Bryostatin induces protein kinase C modulation, Mcl-1 up-regulation and phosphorylation of Bcl-2 resulting in cellular differentiation and resistance to drug-induced apoptosis in B-cell chronic lymphocytic leukemia cells

Bryostatin, a macrocyclic lactone and protein kinase C (PKC) modulator, has been shown to have differentiation and anti-tumor activity against several leukemia cell lines in vitro. In this study, we demonstrated Bryostatin-induced differentiation in B-cell chronic lymphocytic leukemia (B-CLL) cells, characterized by an increase in cell size and a marked up-regulation of CD11c expression. The specific inhibitors of the extracellular signal-regulated kinase (ERK) and protein kinase C pathways, PD98059 and GF 109203X respectively, each completely blocked Bryostatin-induced differentiation of B-CLL cells, suggesting that activation of the ERK pathway plays a direct role in this process in a PKC-dependent manner. Furthermore, Bryostatin reduced both spontaneous and drug-induced apoptosis with chlorambucil, fludarabine and 2-chloro-2'-deoxyadenosine (2-Cda) in B-CLL cells. This resistance was associated with an early up-regulation of the anti-apoptotic protein Mcl-1 and post-translational phosphorylation of Bcl-2 at serine 70. The anti-apoptotic effects of Bryostatin were abrogated by GF 109203X, and to a lesser extent by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, LY294002. Interestingly, the ERK inhibitor, PD98059 inhibited Mcl-1 expression but had little effect on Bryostatin-induced survival suggesting that the ERK pathway predominantly affects differentiation. Taken together these results present an explanation for Bryostatin-induced B-CLL cell survival in which modulation of the PKC pathway couples differentiation with an increase in anti-apoptotic protein expression and calls into question the rationale for its use in the treatment of B-CLL.     

Retinoic acid induction of CD38 antigen expression on normal and leukemic human myeloid cells: relationship with cell differentiation

Differentiation in the hematopoietic system involves, among other changes, altered expression of antigens, including the CD34 and CD38 surface antigens. In normal hematopoiesis, the most immature stem cells have the CD34+CD34 -phenotype. In acute myeloid leukemia (AML), although blasts from most patients are CD38+, some are CD38 -. AML blasts are blocked at early stages of differentiation; in some leukemic cells this block can be overcome by a variety of agents, including retinoids, that induce maturation into macrophages and granulocytes both in vitro and in vivo . Retinoids can also induce CD38 expression. In the present study, we investigated the relationship between induction of CD38 expression and induction of myeloid differentiation by retinoic acid (RA) in normal and leukemic human hematopoietic cells. In the promyelocytic (PML) CD34 -cell lines, HL60 and CB-1, as well as in normal CD34+CD34 -hematopietic progenitor cells RA induced both CD38 expression as well as morphological and functional myeloid differentiation that resulted in loss of self-renewal. In contrast, in the myeloblastic CD34+ leukemic cell lines, ML-1 and KG-1a, as well as in primary cultures of cells derived from CD34+-AML (M 0 and M 1 ) patients, RA caused an increase in CD38+ that was not associated with significant differentiation. Yet, long exposure of ML-1, but not KG-1, cells to RA resulted in loss of self-renewal. The results suggest that while in normal hematopoietic cells and in PML CD34 -cells induction of CD38 antigen expression by RA results in terminal differentiation along the myeloid lineage, in early myeloblastic leukemic CD34+ cells, induction of CD38 and differentiation are not functionally related. Since, several lines of evidence suggest that the CD38 -cells are the targets of leukemic transformation, transition of these cells into CD38+ phenotype by RA or other drugs may have therapeutic effect, either alone or in conjunction with cytotoxic drugs, regardless the ability of the cells to undergo differentiation.     

Arsenic trioxide induces apoptosis in B-cell chronic lymphocytic leukemic cells through down-regulation of survivin via the p53-dependent signaling pathway

Arsenic trioxide (As₂O₃) can induce apoptosis in many tumors. However, the associated mechanisms are not clearly understood. We found that As₂O₃ significantly inhibited the proliferation of WSU-CLL cells and induced apoptosis in dose- and time-dependent manners. WSU-CLL cells treated with 2 μM As₂O₃ showed survivin down-regulation and p53 up-regulation. Survivin siRNA combined with As₂O₃ further inhibited the proliferation of WSU-CLL cells. p53 inhibition by siRNA prevented the down-regulation of survivin by As₂O₃ and prevented the As₂O₃-induced cytotoxicity of WSU-CLL cells. These results suggest that As₂O₃ may be of therapeutic value for chronic lymphocytic leukemia.     

Mechanisms of Interaction Between Interferon Gamma and Antineoplastic Agents on Growth and Differentiation of Leukemic Cells: A Review

INTRODUCTION Combination chemotherapy is commonly used in the treatment of leukemia. The use of biological response modifiers in combination with chemotherapeutic drugs offers a novel therapeutic combination with increased efficacy and attenuation of toxic side effects. The mechanism of interaction between the various biological modifiers themselves and that of the biological modifiers with chemotherapeutic agents is still not completely clear. Interferon gamma (IFNγ) is one of the biological modifiers which exerts both an antiproliferative and differentiating capacity on leukemic cells and also interacts with antileukemic agents in a synergistic manner. Our review summarizes and evaluates the mode of this interaction on leukemic cells.     

Interferon inhibits PWM induced B cell differentiation but not onset of proliferation

The dependence of B lymphocyte differentiation into plasmacytes on anteceding B and T cell proliferation was studied using interferon as a probe. Possible correlations of the effect of interferon on PWM induced T and B cell proliferation and B cell differentiation into either κ or λ light chain immunoglobulin synthesizing plasmacytes have been investigated. The hypothesis that the observed inhibition of the PWM induced formation of plasmacytes by interferon is due to putative enhanced suppressor cell activity resulting from increased T cell proliferation is tested. Human, peripheral blood lymphocytes were exposed to PWM in the presence or absence of human leukocyte interferon. Proliferation was assayed by pulse cytophotometric analysis of cell kinetics, as well as [³H]TdR labelling of S-phase cells. Incidence of plasmacytes was detected by immunofluorescence using κ or λ light chain specific antibody. During continuous [³H]TdR labelling of stimulated cells, interferon inhibited incorporation of precipitable label by 40% at 96 and 144 h. indicating reduced net DNA synthesis by interferon treated cells. The relative fraction of cells in S-phase as well as G₁ ? and G₂ + M- was similar for treated and untreated cells. The fraction of cells rosetting SRBC remained stable for both treated and untreated cells. The size of the interferon treated population was persistently smaller once proliferation began. The time of initiation of proliferation was comparable for treated and untreated cells. Consistent with the findings of others using cell lines, interferon apparently induces a dilation of all cell cycle phases, thereby reducing the rate of proliferation. The same reduction occurred for both T and B cells. Time of initiation of DNA synthesis was, in contrast, not delayed by interferon, suggesting it is specific for events during the proliferative cell cycle. The occurrence of both κ and λ light chain immunoglobulin secreting plasmacytes was inhibited by interferon. The degree of inhibition was comparable for both kinds of plasmacytes detected. While not delaying the onset of DNA synthesis, interferon apparently retards subsequent cell proliferation and inhibits the differentiation of B cells to plasmacytes. The data indicate that active cellular proliferation and B cell differentiation require interferon sensitive events which cells initially recruited from quiescence by PWM do not. The inhibition of the incidence of plasmacytes cannot be attributed to an imbalance of T cell proliferation relative to non-T cells.     

增殖诱导配体和B淋巴细胞刺激因子与B淋巴细胞恶性肿瘤

 肿瘤坏死因子超家族成员增殖诱导配体（APRIL）与B淋巴细胞刺激因子（BlyS）具有较高的同源性。近年来,APRIL／BlyS及其共同受体B细胞成熟抗原（BCMA）和穿膜蛋白活化物（TACI）双受体-双配体系统受到广泛重视。现就APRIL／BlyS及BCMA／TACI双受体-双配体结构、功能及其与B淋巴细胞恶性肿瘤间关系的最新研究进展作简要综述。     

Radiation response of human normal and leukemic hemopoietic cells assayed by in vitro colony formation

The effect of ionizing radiation on the survival of bone marrow cells from patients with acute nonlymphocytic leukemia or from hematologically normal controls was studied using colony formation as an endpoint. A modified agar culture method which incorporated daily feeding with new medium was used to allow the growth of leukemic cell colonies. Analysis of radiation-dose survival curves revealed that normal bone marrow cell populations exhibited a relatively steep slope, with values of D0 ranging from 0.5-1.3 Gy (mean = 0.82 +/- 0.22 Gy). There was essentially no shoulder to the survival curves, with Dq values ranging from less than 0 to 0.3 Gy. The leukemic cells tested displayed survival curves that did not differ qualitatively from those obtained with normal cells, i.e., steep slopes and neglible shoulders. However, the average value of the D0 (0.62 +/- 0.15 Gy) was statistically different (p less than 0.025) than that obtained for the normal cells. The results of these studies may have implications both for the use of radiation therapy for the treatment of malignant hemopoietic diseases, and for total body irradiation prior to bone marrow transplantation.     

Piperazine derivatives of butyric acid as differentiating agents in human leukemic cells

 Butyric acid is a potent antineoplastic agent with a well-documented differentiation activity on a wide variety of tumor cells. However, its clinical development is strongly limited by its very short metabolic half-life. In this study we report on the in vitro effects of new original piperazine derivatives of butyric acid on the induction of differentiation and the growth inhibition of human erythroleukemia K562 cells and myeloid leukemia HL60 cells. 1-(2-hydroxyethyl) 4-(1-oxobutyl)-piperazine (HEPB) and [1-(2-hydroxyethyl) 4-(1-oxobutyl)-piperazine] butyrate (HEPDB) were efficient in acting on the differentiation and proliferation of both cell lines, whereas 1-phenyl 4-(1-oxobutyl)-piperazine (PPB) and 1-(3,4-methylene dioxybenzyl) 4-(1-oxobutyl)-piperazine (POB) acted only on proliferation rates. Such derivatives did not induce significant toxicity in mice. These preliminary results should enable, by the development of new series of piperazine derivatives, a better understanding of the mechanisms of action of butyric acid and its analogues on the coupling of growth and differentiation of neoplastic cells. Received: 1 June 1997 / Accepted: 23 July 1997     

B细胞淋巴瘤干细胞的研究进展

 淋巴瘤干细胞的发生机制十分复杂多样。对于滤泡淋巴瘤（FL）和套细胞淋巴瘤（MCL）来说,有观点认为在骨髓中经V-D-J重排过的淋巴祖细胞（CLP）是其肿瘤干细胞（TSC）的来源;而在弥漫性大B细胞淋巴瘤（DLBCL）和散发性Burkitt淋巴瘤（BL）中,生发中心B细胞是TSC的来源。另一种观点是,表观遗传学改变一次打击使正常造血细胞重新获得干细胞功能,然后经过进一步的染色体易位使这些“前-淋巴瘤干细胞”最终成为淋巴瘤干细胞。分离与鉴定非霍奇金淋巴瘤（NHL）的TSC能够为NHL的发病机制和治疗研究提供新的认识。     

Notch信号通路在B细胞恶性肿瘤中的研究进展

 Notch信号通路是一个进化高度保守的经典信号通路,通过调节细胞的增殖、分化和凋亡而影响细胞的命运。研究表明Notch信号的异常与多种肿瘤的发生发展有关,其在B细胞恶性肿瘤中的作用为双向调节作用,既表现为致瘤的作用,又能抑制肿瘤的发生,同时提示Notch信号可能成为B细胞恶性肿瘤治疗的新靶点。     

Quantitative analysis of bcl-2 expression in normal and leukemic human B-cell differentiation.

Lack of apoptosis has been linked to prolonged survival of malignant B cells expressing bcl-2. The aim of the present study was to analyze the amount of bcl-2 protein expressed along normal human B-cell maturation and to establish the frequency of aberrant bcl-2 expression in B-cell malignancies. In normal bone marrow (n=11), bcl-2 expression obtained by quantitative multiparametric flow cytometry was highly variable: very low in both CD34(+) and CD34(-) B-cell precursors, high in mature B-lymphocytes and very high in plasma cells. Bcl-2 expression of mature B-lymphocytes from peripheral blood (n=10), spleen (n=8) and lymph node (n=5) was significantly higher (P<0.02) in CD23(-) as compared to CD23(+) B cells, independent of the type of tissue analyzed. Upon comparison with normal human B-cell maturation, bcl-2 expression in neoplastic B cells from 144 patients was found to be aberrant in 66% of the cases, usually corresponding to bcl-2 overexpression (63%). Follicular lymphoma (FL) carrying t(14;18) and MALT lymphoma were the only diagnostic groups constantly showing overexpression of bcl-2. Bcl-2 overexpression was also frequently found in precursor B-acute lymphoblastic leukemia (84%), typical (77%) and atypical (75%) B-cell chronic lymphocytic leukemia, prolymphocytic leukemia (two of three cases), mantle cell lymphoma (55%), but not in t(14;18)(-) FL, splenic marginal zone lymphoma, Burkitt lymphoma and multiple myeloma.     

去氢木香内酯对慢性粒细胞白血病K562细胞增殖的影响及其机制

目的探讨去氢木香内酯对慢性粒细胞白血病细胞株K562细胞增殖的影响及其机制。方法用不同浓度去氢木香内酯作用于对数生长期的K562细胞,采用瑞特-吉姆萨染色观察细胞形态,CCK-8法检测细胞增殖情况,流式细胞术检测细胞周期、凋亡情况及细胞表面分化抗原CD14和CD11b表达,蛋白质印迹法检测JAK-STAT通路、细胞凋亡及细胞周期相关蛋白表达。结果不同浓度(4.0、6.0、8.0、10.0、12.0μmol/L)去氢木香内酯作用24h均能抑制K562细胞增殖,与对照组比较,差异有统计学意义(F=109.510,P<0.05)。5.0、10.0μmol/L去氢木香内酯作用24h后,K562细胞凋亡率分别为(16.1±3.8)%、(29.6±4.3)%,较对照组的(3.1±0.5)%升高(F=83.255,P<0.05)。5.0、10.0μmol/L去氢木香内酯作用24h后,K562细胞的G2/M期细胞比例分别为(17.0±3.2)%、(28.8±3.9)%,较对照组的(9.1±2.3)%升高(F=161.598,P<0.05);S期细胞比例分别为(48.1±3.9)%、(61.0±5.4)%,较对照组的(39.6±3.6)%升高(F=192.356,P<0.05)。2.5、5.0μmol/L去氢木香内酯作用72h后,K562细胞的CD14表达率分别为(28.6±3.9)%、(41.1±4.4)%,较对照组的(3.1±0.5)%升高(F=132.811,P<0.05);K562细胞的CD11b表达率分别为(42.4±5.0)%、(61.2±5.7)%,较对照组的(4.2±1.1)%升高(F=179.553,P<0.05)。去氢木香内酯能够降低JAK2、STAT5、cyclinE、CDK2、cyclinA、CDC25C、cyclinB1、CDK1及bcl-2蛋白表达,上调p21及bax蛋白的表达。结论去氢木香内酯能够抑制慢性粒细胞白血病K562细胞增殖,可能是通过细胞周期阻滞、诱导凋亡及分化而实现的。     

Interferon therapy in disseminated renal cell carcinoma

Twenty patients with pulmonary metastases of renal cell carcinomas have been treated in a randomized study with either leucocyte interferon in daily doses of 3 x 10(6) I.U.i.m or irradiation of both lungs in a calculated mean central dose of 10 Gy in 4 weeks in combination with bleomycin and vincristine. Most patients had a short time to progression of their disease. Three patients in the interferon group had complete response (CR) and partial response (PR) and one patient in the other group had PR. The numbers of patients in both arms of the study are too small to allow any conclusions whether beneficial effects are more frequent in one group than in the other, but there is a more favourable tendency in the interferon group.     

外泌体在B细胞肿瘤中的研究进展

外泌体是一种可以携带源细胞多种信息成分,直径30~130 nm的一种生物活性小囊泡。大量研究发现外泌体是由肿瘤细胞或肿瘤微环境细胞分泌的,并通过相互作用影响B细胞肿瘤的增殖、血管生成、耐药和免疫逃逸。同时,外泌体也可作为一种新兴的生物学标志物,用于B细胞肿瘤的诊断和预后判断。另外,外泌体作为天然、无免疫源性的药物载体具有良好的治疗潜能。文章就着重阐述外泌体在B细胞肿瘤诊疗中的研究进展。     

甲异靛对白血病骨髓基质细胞调节白血病细胞增殖的影响

 【摘要】　目的　探讨甲异靛对白血病骨髓基质细胞干预白血病细胞增殖的影响。方法 　利用白血病骨髓单个核细胞培养骨髓基质细胞,并建立白血病细胞和骨髓基质的共培养体系。用锥虫蓝拒染实验测定甲异靛对共培养体系中白血病细胞增殖的影响;流式细胞术检测白血病细胞膜上CXCR4的表达。结果　白血病骨髓基质细胞可抑制白血病细胞的增殖。低浓度的甲异靛（5 μmol／L）可促进白血病和骨髓基质细胞共培养体系中白血病细胞的增殖。白血病细胞膜异常高表达CXCR4,甲异靛可明显抑制HL-60细胞和原代白血病细胞膜上CXCR4的表达,骨髓基质细胞可以促进白血病细胞膜上CXCR4的表达。结论　甲异靛可能通过下调白血病细胞膜上CXCR4的表达起抗白血病作用。     

PI3K signaling pathway in normal B cells and indolent B-cell malignancies

In chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphomas (NHLs), B-cell receptor signaling leads to activation of the phosphatidylinositol 3-kinase (PI3K) pathway. Idelalisib, a PI3Kδ inhibitor was approved in 2014 by the US Food and Drug Administration (FDA) in combination with rituximab for the treatment of patients with CLL for whom single-agent rituximab would be considered appropriate and as a single agent for patients with relapsed small lymphocytic lymphoma (SLL) and relapsed follicular lymphoma (FL). Following its approval, several trials investigating various PI3Kδ inhibitors as single agents or in combination with chemoimmunotherapy or other molecular targeted agents in CLL and indolent NHL (iNHL) have uncovered some severe autoimmune related toxicities. This review discusses and summarizes the biologic basis and the clinical experience of the PI3Kδ inhibitors in indolent B-cell malignancies.     

CGI-100 specific shRNA inhibits proliferation and induces differentiation in leukemia K562 cells

Objective: To investigate the characteristics of CGI-100- knockdown K562 cells and the effect of CGI-100 RNA interference (RNAi) on matrine-treated K562 cells.Methods: Three oligonucleotides targeting CGI-100 gene and a pair of negative control containing the same nucleotide composition with a different sequence were devised and chemically synthesized. The inhibition efficiency of CGI-100 expression by shRNA-CGI-100 in K562 cells was determined using semiquantitative RT-PCR and dot blot hybridization. The effect of CGI-100 RNAi on the growth of K562 cells was examined using MTT assay and cell differentiation was measured by distinct approaches including flow cytometry, benzidine staining and electron microscope. After CGI-100-konckdown K562 cells were incubated with 0.2 mg/ml of matrine or 30 μmol/L of hemin for 48 h, the expression levels of Glycophorin A(GPA)(CD235a) and Growth factor independence-1B mRNA(Gfi-1B mRNA) were measured by RT-PCR and the protein levels of GPA, CD14 and CD15 were detected by flow cytometry.Results: The eukaryotic expression vectors of CGI-100 RNAi were successfully constructed. The K562/shRNA-CGI-100 cell line was established in which the inhibition efficiency of CGI-100 gene expression by shRNA-CGI-100 was 54%. CGI-100-knockdown inhibited the proliferation and induced erythroid differentiation in K562 cells. Compared with the control K562 ceils, the K562/shRNA-CGI-100 cells showed decreased absorbance value detected by MTT assay, decreased enchromation, increased heterochromation, increased percentage of G0/G>1 phase cells, decreased population of S phase cells, decreased PI (proliferation index of cells), and elevated percentage of benzidine-positive cells. Moreover, the sensitivity of K562/shRNA-CGI-100 cells to either matrine or hemin was enhanced and the sensitivity of these cells to matrine was higher than that to hemin. Compared with the control K562 cells, matrine treatment in K562/shRNA-CGI-100 cells resulted in increased inhibitory rate of proliferation, elevated percentage of benzidine-positive cells, obviously up-regulated mRNA expressions of GPA and Gfi-1B, and increased mean fluorescence intensity (MFI) of GPA. No CD14 expression was detected and no statistical significance was found for the detected CD15. Finally, the MFI of GPA increased in K562/shRNA-CGI-100 cells treated with hemin and was 1.7 times less than that in cells exposed to matrine.Conclusion: These results suggest that the function of CGI-100 gene is correlated with the deregulated proliferation and the block of erythroid differentiation in K562 cells and may also be involved in matrine-induced erythroid differentiation in K562 cells.     

Glucocorticoid receptors of normal and leukemic cells: role of proliferation conditions

A published whole-cell binding assay of 3H-dexamethasone (3H-DEX) was combined with cell sedimentation analysis to investigate various factors influencing specific binding of glucocorticoids by leukemic lymphoblasts. Studies with mouse L1210 and human HL-60 cell lines revealed that glucocorticoid (GC) receptors accumulate during G1 phase of the cell cycle. In human lymphoblastic leukemia, the per cell receptor number was highest in cells in S and G2 phase and lowest in small, noncycling cells. In normal human white blood cells, GC receptor content was maximal in large lymphocytes and monocytes while no receptors were present in small lymphocytes. However, the receptor density of large lymphocytes was still 3-4 fold lower than of leukemic lymphoblasts of similar size. In L1210 cells the number of GC receptors decreased considerably in stationary cultures or following inhibition of cell cycle progression by dibutyryl cyclic AMP (dbc-AMP). Receptor content was also reduced in cells growing as ascites tumors in hosts with terminal disease. Accordingly, the receptor content in leukemic blasts appeared highly dependent on cell cycle distribution and on the proliferative status of the tumor cells. These findings may in part explain the large interpatient variation and the conflicting views regarding GC receptor content and prognosis in acute lymphoblastic leukemia.     

Blood lymphocyte characteristics as predictors of prognosis in chronic lymphocytic leukemia of B-cell type

The prognostic information of blood lymphocyte characteristics and clinical findings was assessed in 62 patients with chronic lymphocytic leukemia of B cell type. Bivariate and multivariate survival analyses were performed using age, Rai stage, surface membrane immunoglobulin (smIg) isotype pattern of the leukemic clone, total lymphocyte counts, numbers of proliferating lymphocytes and T cell subpopulations. Rai stages III and IV, high numbers of blood lymphocytes in S-phase (S+) and sm mu isotype were found to be partly independent factors predicting short therapy-free survival. Patients with a sm mu+ leukemic cell clone had a shorter therapy-free and total survival compared to those with sm mu+/delta+ and sm delta+ leukemic cells. Moreover, patients with high numbers of blood S+ lymphocytes had a shorter therapy-free and total survival compared to those with few S+ cells. These prognostic variables were valid also in patients with a low tumour burden (Rai stages 0, I and II) and may thus be of clinical importance as a guideline for therapeutic intervention.