Time sequential expression of markers of apoptosis induced by 1-beta-D-arabinofuranosylcytosine in human T-lymphoblastic leukemia CCRF-CEM cells.

A wide variety of chemotherapeutic agents including 1-beta-D-arabinofuranosylcytosine can induce cell death by apoptosis. However, an appropriate method for early detection of apoptosis that can be clinically applicable to peripheral blood samples freshly obtained from leukemic patients under chemotherapy has not yet been established. We investigated the chronology of ara-C induced apoptosis in CCRF-CEM cells by monitoring the expression of a number of apoptotic markers in a time- and concentration-dependent manner with the aim of estimating the earliest reliable marker of apoptosis to apply to clinical drug sensitivity assays. We employed the following techniques: 1) Pulsed field gel electrophoresis to detect high molecular weight DNA fragmentation; 2) FACS analysis for evaluation of APO 2.7 expression and phosphatidylserine externalization; 3) May-Grunwald-Giemsa staining for studying gross morphology; and 4) TUNEL assay to detect multitudes of terminal 3'-OH groups of oligonucleosomal DNA fragments. At 10 microM ara-C, high molecular weight DNA fragmentation was observed after 4 hours incubation being the earliest marker to manifest. APO 2.7 expression and phosphatidylserine externalization were detected almost simultaneously at 6 hours, with marked similarity in their kinetics. Emergence of apoptotic bodies then followed after 12 hours incubation and, finally, oligonucleosomal DNA fragments were demonstrated by positive TUNEL assay at 48 hours. The results suggest the time-sequential expression of each individual marker and introduce high molecular weight DNA fragmentation assay as the most suitable candidate for early detection of sensitivity of malignant cells to apoptosis-inducing anticancer agents, including ara-C.

T-Cell type acute lymphoblastic leukemia following cyclosporin A therapy for aplastic anemia.

Cyclosporin A (CsA) is used to prevent rejection in transplantation and to treat autoimmune and hematologic diseases such as aplastic anemia. However, the tumor growth-promoting effect of CsA remains controversial. We report the case of a 24-year-old man who developed acute lymphoblastic leukemia of precursor-T-cell origin after 75 months of treatment with CsA for aplastic anemia. The surface antigen phenotype of his leukemic cells was CD2+, CD3+, CD5+, CD7+, CD4-, CD8-, CD10-, CD20-, CD34-, CD41-, and CD56-. Southern blot analysis revealed a monoclonal rearrangement of T-cell receptor-Jgamma nongermline fragments in HindIII digestion.

[Marginal zone lymphoma associated with Sjögren's syndrome and hepatitis C virus infection].

A 64-year-old female was admitted in May 1997, because of salivary gland swelling. Histology of the right parotid gland revealed malignant lymphoma, diffuse medium-sized B-cell type, and she was treated with local radiotherapy and chemotherapy. She was rehospitalized in April 1998, because of recurrence of lymphoma in the stomach and the sigmoid colon. She had splenomegaly and lymphadenopathy (neck and inguinal). Laboratory findings revealed marked elevation of rheumatoid factor and RNA of hepatitis C virus. A diagnosis of Sjogren's syndrome was made by dryness and the histological findings of labial biopsy. Marginal zone B-cell lymphoma mainly consisted of centrocyte-like cells and lymphoepithelial lesions, and CD 20 and IgM-kappa were positive with immunohistochemical staining. Lymphoma involved the gut and spleen. We discuss the correlation of malignant lymphoma with Sjogren's syndrome and HCV infection.

Differentiation induction in non-lymphocytic leukemia cells upon treatment with mycophenolate mofetil.

Inosine 5'-monophosphate (IMP) dehydrogenase catalyzes the rate-limiting reaction of guanine nucleotide biosynthesis and has been implicated in the reaction of cell growth and differentiation. We investigated the ability of mycophenolate mofetil, a prodrug of mycophenolic acid, to induce differentiation in HL-60 and U937 leukemic cells as well as in fresh leukemia cells from patients with non-lymphocytic leukemia. Treatment with mycophenolate mofetil reduced the intracellular guanosine 5'-triphosphate (GTP) levels and induced morphologic and functional differentiation in HL-60 and U937 cells dose-dependently. HL-60 and U937 cells developed macrophage-like cytoplasm as well as the expression of CD11b and CD14 antigens and the ability to oxidize nitroblue tetrazorium (NBT). These changes became evident when the intracellular GTP levels decreased to approximately 20-30% of the untreated control level and were abrogated by the addition of guanosine. In the fresh leukemic cells, differentiation induction was shown in the cells derived from seven of 13 patients. The fresh leukemia cells responding to mycophenolate mofetil revealed significant higher positivity to CD11b, CD14, and NBT before treatment and significantly reduced intracellular GTP levels after treatment compared to the non-responding cells. These findings suggest that mycophenolate mofetil induces differentiation in HL-60 and U937 cells and some fresh leukemia cells with moderate tendency to maturation, by causing a decrease in the intracellular GTP levels. Mycophenolate mofetil could be a promising differentiation inducer in vivo.

[Histiocytic necrotizing lymphadenitis (Kikuchi-Fujimoto's disease) accompanied by hemophagocytosis and salivary gland swelling in a patient with systemic lupus erythematosus].

After 2 years of steroid therapy that had effectively controlled her systemic lupus erythematosus, a 37-year-old woman presented with fever, erythema (face, upper chest), and low CH50. Increased oral steroid (prednisolone from 15 mg to 40 mg) and intravenous methylprednisolone (mPSL) (80 mg for 3 days) alleviated these symptoms except for the fever. Subsequently, the patient's fever worsened and leukocytopenia, abnormal liver function, lymphadenopathy (neck, axilla), and salivary gland swelling developed. Lymph node histology revealed features characteristic of Kikuchi-Fujimoto's disease (KFD). Laboratory examinations showed WBC 600/microliter, Hb 9.5 g/dl, platelets 90,000/microliter, GOT 766 IU/l, GPT 646 IU/l, LDH 4,228 IU/l, TG 1,622 mg/dl, and ferritin 6,330 ng/ml. Serum interferon gamma was also elevated (673 U/ml). Because a bone marrow smear revealed hemophagocytosis, mPSL pulse therapy (1 g for 3 days) was started for treatment of hemophagocytic syndrome. The fever promptly disappeared, and the patient's clinical symptoms resolved within 2 weeks. The abnormal laboratory data related to KFD and hemophagocytosis returned to normal within 4 weeks after the initiation of mPSL pulse therapy. We speculated that the hemophagocytosis and salivary gland involvement in this patient were also symptoms of KFD. This case indicated that corticosteroid pulse therapy is effective for KFD with serious clinical symptoms.

Successful treatment of cytomegalovirus retinitis in a patient with malignant lymphoma: a case report and review of the literature.

A 52-year-old Japanese woman was diagnosed as having angioimmunoblastic T-cell lymphoma (stage IV-B). She received 6 courses of chemotherapy including cyclophosphamide, doxorubicin, vincristine, and prednisolone every two weeks (biweekly CHOP), and was considered to be in partial remission. She complained of loss of visual acuity in her right eye during her last cycle of chemotherapy. Cytomegalovirus (CMV) retinitis was suspected from the characteristic ophthalmoscopic appearance. This diagnosis was further supported by the detection of CMV DNA in blood and antigens in polymorphonuclear leukocytes, a sign of CMV reactivation. Although DNAemia and antigenemia became negative, retinitis remained slightly active despite a 4-week systemic treatment of ganciclovir. Intraocular injection of ganciclovir was started and continued until the retinitis became inactive ophthalmoscopically. The patient received high-dose chemotherapy with peripheral blood stem cell transplantation and achieved complete remission. During the after this therapy no recurrence of CMV infections was observed. This case shows that 1) a quick and accurate diagnosis of CMV retinitis was possible by applying DNAemia and antigenemia and 2) intensive treatment for the CMV infection enabled the accomplishment of cure-oriented chemotherapy of the lymphoma without the recurrence of CMV retinitis.

A novel Philadelphia chromosome-positive cell line with multipotential properties.

A novel Philadelphia chromosome-positive cell line was established from the peripheral blood of a patient with chronic myelogenous leukemia in megakaryoblastic crisis. This cell line, designated TN922 showed the positive phenotypes for myeloid, monocyte-macrophage, erythroid and megakaryocytic markers. The stimulation with phorbol 12-myristate 13-acetate (PMA) increased the expression of megakaryocytic markers including the platelet peroxidase activity, dimethylsulfoxide or transforming growth factor-beta promoted up-regulation of the erythroid markers. Stimulation with PMA, tumor necrosis factor-alpha or interleukin-6 also brought about the expression of monocytoid markers. These findings indicated that TN922 cell line has the property of acting as multipotential progenitor cells. TN922 cells showed gradual growth in the absence of growth factors but the addition of granulocyte/macrophage colony-stimulating factor (GM-CSF) promoted cell growth. The message of GM-CSF was detected in TN922 cells and the neutralizing antibody against GM-CSF receptor alpha-subunit suppressed cell growth. These results indicated that TN922 cell line proliferates in an autocrine secretion of GM-CSF.

Molecular remission induced by fractionated dose-escalating donor leukocyte infusion without graft-versus-host disease in a patient with chronic myelogenous leukemia relapsed after allogeneic bone marrow transplantation.

A 39-year-old male with CML who relapsed 5 years and 8 months after allogeneic bone marrow transplantation achieved complete molecular remission following fractionated dose-escalating donor leukocyte infusions. Acute or chronic graft-versus-host disease (GVHD) did not occur and the patient remained asymptomatic throughout treatment. Since no prophylaxis against GVHD was administered, this case indicated that the graft-versus-leukemia effect is entirely separate from GVHD in certain conditions.

Peripheral blood stem cell collection and transplantation using the Haemonetics Multi Component System.

AIM: To assess clinical usefulness of an intermittent-flow blood cell separator in peripheral blood stem cell (PBSC) collection and transplantation. RESULTS: The Haemonetics Multi Component System (Multi) was used to collect PBSC (52 aphereses in 17 patients). The mean processing blood volume and the mean PBSC yield were 7407 ml and 2.16 x 10(6) CD34+ cells/kg, respectively. When CD34+ cells exceeded 0.3% of the peripheral WBC, more than 2.0 x 10(6) CD34+ cells/kg could be collected by a single apheresis. Eight patients underwent PBSC transplantation after high-dose chemotherapy. Hematopoietic recovery was achieved in a median period of 10 days. CONCLUSIONS: (1) A single-arm, light-weight machine has sufficient capability to collect PBSC. (2) The percentage of CD34+ cells in the peripheral WBC is a good predictor of the CD34+ cell yield of the collection.

[Improvement of bleeding tendency and normalized platelet count increment following splenectomy in a patient with refractory anemia].

A 72-year-old woman with refractory anemia had severe bleeding tendency. Since 1994, platelet transfusions (10 units three times per week) were unable to maintaining her platelet count over 10 x 10(3)/ microliter. Her hemoglobin was decreased to 3.9 g/dl as a result of bleeding from early gastric cancer. At one-hour after posttransfusion corrected platelet count increment (1-hour CCI) was slightly low, as 14 x 10(3)/microliter/m2. A 24-hour posttransfusion CCI (24-hour CCI) and the (24-hour CCI)/(1-hour CCI) ratio were markedly low, as 0.5/microliter/m2 and 0.36, respectively. Anti-HLA antibody was not detected. The ineffectiveness of platelet transfusion was suspected to be highly associated with splenomegaly. Her spleen had been gradually increased in size since the first clinical examination. She underwent both subtotal gastrectomy and splenectomy, while receiving 40 units of platelet transfusion. After splenectomy, the 1-hour CCI and the (1-hour CCI)/(24-hour CCI) ratio markedly improved (76 x 10(3)/microliter/m2 and 0.79, respectively). Cutaneous bleeding halted and there have been no further episodes, despite less frequent platelet transfusions. This is the first report in which bleeding tendency and CCI were improved by splenectomy in a case of myelodysplastic syndrome.

Anti-Leukemia Chemotherapy of High-Risk Myelodysplastic Syndromes.

We evaluated the outcome of anti-leukemia chemotherapy on 42 patients with the high-risk myelodysplastic syndromes (MDS)-refractory anemia with excess of blasts (RAEB), 8 cases; RAEB in transformation (RAEB-T), 18 cases; chronic myelomonocytic leukemia (CMMOL), 6 cases; and leukemic transformation of MDS, 10 cases. The median age was 67 (range 20 to 84). As a remission-induction therapy, 35 patients received low-dose chemotherapy, such as low-dose cytarabine infusion, and seven patients received high-dose combination chemotherapy. The complete remission (CR) rates of the low-dose chemotherapy group and the high-dose combination chemotherapy group were 29% and 57%, respectively, and the overall CR rate was 33%. The median survival durations after induction chemotherapy of the CR group (14 cases), the partial remission (PR) group (11 cases), and non-remission (NR) group (17 cases) were 19 months, 8 months, and 3 months, respectively. As a post-remission consolidation chemotherapy, high-dose combination chemotherapy seemed to be superior to low-dose chemotherapy judging from the median CR duration (16 months versus 4 months), but a long-term disease-free survival is hardly expected, in contrast with results in cases of de novo acute myeloid leukemia.

[Successful treatment of a 74-year-old man with refractory anemia with excess of blasts in transformation (RAEB in T) by low-dose Ara-C injection].

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic disorders characterized by normal or hypercellular but dysfunctional bone marrow. They usually are refractory to empirical therapeutic regimens. Recently the prevalence of MDS in the elderly is increasing and now the syndromes are relatively commonly encountered in elderly patients. Two major causes of death in MDS are progression to acute leukemia (especially in subtypes of RAEB and RAEB in T), and bone marrow aplasia. Since cytoreductive therapy for RAEB or RAEB in T in the elderly is often accompanied by serious adverse complications, such as infection and hemorrhage, special care is necessary. Here we describe successful induction remission in a 74-year-old man with MDS (RAEB in T) by twice daily low-dose cytosine arabinoside injections (10 mg/m2, s.c.), which was well-tolerated, free of serious adverse effects, and seemed to be a useful therapeutic option for elderly patient with RAEB or RAEB in T.

Pharmacokinetic and clinical pilot study of high-dose intermittent ubenimex treatment in patients with myelodysplastic syndrome.

Ubenimex is an orally active aminopeptidase inhibitor with an immunomodulating action. A pharmacokinetic and clinical pilot study of high-dose intermittent ubenimex was performed in patients with myelodysplastic syndrome. Ubenimex (150 mg/body) was administered orally, twice per day (at a 3 hr interval), twice a week, for at least 8 weeks. The concentrations of ubenimex and p-OH ubenimex, another active metabolite, increased gradually up to 7.1 +/- 4.7 micrograms/ml (mean +/- SD) at 5 hr and 0.25 +/- 0.15 mu/ml at 6 hr. The low percentage (4.2%) of the AUC ratio (p-OH ubenimex/ubenimex) suggested minimal metabolism. The plasma half-life of ubenimex was 2.1 +/- 0.7 hr and the total urinary recovery of both was 71.4% in 48 hr. Among 8 patients clinically studied, one achieved good response and another achieved minor response. These findings suggested good bioavailability, and a certain effectiveness of ubenimex in this administration method, which was worth trying, at least in restricted cases, such as cases with refractory disease or with a poor performance status.

Sequential promotion of normal and leukemic hemopoiesis by recombinant human granulocyte colony-stimulating factor during the course of myelodysplastic syndrome.

A 48-year-old man developed refractory anemia with excess of blasts in transformation. Complete response was achieved by low-dose ara-C therapy, but he relapsed 15 months later, with pancytopenia and 13.0% myeloblasts in normocellular marrow. He was treated unsuccessfully with prednisolone, metenolone, and 1-alpha-hydroxyvitamin D3 for 8 weeks. He then developed life-threatening pneumonia and was treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF Filgrastim; 125 micrograms/day s.c.). The pneumonia resolved and, interestingly, he achieved a partial response, with normal blood cell counts and only a few dysmyelopoietic cells in the marrow. However, thrombocytopenia progressed when rhG-CSF administration was tapered. When the dose was increased again, leukemic blasts were found to proliferate. When rhG-CSF was discontinued, blasts rapidly decreased in the peripheral blood. Chromosomal analysis revealed a complex abnormality during the first relapse, a normal 46,XY karyotype during the partial response, and recurrence of the same complex abnormality during leukemic transformation. The stimulation index of marrow mononuclear cells cultured with rhG-CSF increased with disease progression. These findings suggest that rhG-CSF initially stimulated the selective proliferation of normal hemopoietic cells, but the evolution or selection of a leukemic clone responsive to rhG-CSF appears to have occurred subsequently.

DNA damage and cell killing by camptothecin and its derivative in human leukemia HL-60 cells.

Camptothecin (CPT) has been recognized as a topoisomerase I (Topo I) inhibitor. However, the mechanism of cytotoxicity of this agent remains unknown. In the present study, we analyzed the kinetics of Topo I-mediated DNA single-strand breaks and internucleosomal DNA cleavage produced by CPT and its derivative, 7-ethyl-10-hydroxycamptothecin (SN-38), in HL-60 cells. DNA single-strand breaks were detected using alkaline sucrose gradient centrifugation when HL-60 cells were incubated with 10 microM CPT or 10 microM SN-38 for 30 min. These DNA single-strand breaks were rapidly repaired after drug removal, while the cytotoxic action of these drugs was sustained. Treatment of HL-60 cells with CPT or SN-38 for 3 h produced extensive degradation of DNA. Agarose gel electrophoresis showed a ladder of DNA fragments consisted of multimers of approximately 200 base pairs, characteristic of apoptosis. Interestingly, this type of DNA fragmentation was also induced within 4 h after repair of DNA single-strand breaks, and subsequently loss of cell viability was observed. When zinc ion, a potent inhibitor of endonuclease, was added to drug-free medium after treatment with CPT or SN-38, internucleosomal DNA cleavage was abolished. Furthermore, addition of zinc ion reduced the loss of cell viability. These data suggest that Topo I-mediated DNA single-strand breaks may be necessary but are not sufficient for cell death, and the endonuclease involved in induction of internucleosomal DNA cleavage may play an important role in HL-60 cell death induced by Topo I inhibitor.

[Treatment of myelodysplastic syndromes with cytokines].

To evaluate the effects of colony-stimulating factors (CSFs) on pathological cells from myelodysplastic syndromes (MDS), CD34-positive cell fractions (mainly blast cells) from nineteen MDS patients and four normal volunteers were successfully enriched by the use of immunomagnetic beads, and their responsiveness to CSFs was examined in short-term liquid suspension cultures. Considerable parts of blast cells from MDS patients remained immature as compared with the favorable maturation of normal blast cells, especially in high-risk MDS group that included two prominent cases with a remarkable blast cell growth without maturation induction by CSFs. Short-term clinical administrations of G-CSF often brought about hematological improvements on MDS patients. Other cytokines are also reported to be of use on some patients. However, a few cases progressed to overt leukemia in relation to G-CSF treatment. Much care should be taken with in vivo application of CSFs to high-risk MDS patients, although there may be no effective clue to foresee the risk of leukemic transformation yet.

A high proportion of early response genes are constitutively activated in T cells by HTLV-I.

Immortalization of T cells by HTLV-I is mediated by the X region of the virus and probably involves transactivation of cellular genes. We show that T cells transformed by HTLV-I constitutively express a high proportion of early response genes that are normally transiently induced following antigenic or mitogenic activation of T cells. Thus, HTLV-I-infected T cells display an 'early activation' phenotype that is distinct from the gene expression pattern of continuously dividing T cells. Ten early response genes representing a diverse array of functional categories were assayed. Four DNA-binding proteins/transcription factors including the p50 subunit of NF-kappa B were evaluated. A protein(s) encoded by the X region of HTLV-I appeared to contribute to up-regulated expression of most, if not all, of the early response genes. For those genes that could be assayed, increased transcriptional rates, but not substantial changes in mRNA half-life, were demonstrated in the presence of pX-encoded proteins, suggesting that the transcriptional transactivator, Tax, affects the induction or maintenance of transcription for these mitogen-inducible genes. Therefore, Tax may mimic or interact with a component(s) of the signal transduction pathway activated by antigen or mitogen treatment. These data demonstrate that early response genes, some of which probably play roles in initiating or maintaining cellular proliferation, are frequent targets of HTLV-I activation.

[Blastic crisis of primary myelofibrosis associated with multiple myeloblastomas ].

The patient is a 71-year-old female who underwent splenectomy due to splenomegaly 32 months after diagnosed as having primary myelofibrosis. On examination she was found to have massive skin nodules, lymph nodes swelling and an enlarged liver with an abnormal hematologic profile as follows: RBC count 3.68 x 10(6)/microliters; WBC count 151 x 10(3)/microliters with 11% blasts; and platelet count 42 x 10(3)/microliters. The bone marrow aspirate showed a hypocellular marrow with 19.2% blasts. Histological examination of the skin nodules revealed that they were myeloblastomas, thus suggesting leukemic transformation of primary myelofibrosis. Her WBC count dropped to about 20 x 10(3)/microliters through treatment with vindesine, cyclophosphamide, 6-mercaptopurine and prednisolone, but it did not drop further. Treatment with dexamethasone remarkably regressed the myeloblastomas, but she died of heart failure 4 months after diagnosis of leukemic transformation of primary myelofibrosis. The autopsy findings showed the formation of numbers of myeloblastomas in both the systemic fatty tissue and dura mater as well as extramedullary hematopoiesis in liver and lymph nodes. A rapid development of splenomegaly in a patient with primary myelofibrosis seems to be associated with leukemic transformation.