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In diffuse large B-cell lymphoma (DLBCL), a CD20-negative relapse is clinically significant because it is associated with chemo-refractory phenotypes and loss of a therapeutic target. The alteration of the CD20 gene is reported as infrequent in CD20-negative relapse in B-cell lymphoma. We established a DLBCL cell line with loss of CD20 expression (SD07) from a patient at CD20-negative relapse. She was initially diagnosed with CD20-positive DLBCL and received repeated immuno-chemotherapy that included rituximab. SD07, which has an immunoglobulin κ rearrangement identical to that of lymphoma cells at CD20-negative relapse, showed homozygous deletion of the CD20 gene with loss of the copy number of 11q12. SD07 is the first case in which it is proven that the loss of CD20 expression in relapsed DLBCL is the result of deletion of the CD20 gene. Deletion of the CD20 gene is a molecular mechanism of CD20-negative relapse in a subset of DLBCL.

We analyzed a case with the blastoid variant of mantle cell lymphoma (MCL-BV), a rare subtype of B-cell lymphoma, presenting with marked hypercalcemia at diagnosis. Enzyme-linked immunosorbent assay (ELISA) showed elevated serum levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1alpha (MIP-1alpha), and type I collagen telopeptide, but not parathyroid hormone, calcitriol or parathyroid hormone-related peptide at diagnosis, suggesting local osteoclastic hypercalcemia in this case. By reverse transcription polymerase chain reaction (RT-PCR) analysis, we found predominant expression of mRNA for MIP-1alpha in addition to those for receptor-activator of nuclear-factor kappa B ligand (RANKL), TNF-alpha, and IL-6 in lymphoma cells obtained from the patient. Furthermore, recombinant MIP-1alpha significantly stimulated (3)H-thymidine uptake by isolated MCL cells in vitro. Treatment with intravenous fluids, bisphosphonate, and methylprednisolone followed by combination chemotherapy promptly corrects the hypercalcemia and successfully induced complete remission, which was accompanied by a decrease of these cytokines in the serum, including MIP-1alpha. In the present case, MIP-1alpha, an osteoclast-activating factor produced by mantle lymphoma cells, may contribute to the development of hypercalcemia. It likely acts through RANKL expression in tumor cells and/or stroma cells, as indicated in multiple myeloma (MM) and adult T-cell leukemia/lymphoma (ATLL). Furthermore, MIP-1alpha is also involved in the development of an aggressive phenotype on MCL by stimulating proliferation of these lymphoma cells. In summary, the present study demonstrated that MIP-1alpha is an important factor in the development of both hypercalcemia and an aggressive phenotype in some types of B-cell lymphoma.

Patients with Ph chromosome negative myeloproliferative disease (Ph-MPD) have an increased risk of vascular complications. It remains controversial whether patients with the JAK2 V617F mutation (V617F) exhibit increased risk, while recent growing evidence has shown a critical role for V617F in clonal erythropoiesis in Ph-MPD. We studied 53 patients with Ph-MPD especially in relation to megakaryopoiesis, the thrombotic complications and the presence of V617F. Using novel mutation-specific PCR which is a highly sensitive PCR-based assay for detection of JAK2 mutated allele(s), we identified V617F in 38 Ph-MPD, which include 13 polycythemia vera (PV), 23 essential thrombocythemia (ET) and 2 chronic idiopatic myelofibrosis. The numbers of megakaryocytes were significantly increased in PV and ET patients with V617F, but the platelet counts were slightly lower. Although statistically not significant, the incidence of thrombotic events was higher in the group with V617F compared to in those without the mutation. Agonist-induced in vitro platelet aggregation and platelet adhesion were not affected by the presence of this mutation. Nonetheless, we found a hypercoagulable state in Ph-CMPD with V617F by employing whole blood thromboelastography. It suggests pre-thrombotic tendencies in CMPD are complex and JAK2 V617F mutation might have a role in vivo blood coagulation by altering not only the number, but function(s) of all three myeloid cells, including red blood cells, white blood cells and platelets in Ph-CMPD.

Senile EBV-associated B-cell lymphoproliferative disorder (LPD) was proposed as a new disease entity in 2003. This condition has a high incidence in elderly people without underlying immunodeficiencies, and is characterized by EBV-positive B-cell proliferation with a polymorphic composition. Histologically, the disease has two subtypes. The polymorphic LPD (PLPD) subtype has a preferable prognosis, whereas the large cell lymphoma (LCL) subtype involves aggressive disease progression. Reported herein is a case of senile EBV-BLPD with indolent clinical features and PLPD subtype in the initial phase that recurred as an aggressive lymphoma 3 years after the initial diagnosis. In the recurrent phase, Southern blotting confirmed monoclonal proliferation of large lymphoid B-cells. In both the initial and recurrent phases, polymerase chain reaction (PCR) yielded a single discrete band of a similar size due to an immunoglobulin heavy-chain gene rearrangement, indicating that the large lymphoid B-cells retained identical monoclonality throughout the histological progression and over the whole clinical course. These results suggest that the PLPD subtype is a histological finding in early phase senile EBV-BLPD and that the LCL subtype reflects the progressive phase of the disease.

Allogeneic stem cell transplantation (allo-SCT) is used as curative therapy for malignant lymphoma, and reduced-intensity hematopoietic stem cell transplantation (RIST) is sometimes performed to avoid the toxicity and mortality associated with myeloablative allo-SCT. RIST is generally preferred for elderly patients with malignant lymphoma. A 62-year-old woman with follicular lymphoma (FL) involving bone marrow (BM) suffered relapse after autologous SCT. RIST was performed; cells were from an unrelated, fully human leukocyte antigen-matched donor. To study the hematopoietic reconstitution, BM biopsy specimens that were obtained at different times after RIST, were evaluated. Engraftment of donor cells was observed on days 19 and 48 after RIST, and residual FL in BM had completely disappeared by day 73 after RIST. This is the first report to document histological BM regeneration after RIST and disappearance of FL involving the BM.

Purpose The aim of the study was to analyze the methylation status of the promoter regions of p15 and p16 and to assess the prognostic significance of promoter hypermethylation in diffuse large B-cell lymphoma (DLBCL). Experimental design DLBCL was diagnosed by morphology and immunohistochemical analysis according to the World Health Organization (WHO) classification. The methylation status of CpG islands in the p15 and p16 promoters was analyzed by methylation-specific polymerase chain reaction in 49 DLBCLs. Results Hypermethylation of the p15 and p16 promoters was detected in 20 (41%) and 22 (45%) of the 49 DLBCLs, respectively. Among all patients with DLBCL, there was no significant difference in the overall survival between those with hypermethylated and unmethylated p15 (P = 0.442) or between those with hypermethylated and unmethylated p16 (P = 0.468). Therefore, methylation was analyzed in combination with evaluation of clinical features using the international prognostic index (IPI). In the high-intermediate-risk and high-risk groups, patients with hypermethylated p16 had significantly lower survival rates than those of patients in the same risk group with unmethylated p16 (P = 0.010). Conclusions Our results suggest that hypermethylation of the p16 promoter indicates a poor prognosis in high-intermediate-risk and high-risk DLBCL patients, and may be a useful marker for selection of appropriate treatment when used in conjunction with the IPI.

Rituximab is widely used for the treatment of B-cell non-Hodgkin's lymphoma (NHL), and encouraging results have been obtained. However, some CD20-positive NHL show minimal response to rituximab, indicating that the treatment effect depends on the presence or absence of an unidentified factor. We analyzed the relationship between the effect of rituximab plus chemotherapy and expression of Ki-67, p53 and bcl-2 and several clinical variables in cases of B-NHL, particularly follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). Forty-four patients were included in the present study, and the overall treatment response rate was 68%. Twelve of 30 patients (40%) achieved a complete response, five (16%) reached an unconfirmed complete response and 13 (43%) achieved a partial response. A high serum lactate dehydrogenase level and International Prognostic Index of high or high intermediate risk were associated with a decreased response in the case of FL. Immunohistochemical assays were performed in 18 FL patients (55%) and 15 DLBCL patients (45%). Significant correlation was found between an inferior response to treatment and high Ki-67 expression in the cases of FL (P = 0.006). p53 and bcl-2 expression did not correlate significantly with the response rate. The cell cycle appears to be an important factor in the efficacy of rituximab treatment. Ki-67 expression might be a predictor of efficacy of rituximab plus chemotherapy.

We present the case of a 66-year-old man with follicular lymphoma who developed acute respiratory distress syndrome (ARDS) during a third infusion of rituximab. High fever, tachypnea, and progressive hypoxemia accompanied by massive bilateral pleural effusions appeared suddenly approximately 3 hours after the third infusion was started, although the 2 prior infusions of rituximab had produced only mild adverse effects. The patient was treated successfully with high-dose methylprednisolone and 3 days of mechanical ventilatory support. No evidence was obtained to indicate that the ARDS had been caused by either cytokine release or tumor lysis, and serum human antichimeric antibody was not detected. Although the cause of ARDS was not confirmed, our experience in this case suggested that an anaphylactic reaction induced by repeated infusion of rituximab was involved in the onset of pulmonary disease. Although ARDS is rarely seen with rituximab infusion, careful management is required for safe administration of the newly developed rituximab therapy. This management includes monitoring biological reactions not only during the initial infusion but also during subsequent infusions of the antibody.

We previously found that expression of the Mm-1 cell-derived transplantability-associated gene 1b (MmTRA1b)/phospholipid scramblase 1 gene was markedly induced during the granulocytic differentiation of human myeloid leukemia cells. To evaluate the role of MmTRA1b expression in human myeloid leukemia, we investigated the relative levels of MmTRA1b transcripts in 81 patients with acute myelogenous leukemia (AML) by the reverse transcriptase polymerase chain reaction. The expression of MmTRA1b in AML-M1, -M5a and -M5b was significantly lower than that in normal bone marrow cells. The levels of MmTRA1b expression in AML-M2 and -M4 varied among patients. Higher MmTRA1b mRNA levels were associated with significantly longer overall survival in AML, especially in AML-M4 patients, independent of chromosomal aberrations such as t(8;21) and inv(16). The present results suggest that the MmTRA1b mRNA level is a new prognostic factor for AML, especially the AML-M4 subtype.

More than one histological type of malignant lymphoma can occur simultaneously in an individual. The entity is classified as either composite or discordant lymphoma. Both types of lymphoma, particularly discordant lymphoma comprised of extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue lymphoma (MALT-L) and follicular lymphoma (FL), are rare. We report a case of discordant lymphoma comprising MALT-L in the stomach and FL in the parotid gland. The patient was a 50-year-old Japanese woman who visited the University Hospital of Showa (Tokyo, Japan) because a barium study showed erosive gastric lesions. A gastro-intestinal endoscopy was performed 2 months after the barium study, which showed irregular erosions throughout the stomach body. A gastric biopsy showed MALT-L, and Helicobacter pylori (H. pylori) infection was confirmed. The patient had noticed a painless and elastic hard tumor mass of about 2 cm in diameter in the area of the left parotid gland 6 months before the barium study. We removed the parotid gland tumor and diagnosed it as FL 6 months after the barium study. We were able to diagnose the MALT-L and FL by morphological, immunohistochemical and molecular analyses of paraffin-embedded sections. This appears to be the first reported case of MALT-L and FL occurring together as a discordant lymphoma.

Objective We previously cloned a human normal counterpart (MmTRA1b/phospholipid scramblase 1) of the mouse leukemogenesis-associated gene MmTRA1a. MmTRA1b gene expression was increased during differentiation of human monoblastic leukemia U937 cells using some differentiation inducers but not 1α,25-dihydroxyvitamin D 3 (a typical monocytic differentiation inducer). To further elucidate the role of human MmTRA1b gene expression in the differentiation of myelogenous leukemia cells, we measured MmTRA1b gene expression in several myeloid leukemia cell lines and primary leukemia cells. Materials and Methods The expression of MmTRA1b mRNA was determined by semiquantitative reverse transcriptase polymerase chain reaction. Results Expression of the MmTRA1b gene was markedly induced during granulocytic differentiation of promyelocytic leukemia NB4 and HT93 cells induced by all-trans retinoic acid (ATRA). The level of MmTRA1b mRNA was significantly increased during differentiation toward granulocytes, but not monocytes/macrophages, in bipotential myeloid leukemia HL-60 cells. The level of MmTRA1 mRNA was not increased during erythroid differentiation induced by hemin in erythroid leukemia K562 and HEL cells or during megakaryocytic differentiation induced by 12- O -tetradecanoylphorbol-13-acetate in K562 cells. Expression of the MmTRA1b gene also was not induced when apoptosis of NB4 cells was induced by antileukemic drugs. ATRA-induced differentiation of antisense MmTRA1b-transfected NB4 cells was significantly suppressed. On the other hand, ATRA induced the differentiation of MmTRA1b-transfected NB4 cells more efficiently than that of mock-transfected cells. MmTRA1b mRNA also was clearly induced in ATRA-treated primary acute promyelocytic leukemia cells during granulocytic differentiation. Conclusion MmTRA1b mRNA was specifically induced during granulocytic differentiation of acute promyelocytic leukemia cells and was associated with induction of their differentiation.

We analyzed CD34 positive cells in peripheral blood stem cell harvest (PBSCH) using flow cytometry. PBSCH from CD34 positive acute myelogeous leukemia (AML-M2) patient contained 1.87% CD34 positive cells, of which 1.21% was represented by MRD.PBSCH from CD34 positive acute lymphoblast leukemia (ALL) patient contained 3.14% CD34 positive cells, of which 0.11% was accounted for by minimal residual disease (MRD). If PBSCH from CD34 positive acute leukemia patient is analyzed for CD34 monoclonal antibody alone, the presence of CD34 positive MRD may escape attention so that CD34 positive hematopoietic progenitor cells may be overestimated. To avoid this risk, it is necessary to analyze PBSCH using both CD34 monoclonal antibody and characteristic markers of leukemia cells that were found pre-treatment.

Expression of the cyclin kinase inhibitor, p21, is regulated both transcriptionally and posttranscriptionally by the ubiquitin-proteasome degradation pathway. Recently, we reported that DNA damage is required for efficient p21 expression by demonstrating that enhanced p21 mRNA expression induced by DNA damage results in increased p21 protein, but enhanced p21 mRNA without DNA damage does not. In addition, we demonstrated that DNA damage suppressed the ubiquitination of p21. In this study, we analyze the link between p21 stabilization and DNA damage. Enhanced p21 protein expression in ML-1 cells resulting from 15 Gy gamma-irradiation was diminished by Wortmannin or LY294002 pretreatment of cells. However, the levels of p21 mRNA were not affected by inhibitor pretreatment. Wortmannin or LY294002 pretreatment reduces p53 expression after gamma-irradiation to a lesser degree than that of p21. In addition, we examined the involvement of DNA-PK, whose activity is inhibited by Wortmannin or LY294002, in p21 stabilization using the SCID fibroblast cell line and a DNA-PK targeting ML-1 cell line. Accumulation of p21 protein by gamma-irradiation was similar to that of DNA-PK intact cells and was reduced by Wortmannin or LY294002 pretreatment. Involvement of another DNA damage detecting enzyme, the ATM gene product, whose activity is also inhibited by Wortmannin or LY294002, was evaluated. ATM deficient cells induced p21 after gamma-irradiation, gamma-irradiation-induced p21 protein was diminished by pretreatment of cells with Wortmannin or LY294002. We conclude that the p21 stabilization mechanism functions after gamma-irradiation, was sensitive to Wortmannin or LY294002, and required neither DNA-PK nor ATM gene product for activity.

The differentiation inhibitory factor nm23 can inhibit the differentiation of murine and human myeloid leukemia cells. We recently reported that nm23 genes were overexpressed in acute myelogenous leukemia (AML), and a higher level of nm23-H1expression was correlated with a poor prognosis in AML, especially in AML-M5 (acute monocytic leukemia). To evaluate the importance ofnm23 expression as a prognostic factor in AML, we compared it with other putative prognostic factors in AML. An analysis of the correlation between nm23 expression and the clinical parameters of 110 patients with AML demonstrated that increased nm23-H1mRNA levels were associated with resistance to initial chemotherapy and with reduced overall survival. Multivariate analysis using Cox's proportional hazard model also showed that elevated nm23-H1mRNA levels significantly contributed to the prognosis of patients with AML. Especially in AML-M5, nm23-H1 status was the most important prognostic factor. Furthermore, to determine whether we can apply the results observed in AML to other hematologic malignancies, we investigated the relative levels of nm23-H1 and nm23-H2transcripts in 149 patients with hematologic neoplasms, including 110 with de novo AML, 9 with de novo acute lymphoblastic leukemia, 14 with myelodysplastic syndrome, 16 with chronic myelogenous leukemia (CML), and 5 normal subjects by the reverse transcriptase-polymerase chain reaction. Expression of nm23-H1 was significantly higher in all the hematologic neoplasms, except CML in chronic phase, than in normal blood cells. nm23 may have a prognostic effect in these hematologic malignancies as well as in AML.

Profound immunodeficiency in multicentric Castleman's disease has already been elucidated. In the present study, we investigated CD45RO and Fas antigen expression and apoptosis by T cells in three patients with this disease. T cell expression of CD45RO and Fas antigen was increased in two of the three patients, indicating in vivo lymphocyte activation. Apoptosis of T cells from the two patients with increased CD45RO and Fas antigen expression occurred after overnight culture in the presence of pokeweed mitogen. Occurrence of apoptosis was indicated by DNA laddering and nuclear chromatin condensation. Peripheral blood mononuclear cells from the two MCD patients revealed spontaneous apoptosis following 3 days of culture by quantitative assay using flow cytometry. These findings show that T cells are activated in some patients with multicentric Castleman's disease and suggest that this activation may promote apoptosis.

Angelmicin B is a new microbial substance which inhibits src tyrosine kinase activity and oncogenic signal transduction. We investigated the effect of angelmicin B on the proliferation and differentiation of the HL-60 human myeloid leukemia cell line. Angelmicin B caused the dose-dependent inhibition of cell proliferation and induction of differentiation along the myelomonocytic pathway, as determined by morphological changes, nitroblue tetrazolium (NBT) reduction, and non-specific esterase and lysozyme activities at concentrations ranging from 0.1 to 0.5 microgram/ml. Also, it induced significantly the differentiation of mouse myeloid leukemia M1 cells. A similar concentration of angelmicin B inhibited the growth of the myeloid leukemia cell lines K562, HEL, KU812, ML-1, U937 and THP-1, but did not induce differentiation of these cells significantly. The differentiation of HL-60 cells was enhanced by combined treatment with angelmicin B and 1 alpha, 25-dihydroxyvitamin D3 (VD3), retinoic acid or tumor necrosis factor-alpha (TNF alpha). Angelmicin analogs (A1, A2, B, C and D) had almost equivalent effects on the differentiation of HL-60 cells, although angelmicins C and D inhibited src tyrosine kinase activity less than the other analogs. The effective concentrations of angelmicin B in src kinase inactivation was about 100-fold higher than those required for the growth inhibition and differentiation induction. These findings indicate that the differentiation-inducing activity of angelmicins is not associated with their src kinase-inhibiting activity, and may be associated with the modulation of other signal pathway(s).

A 77 year-old woman was admitted to the hospital because of nasal obstraction on March 1994. Tumorectomy of the nasopharyngeal tumor disclosed non-Hodgkin's lymphoma (LSG : diffuse, medium sized). The patient was treated with local radiotherapy to nasopharyngeal region and combined chemotherapy (2 courses of CHOP) to reduce residual tumor. On July, the pericardial effusion appeared and the large granular lymphocyte (LGL) like lymphoma cells were observed in the effusion. Flow cytometic analysis of these cells showed that they expressed CD 2, CD 7, CD 56 and HLA-DR, but did not express CD 3. T-cell receptor gene (TCR beta) rearrangement was not observed and natural killer activity was detected in these lymphoma cells. The patient was treated with ProMACE and the pericardial infusion of methotrexate, carboplatin and prednisolone, but the patient died of heart failure. Monoclonarity of lymphoma cells in the pericardial effusion was determined by southernblot analysis, using the terminal repeat of Epstein-Barr virus (EBV) for probe. It was suggested that EBV participated in tumorgenesis in this case.

A patient with myelodysplastic syndromes (MDS) developed eosinophilia during treatment with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). To study the mechanism of this eosinophilia, we investigated the proliferation of eosinophil colony-forming units (CFU-Eo) in nine patients and four healthy controls. Eosinophil clusters increased significantly in the patients (P < 0.01) compared with controls, but eosinophil colonies were not different between controls and MDS patients. In addition, the eosinophil clusters were significantly increased with rhGM-CSF in MDS patients compared with controls, although serum GM-CSF concentrations were similar in both groups. These results suggest that eosinophil clusters are increased in MDS either through abnormal progenitor proliferation or hypersensitivity to GM-CSF.

We have screened for mutations in the Cip1/Waf1 gene using Southern blot analysis and the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method in diverse human tumors. Seven of 102 (7%) human tumor samples were identified to have point mutations within the coding region of the Cip1/Waf1 gene. Two of the seven mutated cases showed gene rearrangements. These results suggest that the frequency of genetic alterations in the Cip1/Waf1 gene is relatively low in comparison with several known tumor suppressor genes.

A 44-year-old male patient was diagnosed with acute lymphoblastic leukemia (CD10+, CD19+, CD20 weak) and underwent unrelated bone marrow transplantation (uBMT) with a conditioning regimen of cyclophosphamide plus total body irradiation during first complete remission (CR). Twenty months post-uBMT, the serum creatinine level (Cre) increased gradually, up to ≥ 1.5 mg/dl at 23 months. Since the increase in Cre was observed continuously, imaging examinations were performed and showed significant bilateral enlargement of the kidneys. Renal biopsy showed diffuse invasion of TdT, CD10 and CD19 positive lymphoid cells in the tubulo-interstitial region. Since leukemia cells were observed in the bone marrow, it was diagnosed as relapse in the bone marrow and kidney. Following reinduction chemotherapy, both kidneys returned to normal size. The patient entered into a second CR, but relapse occurred 6 months thereafter. The patient underwent uBMT again with a reduced-intensity conditioning regimen and CR has been maintained up to 5 months post-second uBMT. Although it is considered rare for relapse to occur with diffuse enlargement of both kidneys, as shown in this case, it is important to confirm the state of the kidney by performing blood tests and image diagnosis during the early phase, when renal dysfunction of an uncertain cause occurs after transplantation.

Recently an increase of the elderly patients with hematological malignancies has been pointed out. We analyzed second malignancies in elderly patients with hematological malignancies (95 age 65 or more), and made a comparative study with non-elderly case for the past 5 years. Second malignancies were observed in 26 cases out of the total of 282 hematological malignancies (9.2%). The percentage of patients with second malignancies in the elderly group (19/95; 20%) was significantly higher than that of the non-elderly group (7/187; 3.7%). Among the all kinds of hematological malignancies, the second malignancies were mainly observed in cases with myelodysplastic syndrome and chronic myelo-proliferative disorder. Colon carcinoma, gastric carcinoma and lung carcinoma accounted for nearly half of all the second malignancies. On 11 of the 26 cases with second malignancies, the first malignancies had been treated with some anti-cancer drug such as alkylating agents. Development of a second cancer was greater in cases in which the first hematological malignancy was treated with alkylating agents more than in cases in which the first carcinoma was not treated with alkylating agents.

We report a case of primary pulmonary classical Hodgkin lymphoma (CHL) in a 58-year-old woman. Twelve years ago, the patient complained of slight fever and weight loss. A mass of about 5 cm in diameter was seen in the right lung on radiography and computed tomography (CT). Right total pneumonectomy and resection of mediastinal lymph nodes were performed. A pathological examination led to a strong suspicion of Hodgkin disease (HD) (now referred to as CHL), but a definite diagnosis could not be made at the time. Six years later, a chest CT showed a tumor around the ascending aorta, which was treated successfully by radiation therapy. Six years later, the chest CT revealed a tumor in the anterior mediastinum. CHL was diagnosed based on an immunohistochemical re-examination of lung specimens resected 12 years earlier and CT-guided fine needle tumor biopsy specimens of the second recurrent tumor in the anterior mediastinum were compatible with the recurrence of CHL. Therefore, we diagnosed this case as primary pulmonary CHL that later relapsed in the mediastinum. The tumor size was reduced by radiation therapy and the patient is currently under observation as an outpatient.

A multi-institutional study was conducted to assess efficacy and safety of biapenem (BIPM), a carbapenem antibiotic, as an initial-stage therapeutic agent for febrile neutropenia (FN) in patients with hematopoietic diseases. A total of 216 patients from 25 medical institutions were enrolled in this study; of these, 204 were included in the safety analysis and 178 in the efficacy analysis. The combined (excellent and good) response rate was 67.9%, and antipyretic effect (subsidence + tendency to subsidence) was achieved within 3 and 5 days of treatment in 67.3 and 75.9% of patients, respectively. Thus, the clinical responses were gratifying. A response rate of 61.7% (37/60) was observed even in high-risk FN patients in whom neutrophil counts prior to and at 72 h after the start of BIPM were ≤100/μl. BIPM is considered to be a highly promising drug, with prompt onset of clinical benefit, as an initial-stage therapeutic agent for the treatment of FN in patients with hematopoietic diseases.

We report a case of B-cell prolymphocytic leukemia (B-PLL) that was treated successfully with splenic irradiation (SI). An 86-year-old man underwent a medical examination for lumbago and general fatigue at another hospital in June 2007. A compressed lumbar fracture and splenomegaly were found using computed tomography (CT). Thereafter, the patient consulted our hospital because of leukocytosis. Peripheral blood showed hemoglobin level 9.8 g/dl and white blood cell count 38.1x10(9)/l with 91% atypical cells. Surface marker analysis demonstrated that atypical cells were positive for CD20, CD22, FMC7, surface IgM, surface IgD and kappa, but were negative for CD5, TdT and lambda. The morphology of these cells was compatible with prolymphocytes with prominent nucleoli and condensed nuclear chromatin. A diagnosis of B-PLL was made. SI (total dose 20 Gy) was chosen for the treatment and a single course of SI was very effective without causing any significant adverse events. This case demonstrates that SI may remain valuable for the treatment of B-PLL in an elderly patient.

A 66-year-old woman presented with anemia in January 2006. She was admitted to our Department in February, after laboratory data showed pancytopenia and bone marrow biopsy reticulin fibrosis. The results of the diagnostic work-up, which included the anti-SS-A antibody, anti-SS-B antibody positivity and salivary gland scintigraphy, Schirmer test and Rose Bengal test, supported the classification criteria of Sjögren syndrome. Due to secondary myelofibrosis accompanied by Sjögren syndrome, she was started on prednisolone (PSL) and recovered completely from the anemia and thrombocytopenia. After the PSL was tapered, a recent follow-up indicated that the peripheral blood had normalized with the PSL therapy. As a causal disease of autoimmune myelofibrosis in collagen disease, systemic lupus erythematosus occurs frequently. This patient is considered to be a rare case in whom secondary myelofibrosis was accompanied by Sjögren syndrome.

To study hematopoietic reconstitution in umbilical cord blood transplantation (CBT), bone marrow (BM) histology was investigated in 35 biopsies after bone marrow transplantation (BMT) and in 40 biopsies after CBT. BM biopsies were obtained at different times after transplantation and were evaluated for cellularity, number of megakaryocytes and CD34-positive cells, and fibrosis. In biopsies up to 29 days after BMT, cellularity was increased and megakaryocytes were observed, but at 29 days after CBT, biopsies showed severe cellular depletion and almost no megakaryocytes. In addition, fewer CD34-positive cells were observed after CBT compared to after BMT. After day 30 after CBT, hematopoietic recovery of the BM was gradually observed and after day 100 after transplantation, no essential differences were observed between BMT and CBT. Hematopoietic recovery of the BM after CBT was delayed compared to that after BMT, but engraftment of donor cells after CBT was also observed in histopathologically. To the best of the authors' knowledge this is the first histopathological description of BM reconstitution after CBT.

Cotylenin A, which has been isolated as a plant growth regulator, potently induces the differentiation of human myeloid leukemia cells. Treatment of HL-60 cells with a combination of transforming growth factor (TGF)-beta and 1alpha, 25-dihydroxyvitamin D(3) (VD3) resulted in increased differentiation compared to separate treatments, but TGF-beta did not affect the cotylenin A-induced differentiation of HL-60 cells. It is possible that the signal transduction pathway used by cotylenin A for inducing the differentiation of leukemia cells is the same as that used by TGF-beta. However, cotylenin A did not affect the expression of TGF superfamily or Smad genes in HL-60 cells. Treatment with neutralizing anti-TGF-beta antibody or an inhibitor of TGF-beta signaling did not inhibit cotylenin A-induced differentiation, although VD3-induced differentiation was significantly suppressed by these treatments. The subcellular distribution of Smad3 was also unaffected by cotylenin A. These results suggest that the cotylenin A-induced differentiation of leukemia cells is independent of the TGF-beta signaling system, although TGF-beta acts as an autocrine mediator of the growth arrest and differentiation of leukemia cells induced by VD3 and other inducers.

Transferrin receptor 1 (TfR1) is a type II membrane protein that mediates cellular iron uptake. Transferrin receptor 2(TfR2), another receptor for transferrin (Tf), has recently been cloned. We examined expression levels of TfR1, TfR2-alpha (membrane form) and TfR2-beta (non-membrane form) transcripts in cells from 67 patients with de novo acute myeloid leukaemia (AML) using reverse transcription-polymerase chain reaction (RT-PCR), and correlated the results with a variety of clinical features and disease outcomes of these patients. Significant correlations were noted between the levels of both TfR1 and TfR2-alpha (r = 0.771, P0.001) and TfR1 and TfR2-beta (r = 0.534, P0.001). Unexpectedly, initial white blood cell (WBC) counts were inversely correlated with levels of expression of either TfR1(r = -0.357, P = 0.003), TfR2-alpha (r = -0.486, P0.0001), or TfR2-beta (r = -0.435, P = 0.0003). Only TfR2 expression was significantly associated with either serum iron (r = -0.270, P = 0.045) or serum ferritin (r = -0.364, P = 0.008). Multivariate analyses using Cox's proportional hazard model showed that elevated TfR2-alpha, but not TfR1 or TfR2-beta mRNA levels significantly contributed to a better prognosis for AML patients. Furthermore, a group with high expression levels of both TfR2-alpha and TfR2-beta survived significantly longer than a group without high expression of both of them (P0.01 by log-rank). The present study suggests that (i) TfRs-independent iron uptake might have an important role in in vivo proliferation of AML cells; (ii) expression of TfR2 (especially the alpha form) is a novel prognostic factor for patients with AML.

A 21-year-old man was admitted to our hospital for acute hepatitis of unknown cause. His liver function improved with rest, but worsened 2 months later. He developed a high fever and pancytopenia. The serum level of cytokines including TNF-alpha, IFN-gamma, IL-6, and M-CSF was elevated, and hemophagocytes were seen in bone marrow. These findings suggested a hemophagocytic syndrome-like state. With prednisolone, gamma-globulin, and G-CSF, the high fever disappeared and the patient's liver function gradually recovered. However, the severe pancytopenia persisted. The bone marrow became acellular with a small number of hemophagocytes, and hepatitis-associated aplastic anemia was diagnosed. After immunosuppressive therapy with ATG, CyA and G-CSF was started, and the patient showed hematopoietic reconstitution. The bone marrow CD4+/CD8+ lymphocyte ratio recovered to within the normal range, and the serum cytokines including TNF-alpha and IFN-gamma decreased. The increase in serum cytokines, particularly TNF-alpha and INF-gamma, as well as the presence of activated T cells associated with the preceding hemophagocytic syndrome-like state may have predisposed this patient to aplastic anemia.

A 22-year-old female was diagnosed as having acute lymphoid leukemia (ALL) in February 1995, from the findings of peroxidase negative, CD10+, CD19+, TdT+ and rearrangement of IgH and TCR beta. AdVP (doxorubicin, vincristine and prednisolone) therapy achieved a complete remission (CR). Bone marrow transplantation had to be abandoned because of the lack of an HLA-identical donor. Intensification therapy was thus carried out repeatedly. In June 1998, myeloblast with Auer rods, peroxidase positive, CD13+, CD33+ and HLA-DR+, appeared. The patient was diagnosed as having lineage switch acute myeloid leukemia (AML) from ALL. Though A-DMP (cytosine arabinoside, daunorubicin, 6-mercaptopurine) therapy was resistant, AdVP therapy led to a CR. The patient died of cardiotoxicity from anthracyclines in February 1999. From the results of the Ramasamy method using the clonal rearrangements of the Ig heavy chain gene locus, the origin of the pathological cells of ALL and AML was indicated to be the same leukemia clone.

Cyclin A (A2) and cyclin A1 are members of the G2 cyclins, which are involved in the control of G2/M and G1/S transitions as well as mitosis. Human cyclin A1 was cloned as an A-type cyclin that is highly expressed in acute myeloid leukaemia (AML). The clinical significance of these cyclins in myeloid leukaemia remains to be clarified. We investigated the relative levels of these transcripts in 80 patients with de novo AML. Correlations with clinical parameters showed that the initial white blood cell count and serum lactate dehydrogenase levels were inversely associated with cyclin A (A2) mRNA levels (r = -0.276, P = 0.019) and cyclin A1 mRNA levels (r = -0.241, P = 0.042) respectively. They were independently associated with increased overall survival [P = 0.035 for cyclin A (A2) and P = 0.016 for cyclin A1]. Multivariate analysis using Cox's proportional hazard model showed that elevated cyclin A1 mRNA levels contributed significantly to the better prognosis of patients with AML. Furthermore, the analysis of survival probability showed that the group with high levels of both cyclin A (A2) and A1 survived significantly longer than the group with low expression of both these cyclins (P = 0.002). These data indicate that high expression levels of both cyclin A (A2) and A1 are associated with good prognosis in AML patients.

Some follicular lymphomas histologically transform into diffuse aggressive lymphomas, the prognosis of which is poor. There are, however, no reliable histological criteria for predicting which cases will later undergo such transformation. In low-grade B-cell lymphomas, follicular dendritic cells form dense mesh-like networks that contain accumulating neoplastic B-cells. These are rare in high-grade lymphomas. We immunohistochemically analyzed CD21-positive follicular dendritic cells in 32 follicular lymphomas, including 3 transformed lymphomas, in addition to immunohistological study using P-glycoprotein, p53, and Ki-67. We found that the mesh-like networks in follicles are more clearly defined in low-grade lymphomas than in high-grade lymphomas (p = 0.015). Neoplastic follicles in 2 transformed lymphomas lost the networks of follicular dendritic cells before transformation despite the existence of morphologically clear follicles. This differed from the non-transformed cases of the same cytological grades. Prognosis was statistically better for patients with low-grade tumor than for those with high-grade tumor (p = 0.026), and there was a trend toward poorer survival among CD21-negative cases (p = 0.186). P-glycoprotein, p53, and Ki-67 expressions did not provide sufficient information to predict the transformation of follicular lymphoma. The presence of CD21-positive follicular dendritic cells in neoplastic follicles might help predict the potential of follicular lymphoma to transform to diffuse large B-cell lymphoma.

A 16-year-old girl was admitted to our hospital because of high fever, abdominal pain, and jaundice. Abnormal lymphocytes and hemophagocytic cells had infiltrated the bone marrow. Laboratory data revealed a severe type of hemophagocytic syndrome accompanied by an initial Epstein-Barr virus (EBV) infection. Persistent EBV infection was identified by polymerase chain reaction (PCR) detection of EBV-DNA in peripheral blood and bone marrow mononuclear cells. The limited efficacy of initial treatment with high-dose gamma-globulin, plasmapheresis, and high-dose methylprednisolone prompted us to administration of T-COP-E (VP-16). Two courses of T-COP-E improved the patient's clinical symptoms and laboratory data; however, marked splenomegaly remained. In addition, fever and serum increase of lactate dehydrogenase (LDH) and cytokines such as gamma-interferon recurred shortly after chemotherapy. On day 53 after diagnosis, the patient underwent laparoscopic splenectomy. The resected spleen weighted 420 g and abnormal lymphocytes in the spleen were positive for CD 8 and negative for CD 56. In situ hybridization revealed EBV-encoded small RNAs (EBERs) in the abnormal lymphocytes. Clinical symptoms including high fever disappeared shortly after the splenectomy, and laboratory data returned to normal. Lymphocytosis after the splenectomy was not observed. We continued out patient monitoring of the case, and 16 months after diagnosis, EBV-DNA in peripheral blood mononuclear cells was not detected, even by PCR.

The various spare ability of the internal organs declines in the elderly ITP. There are not a few who have some complications such as diabetes mellitus and cardiovascular disease. So, various restrictions are often taken in the treatment. PSL is the effective drug most securely against ITP. However, it is necessary in elderly ITP to reduce the amount of PSL, because the various side effects including the decline of QOL. On the other hand, the elimination therapy against Helicobacter pylori is carried out for only one week and remission lasts for a long time, and side effect is little. This elimination therapy is useful treatment for elderly ITP. It is important to improve and elevate QOL of the patient.

A 73-year-old man noticed a subcutaneous tumor on the left upper palpebra from April 1998, but did not seek therapy for it. Facial subcutaneous tumors appeared from November 1999, and multiple tumors appeared on the skin of the chest and both upper arms from January 2000. Tumor biopsy revealed that these tumors were non-Hodgkin lymphoma showing CD19 (+), CD20 (+), CD5 (+), CD10 (-), smIgM (+), sm lambda (+) and cyclin D1 (+). The karyotype was t(11;14) (q13;q32), but bcl-1 gene rearrangement was not detected. On the basis of these data, primary mantle cell lymphoma (MCL) of the subcutis was diagnosed. The patient underwent eight courses of THP-COP therapy, and complete remission was achieved. Primary subcutaneous B-cell lymphoma, especially MCL, is rare. MCL is aggressive and difficult to cure; the median survival of patients is 3 to 5 years, and the 5-year survival is 30%. However, the present patient showed a good response to chemotherapy, and complete remission has continued for 17 months since the MCL was first diagnosed.

The active form of vitamin D3, 1,25(OH)2D3, inhibits proliferation and induces differentiation of a variety of malignant cells. A new class of vitamin D3 analogs, having 2 identical side chains attached to carbon-20, was synthesized and the anticancer effects evaluated. Four analogs were evaluated for their ability to inhibit growth of myeloid leukemia (NB4, HL-60), breast (MCF-7), and prostate (LNCaP) cancer cells. All 4 analogs inhibited growth in a dose-dependent manner. Most effective was 21-(3-methyl-3-hydroxy-butyl)-19-nor D3(Gemini-19-nor), which has 2 side chains and removal of the C-19. Gemini-19-nor was approximately 40 625-, 70-, 23-, and 380-fold more potent than 1,25(OH)2D3 in inhibiting 50% clonal growth (ED50) of NB4, HL-60, MCF-7, and LNCaP cells, respectively. Gemini-19-nor (10−8 M) strongly induced expression of CD11b and CD14 on HL-60 cells (90%); in contrast, 1,25(OH)2D3 (10−8 M) stimulated only 50% expression. Annexin V assay showed that Gemini-19-nor and 1,25(OH)2D3 induced apoptosis in a dose-dependent fashion. Gemini-19-nor (10−8 M, 4 days) caused apoptosis in approximately 20% of cells, whereas 1,25(OH)2D3 at the same concentration did not induce apoptosis. Gemini-19-nor increased in HL-60 both the proportion of cells in the G1/G0 phase and expression level of p27kip1. Moreover, Gemini-19-nor stimulated expression of the potential tumor suppressor, PTEN. Furthermore, other inducers of differentiation, all-trans-retinoic acid and 12-O-tetradecanoylphorbol 13-acetate, increased PTEN expression in HL-60. In summary, Gemini-19-nor strongly inhibited clonal proliferation in various types of cancer cells, especially NB4 cells, suggesting that further studies to explore its anticancer potential are warranted. In addition, PTEN expression appears to parallel terminal differentiation of myeloid cells.

We analyzed G1 accumulation induced by the iron chelator deferoxamine B mesylate (DFO) compared it with that caused by etoposide and cytosine arabinoside (AraC). The results showed that p53 protein increased with all three treatments without an increase in p53 mRNA. After treatment for 3 or 6 h, p21 mRNA increased with 10 −4 DFO to 159% or 556% of pretreatment levels, to 509% or 391% with 10 −5 etoposide, and to 263% or 304% with 10 −5 AraC. Induction of p21 protein was not observed with fluorescence activated cell sorting and Western blot analysis after treatment with DFO or AraC. Treatment with DFO did not cause any change in levels of CDK4 mRNA or protein, whereas etoposide or AraC treatment did diminish CDK4 protein. Enzyme linked immunosorbent assay for pRB and its phosphorylation, which reflects CDK4 activity, revealed that treatment with DFO did not change the amount of pRB or the phosphorylation status. Results of this investigation show that the mechanism of G1 accumulation induced by DFO involves a p53-independent pathway and that expression of p21 protein may be regulated posttranscriptionally.

Although 1alpha,25(OH)2D3 induces HL-60 cells to differentiate into monocytes, whether or not these monocytes revert to native promyelocytes is not clear. To investigate this question, HL-60 cells were treated with 1alpha,25(OH)2D3, and divided into CD14-positive and -negative cells with a cell sorter. These two populations were cultured with or without 1alpha,25(OH)2D3. Sorted CD14-positive HL-60 cells, treated with 1alpha,25(OH)2D3 for 7 days, reverted to CD14-negative cells and promyelocyte-like cells if 1alpha,25(OH)2D3 was removed from the medium. We conclude that the 1alpha,25(OH)2D3-induced differentiation of HL-60 cells into monocytes is reversible, and continuous administration of 1alpha,25(OH)2D3 is required for the differentiation of HL-60 cells.

The Epstein-Barr virus (EBV) genome was detected by polymerase chain reaction (PCR) in mononuclear cells from bone marrows with diverse types of hematopoietic malignancies. Viral repeated sequences (BamHI-W region) were detected in 42 of 82 (51%) hematopoietic malignancies, including polycythemia vera, but not in nonneoplastic cases. EBV-positive cases were found to consist of various histological types. We did not detect any EBV PCR product in the peripheral blood. The EBV BamHI-Y, -H region, encoding EBV nuclear antigen 2 DNA, which is a single-copy gene in the viral genome, was detected in only 13 of 42 BamHI-W-positive cases, suggesting that the copy number of the EBV genome differed in each case. In all cases, the PCR band was verified by Southern blot hybridization using specific EBV probes. Whether the infected virus is an etiologic agent of the malignancy or merely a latent infection cannot be determined by the PCR assay performed under these conditions. These results, however, suggest that a novel form of EBV latent infection is present in the bone marrow of patients with hematopoietic malignancies.

Differentiation inhibitory factor (nm23 protein) inhibited the induction of differentiation of mouse myeloid leukemia M1 and WEHI-3BD+ and human erythroleukemia HEL, KU812, and K562 cells. Block of differentiation may be associated with the aggressive behavior of leukemia. To examine the role of nm23 in human myeloid leukemia, we investigated the relative levels of nm23-H1, nm23-H2, and c-myc transcripts in 42 patients with acute myelogenous leukemia (AML), and in 5 with chronic myelogenous leukemia at chronic phase by reverse transcriptase polymerase chain reaction. The expression of nm23-H1 and -H2 but not of c-myc in AML was significantly higher than that in normal blood cells. Among AMLs, acute monocytic leukemia (presentation with AML-M5 morphology) was especially associated with elevated nm23-H1 and -H2 mRNA levels. On the other hand, the elevated levels of c-myc expression in AML-M5 were less evident. An analysis of correlation between nm23 expression and clinicopathological parameters showed that resistance to initial chemotherapy is associated with increased nm23-H1 mRNA levels and that a high initial white blood cell count is associated with increased nm23-H2 mRNA levels. Elevated nm23-H1 mRNA levels were associated with significantly reduced the overall survival of AML, especially of AML-M5 patients. The present results indicate that nm23-H1 and -H2 are overexpressed in AML and especially nm23-H1 gene expression predicts the prognosis of AML, especially of AML-M5.

We studied spontaneous cytokine production by peripheral blood mononuclear cells (PBMC) obtained from 14 patients with aplastic anemia (AA) and 28 various myelodysplastic syndromes (MDS). The levels of interleukin-6, interleukin-1 beta, and tumor necrosis factor-alpha in cultured PBMC were measured by ELISA. The average levels of these cytokines were higher in AA or in refractory anemia (RA) than in RA with excess of blasts (RAEB) or in RAEB in transformation (RAEB-T). Marked cytokine overproduction was observed in RA as well as in AA. High cytokine levels were observed in hypocellularity and low blast cell counts in the bone marrow. These results may suggest that the increase of cytokines may be a reactive response in hypocellular bone marrow.

Cells with monocyte/macrophage lineage expressing receptor activator of NF-κB (RANK) differentiate into osteoclasts following stimulation with the RANK ligand (RANKL). Cell adhesion signaling is also required for osteoclast differentiation from precursors. However, details of the mechanism by which cell adhesion signals induce osteoclast differentiation have not been fully elucidated. To investigate the participation of cell adhesion signaling in osteoclast differentiation, mouse bone marrow-derived macrophages (BMMs) were used as osteoclast precursors, and cultured on either plastic cell culture dishes (adherent condition) or the top surface of semisolid methylcellulose gel loaded in culture tubes (non-adherent condition). BMMs cultured under the adherent condition differentiated into osteoclasts in response to RANKL stimulation. However, under the non-adherent condition, the efficiency of osteoclast differentiation was markedly reduced even in the presence of RANKL. These BMMs retained macrophage characteristics including phagocytic function and gene expression profile. Lipopolysaccharide (LPS) and tumor necrosis factor -αTNF-α activated the NF-κB-mediated signaling pathways under both the adherent and non-adherent conditions, while RANKL activated the pathways only under the adherent condition. BMMs highly expressed RANK mRNA and protein under the adherent condition as compared to the non-adherent condition. Also, BMMs transferred from the adherent to non-adherent condition showed downregulated RANK expression within 24 hours. In contrast, transferring those from the non-adherent to adherent condition significantly increased the level of RANK expression. Moreover, interruption of cell adhesion signaling by echistatin, an RGD-containing disintegrin, decreased RANK expression in BMMs, while forced expression of either RANK or TNFR-associated factor 6 (TRAF6) in BMMs induced their differentiation into osteoclasts even under the non-adherent condition. These results suggest that cell adhesion signaling regulates RANK expression in osteoclast precursors.

To study the pathogenesis of multicentric Castleman's disease (MCD), IL-6 producing cells and immune function were investigated in four MCD patients. The expression of IL-6 mRNA in one MCD lymph node was analysed by in situ hybridization. IL-6 mRNA expressing cells were scattered in the interfollicular areas and did not resemble plasma cells. Spontaneous IL-6 production was detected in the culture supernatants of peripheral blood mononuclear cells (PBMNC) from four patients. The IL-6 producing cells among the PBMNC were found to be monocytes by both in situ hybridization and immunohistochemistry. We evaluated immune function in four MCD patients. These studies show: (1) a negative PPD skin test in 3/4 patients, (2) decreased IL-2 production in 3/4 patients, (3) decreased T cell colony formation in 3/4 patients, (4) decreased NK activity and NK cell number in 2/4 patients, (5) increased soluble IL-2 receptor in 4/4 patients, and (6) decreased CD4/CD8 ratio in 3/4 patients. These results show that MCD resembles, in several ways, acquired immunodeficiency syndrome (AIDS).

The molecular basis of the Philadelphia chromosome (Ph1) is a structurally altered c-abl (bcr/abl) gene which encodes an abnormally large protein with protein tyrosine kinase activity. Herbimycin A, an inhibitor of tyrosine kinase, preferentially inhibited the growth of Ph1-positive acute lymphoid leukemia (ALL) cell lines, as well as Ph1-positive chronic myeloid leukemia (CML) cell lines. Although noncytotoxic concentrations of herbimycin A induced erythroid differentiation of two CML-derived cell lines, K562 and KU812, in a previous study, the differentiation-inducing effect of herbimycin A on Ph1-positive ALL cell lines was less strong. Herbimycin A enhanced some differentiation-associated properties of one Ph1-positive ALL cell line, L2, but the effect of herbimycin A on the other Ph1-positive ALL cell lines was cytotoxic rather than cytostatic (differentiation-inducing). Several derivatives of herbimycin A were synthesized and their effects on the cell proliferation of Ph1-positive CML and ALL cell lines were examined. The sensitivities of the Ph1-positive cell lines to herbimycin A derivatives were different from the data on the rat kidney cell line infected with Rous sarcoma virus (v-src) derived from a previous study, suggesting bcr/abl kinase may differ in sensitivity from other tyrosine kinases. Moreover, the sensitivities of the ALL cell lines were not the same as those of the CML cell lines. These results suggest that a specific inhibitor of bcr/abl kinase could be an effective antileukemic agent against Ph1-positive CML or ALL.

We investigated the effect of transforming growth factor-beta 1 (TGF beta) on the proliferation and differentiation of cultured acute promyelocytic leukemia (APL) cells with the chromosomal t(15;17) translocation obtained from four patients to determine the role of TGF beta on growth and differentiation of APL cells. DNA synthesis, determined by 3H-thymidine uptake, was inhibited in the presence and absence of granulocyte colony-stimulating factor (G-CSF) in a dose-dependent manner by TGF beta in APL cells obtained from three of the four cases. TGF beta and G-CSF did not significantly affect the differentiation of APL cells, but all-trans retinoic acid (RA) induced morphological and functional differentiation in all APL cells tested. G-CSF markedly enhanced RA-induced granulocytic differentiation in APL cells obtained from all four cases. In cells in which TGF beta inhibited DNA synthesis, it also inhibited RA-induced granulocytic differentiation of APL cells and, to a greater degree, granulocytic differentiation induced by RA plus G-CSF. These results suggest that TGF beta is a negative regulator of the proliferation and differentiation of APL cells. The significance of TGF beta as an endogenous regulator in differentiation therapy with RA of APL patients is discussed.