in liquid suspension culture by combined treatment with granulocyte colony-stimulating factor (G-CSF) plus low-dose cytosine arabinoside (Ara-C; 5-10 ng/ml) in 25 patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) in leukemic transformation. Culture with G-CSF alone showed leukemic cell growth stimulation in 15 out of the 25 cases (60%) and induction of cell differentiation in 19 out of the 25 cases (76%), respectively. In 23 cases (92%), either growth stimulation and/or differentiation induction of leukemia cells was observed in response to G-CSF. This suggests that most myeloid leukemia cells are able to respond to G-CSF stimulation. In addition, treatment of cells with low-dose Ara-C alone resulted in the enhancement of myeloid specific antigens expression in 16 cases (64%). Treatment of leukemia cells with higher concentrations of Ara-C (over 50 ng/ml) alone resulted in cytocidal effects but not in the induction of differentiation. Furthermore, 15 cases (60%) showed pronounced myeloid differentiation of leukemia cells after combined exposure to G-CSF plus low-dose Ara-C as compared with cells treated with either G-CSF or Ara-C alone. The enhanced effect of differentiation induction by combining G-CSF plus low-dose Ara-C was also observed in a murine myeloid leukemia cell line WEHI-3B in vitro. These data suggest that treatment with G-CSF plus low-dose Ara-C is capable of inducing differentiation of leukemic cells in vitro, and also appears to be useful for the differentiation-based therapy of patients with AML and MDS.

MALT lymphoma originating in breast and uvula.

A case of marginal zone B cell lymphoma of MALT type arising in the uvula and breast is reported. The patient, a 30-year-old woman who delivered a child and lactated in 1997, was suffering from Sjögren syndrome (SS). She was diagnosed with MALT lymphoma after a biopsy of the right breast and uvula. To investigate the relationship of the delivery, lactation and MALT lymphoma, we examined the immunohistochemical analysis of hormone receptors. As a result, lymphoid cells of the breast were stained with anti-progesterone receptor antibodies in the cytoplasm. Consequently, the MALT lymphoma of the uvula appeared to be associated with SS. Moreover, hormones such as progesterone may have influenced the breast involvement of MALT lymphoma in our case.

Decline in Antibiotic Enzyme Activity of Neutrophils Is a Prognostic Factor for Infections in Patients with Myelodysplastic Syndrome

We used flow cytometry to measure the activities of cathepsin G and elastase. The activity of elastase in neutrophils from patients with myelodysplastic syndrome (MDS) was significantly lower than that in neutrophils from the control group (P<.001). Patients with low elastase activity were significantly susceptible to infection (P<. 05). Our study suggests that analyzing antibacterial enzymes is useful in evaluating the prognosis of patients with MDS.

Vitamin K2 therapy for a patient with myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is an acquired stem cell disorder most likely arising from a genetic lesion in a single mulipotent hematopoietic stem cell that often progresses to overt acute myeloid leukemia (post-MDS AML). The syndrome predominantly affects the elderly, with a median age of 60 to 70 years. Intensive chemotherapy achieves an acceptable response rate, but the high incidence of treatment-related death and the short duration of complete remission mean that there is no survival benefit.1 High-dose chemotherapy supported by allogenic bone marrow transplantation is the only potentially curative treatment for MDS, however, such therapy can only be offered to a few younger patients. Since the prognosis of MDS in the elderly is poor, establishment of a novel strategy for treating these patients has been an urgent issue. We have previously reported that vitamin K2 (VK2) analogs such as menaquinone (MK)-3, -4 and -5, but not vitamin K1, have a potent apoptosis-inducing effect on various types of primary cultured leukemic cells, including post-MDS AML cells.3 In addition, we recently demonstrated that this apoptosis-inducing effect is rather selective for blast cells, as assessed by flow cytometry of whole marrow mononuclear cells from patients who had refractory anemia with excess of blast (RAEB) or RAEB in transformation. In contrast, VK2 was reported to show some differentiation-inducing activity on acute myeloid leukemic cell lines such as HL-60 and U937 in vitro. MK4 has been used clinically for the treatment of the patients with osteoporosis in Japan. The safety of daily doses of MK4 up to 135 mg orally over 24 weeks has already been confirmed in a late phase II trial for osteoporosis.6 These findings suggested that VK2 may be useful for the treatment of patients with MDS in blastic transformation. A 65-year-old man was referred to our department because of thrombocytopenia and anemia. Peripheral blood examination showed leukoerythroblastosis with morphological abnormalities such as hypogranulation and pseudo-Pelger nuclear abnormality of neutrophils, giant platelets, micromegakaryocytes, and binuclear erythroblasts with megaloblastic change. He was diagnosed as having MDS with myelofibrosis by bone marrow trephine. The percentage of blast cells in his peripheral blood soon reached over 30%, indicating progression to the overt acute leukemic phase (Table 1). As we also found previously in other MDS patients, culture of his peripheral mononuclear cells in the presence of 3 and 10 mm MK4 for 72 h resulted in selective elimination of CD34++/CD33dull+ blast cells in a dose-dependent manner (Figure 1). Because his fibrotic marrow made him unable to tolerate intensive chemotherapy, oral administration of 90 mg of MK4 daily was started after obtaining informed consent. Six weeks after initiation of MK4 therapy, the peripheral blast cell count has significantly decreased and the platelet count has increased from 31 × 109/l to 133 × 109/l (Table 1). Thereafter, the dose of MK4 was reduced to 45 mg/day, and treatment was continued. This has resulted in maintaining a good per-