Adenosine 3′,5′-monophosphate protein kinase isoenzyme distribution in lymphocytes from normal individuals and patients with chronic lymphocytic leukaemia

Two major isoenzymic forms of adenosine 3′,5′-monophosphate (cyclic AMP)-dependent protein kinase were resolved by DEAE cellulose chromatography of cytosols of normal human lymphocytes and of abnormal (colchicine ultrasensitive) lymphocytes from patients with chronic lymphocytic leukaemia (CLL). With only one exception the activity of type I protein kinase exceeded that of type II in normal lymphocytes, whilst in CLL the activity of the type II isoenzyme exceeded that of the type I. The total activity of protein kinase was approximately the same in normal and CLL lymphocytes. This difference in isoenzyme distribution may reflect the immaturity of CLL lymphocytes.     

Properties of lymphocyte 5'nucleotidase in normal subjects and patients with chronic lymphocytic leukemia

Heterogeneity for 5'nucleotidase has been demonstrated in chronic lymphocytic leukemia [12]. The enzyme is not detectable in the lymphocytes from the majority of patients with this disorder, but normal and even supranormal levels are found in some cases [17]. In the present studies, the properties of this ecto-nucleotidase were investigated in unhomogenized lymphocytes isolated from the blood of normal subjects and patients with chronic lymphocytic leukemia. The activity was found to have a pH optimum of 8.0-8.5 and a preference of 5'ribo- over 5'deoxyribonucleotides. The enzyme was inactive towards 2',3'AMP. The Km for AMP showed a broad range from 19 to 210 microM in unhomogenized lymphocytes. An unexpected relationship was observed between sp. act. and this Km in that higher Km values were noted with cells from subjects with high lymphocyte 5'nucleotidase sp. act. and low Km values in lymphocyte preparations with low sp. act. When plasma membranes were prepared, a narrow range of low Km values unrelated to sp. act. was noted. The change in Km was not due to Pi concentration or nucleotide effects. The ecto-enzyme was inhibited by alpha, beta-methylene adenosine diphosphate, while cytosol phosphatase activity was not inhibited by this compound. Heat stability studies revealed a 45% loss in 5'nucleotidase activity after incubation for 20 min at 65 degrees C. A comparison of the substrate preference, Km values, effect of inhibitor and subcellular localization revealed no differences between the enzyme from patients with chronic lymphocytic leukemia and from normal subjects. This finding is consistent with the suggestion that the undetectable or supranormal levels observed in the lymphocytes of patients with this disorder stems from variations in the percentage of cells having 5'nucleotidase rather than changes in the enzyme proper.     

γ-Linolenic Acid Induces Apoptosis in B-Chronic Lymphocytic Leukaemia Cells in Vitro

Gamma linolenic acid (GLA) is cytotoxic to many types of human cancer cells. Most chemotherapeutic agents are cytotoxic by inducing apoptosis. We examined the apoptotic activity of GLA on purified B-cells isolated from patients with B-cell chronic lymphocytic leukaemia (B-CLL) and from normal individuals. GLA significantly increased the degree of apoptosis in B-CLL B-cells after 24 hours of culture. The mean percentage of cells undergoing apoptosis when cultured in medium alone (spontaneous apoptosis) was 20% (range: 7 to 31%) (n=25) and in the presence of GLA (5μg-60μg) was: 42%-95%. In the presence of GLA 5μg/ml and dexamethasone the degree of apoptosis was 86% (range: 72 to 100%). GLA induced apoptosis in B-CLL B-cells at a higher level than that observed with normal B-cells at all lower concentrations tested 5, 10 and 15μg/ml: P=0.045; 0.027 and 0.022, respectively. At 30μg/ml of GLA, no significant difference in the percentage of cells displaying apoptosis between B-CLL and normal B-cells was observed (P=0.075). GLA induced apoptosis in B-CLL T-cells at both 10 and 30μg/ml. The degree of apoptosis in normal T-cells with GLA was also significant at the higher concentration of 30μg/ml. Interleukin 4 (IL4), a viability factor in B-CLL, and vitamin E, an anti-oxidant, protected B-CLL B-cells against GLA (20μg/ml)-induced apoptosis. These results demonstrate that GLA induces apoptosis in B-CLL B-and T-cells cells in-vitro and that they are more susceptible to GLA-induced apoptosis than normal peripheral blood B-and T-cells.     

High levels of 5′-nucleotidase activity in blastic chronic myelogenous leukemia with common all-antigen

The activity of 5′-nucleotidase (5′-N) on the surface of leukemia cells was determined in 44 patients with blast crisis of chronic myelogenous leukemia (CML) and in 52 patients with acute myeloblastic (AML) or acute lymphoblastic (ALL) leukemia. The phenotype of blast cells was classified according to immunological surface markers, including the common ALL-antigen (cALLA). High 5′-N activities were found in the presence of cALLA, whereas low to undetectable levels were characteristic for cALLA-negative leukemias, the difference being highly significant (p 0.0001). While there was a certain overlap of 5′-N levels between cALLA-positive ALL and cALLA-negative AML, no overlap of 5′-N was observed between cALLA-positive lymphoid and cALLA-negative myeloid blast crisis of CML. Thus, 5′-N affords an additional easily-testable biochemical marker that may have its diagnostic and prognostic value especially in the case of blastic chronic myelogenous leukemia.     

Lymphocytes aminergic binding changes in chronic lymphocytic leukaemia

β-Adrenergic and dopaminergic binding were studied on intact normal human lymphocytes and abnormal lymphocytes from patients with chronic lymphocytic leukaemia (CLL). For both aminergic receptors, a significant difference was found between the two groups. Our results suggest that membrane alterations in the unregulated cellular proliferation may modify several hormone receptors.     

Rituximab anti-CD20 monoclonal antibody induces marked but transient reductions of peripheral blood lymphocytes in chronic lymphocytic leukaemia patients

Rituximab has been recently proposed as an effective non-chemotherapeutic option for patients with follicle centre lymphoma (FCL). However, less is known on its role in chronic lymphocytic leukaemia (CLL). We thus decided to assess its effectiveness on a panel of 7 patients with refractory or relapsed CLL. Mild (5 patients) or severe (1 patient) adverse reactions were observed during the first hours of Rituximab infusion, almost exclusively at the first course. Symptoms rapidly subsided with temporary drug withdrawal and low dose steroids. All patients could receive the whole scheduled treatment. A striking reduction of peripheral blood (PB) lymphocyte counts was observed in all patients (median 93%; range 57–99%). However, Rituximab was poorly effective towards nodal and splenic disease. Patients required additional treatment after a median time of 70 d (range: 20–180 d). Our data show that Rituximab delivery in CLL patients is feasible and has an acceptable toxicity, although it probably does not represent an ideal treatment option when delivered using schedules originally designed for FCL patients. However, responses observed at PB level suggest that Rituximab has an activity which is not negligible and deserves further investigation in CLL. Future approaches will be directed to the development of alternative schedules which may include dose intensification, combination of Rituximab and chemotherapy, andin vivo purging of periphral blood progenitor cell harvests for autografting procedures.     

Extracellular 5′‐nucleotidase (CD73) promotes human breast cancer cells growth through AKT/GSK‐3β/β‐catenin/cyclinD1 signaling pathway

To identify the role and to explore the mechanism of extracellular 5′‐nucleotidase (CD73) in human breast cancer growth, CD73 expression was measured firstly in breast cancer tissues and cell lines, and then interfered with or over‐expressed by recombinant lentivirus in cell lines. Impacts of CD73 on breast cancer cell proliferation and cell cycle were investigated with colony formation assay, CCK‐8 and flow cytometry. The relationship between CD73 and AKT/GSK‐3β/β‐catenin pathway was assessed with adenosine, adenosine 2A receptor antagonist (SCH‐58261), adenosine 2A receptor agonist (NECA), CD73 enzyme inhibitor (APCP) and Akt inhibitor (MK‐2206). Moreover, the effect of CD73 on breast cancer growth in vivo was examined with human breast cancer transplanting model of nude mice. The results showed that the expression of CD73 was high in breast cancer tissues and increased with advanced tumor grades and lympho‐node status. CD73 expression was higher in more malignant cells, and CD73 overexpression promoted breast cancer cell proliferation in both in vivo and in vitro. It activated AKT/GSK‐3β/β‐catenin/cyclinD1 signaling pathway through CD73 enzyme activity and other mechanism.     

The fatty acyl chain composition of human normal and leukaemic lymphocytes and its modulation by specialised hydrogenation

Thirty species of fatty acyl chain have been quantitatively identified in human normal peripheral blood lymphocytes (four donors) and lymphocytes circulating in eight patients with chronic lymphocytic leukaemia (CLL). Towards the aim of influencing cell behaviour by lowering membrane fluidity, reaction conditions for catalytic hydrogenation at physiological temperature and pH have been established that effect reduction of the unsaturated species, and preferentially the polyunsaturated forms, but this has not yet been accomplished without killing the cells. That saturation of ethylenic linkages per se is the cause of death is indicated by separate findings showing that the lymphocytes are capable of withstanding hydrogen gas at the requisite high pressure (9 atm.) or exposure alone to the rhodium catalyst [chlorotris (sodium diphenylphosphinobenzene-m-sulphonate)-rhodium(I) tetrahydrate]. It remains feasible that future use of these two agents in combination under milder conditions to produce much lower degrees of hydrogenation than those reported here will permit the cells to survive.Concerning fatty acyl chain composition, the lymphocytes from most of the patients exhibited an inversion in the level of palmitic and stearic acid. A consistently abnormal pattern exhibited by the patients was a rise in oleic acid and a fall in arachidonic acid content. This same alteration has been demonstrated elsewhere in transformed/neoplastic cell types and hence it could well represent phenotypic expression in the CLL lymphocyte of malignant change.Fatty acyl chain composition remained unchanged in lymphocytes reconstituted after cryopreservation in liquid nitrogen.     

脐血来源CIK细胞的体外扩增培养

目的 研究人脐血来源的细胞因子诱导的杀伤细胞（cytokine-induced killer,CIK）体外培养扩增,并检测其功能。方法 应用Ficoll-Hypaque离心获得界面细胞,贴壁培养2h,获得悬浮单个核细胞,体外以重组人白介素-1、重组人白介素-2、γ-干扰素和CD3抗体诱导培养15d。在CIK发育过程中,在光镜下观察其生长情况,应用流式细胞仪检测CIK表型。采用MTT法检测CIK对肿瘤细胞的杀伤性。结果 CIK前3d细胞扩增不明显,在培养4d后,细胞增殖,呈团,可观察到不规则形的细胞．细胞体积增大,胞质少、胞核大、圆．有时可观察到细胞分裂相。培养12d后CIK细胞高表达CD3^＋CD56^＋,CD3^＋CD8^＋细胞缓慢增长,CD3、CD4细胞有所增加,在d7之后有所下降,CD3^＋细胞维持高水平且变化不明显。CIK细胞对2种来源于不同组织的肿瘤细胞均产生了明显的杀伤性。结论 人脐血经重组人白介素-1、重组人白介素-2、γ-干扰素和CD3抗体体外诱导培养,能诱导出CIK,并对恶性肿瘤细胞有明显的杀伤活性。     

Phorbol ester-induced loss of colchicine ultrasensitivity in chronic lymphocytic leukaemia lymphocytes

On exposure to the phorbol ester 12-O-tetradecanoyl-13-acetate (TPA) the pathological (non-dividing) lymphocytes of B-cell chronic lymphocytic leukaemia (CLL) lose their characteristic ultrasensitivity to the cytocidal action of colchicine in vitro. They are no longer killed in 1 day by the drug at 10(-6)M-concentration. The effect was the same whether the cells were incubated in the continuous presence of TPA, or subjected instead to pulse-treatment with it (for as little as 5 min.). Colchicine at one thousand times greater concentration was now needed to kill the cells. CLL lymphocytes already primed to undergo interphase death by pretreatment with colchicine could be prevented from doing so by early addition of TPA. A marked proportion of those CLL lymphocytes destined to undergo early spontaneous death in vitro in the absence of colchicine could be prevented from doing so by TPA. The loss of colchicine ultrasensitivity applied to cells which had not yet undergone TPA-induced morphological transformation to blast-like cells or differentiation to cells containing abundant cytoplasmic immunoglobulins (CIg). These transformed cells materialised in greatest incidence (70-80%) after 3 days of culture, an observation in agreement with others workers.     

石斛多糖增强脐带血和肿瘤病人外周血LAK细胞体外杀伤作用的研究

目的：探讨石斛多糖（ｄｅｎｄｒｏｂｉｕｍ ｃａｎｄｉｄｕｍ ｐｏｌｙｓａｃｃｈａｒｉｄｅ，ＤＣＰ）与ｒＩＬ－２联合应用对脐带血ＬＡＫ细胞（ＣＢ－ＬＡＫ）和肿瘤病人外周血ＬＡＫ细胞（ＰＢ－ＬＡＫ）体外杀伤肿瘤细胞作用的影响。方法：应用＾３Ｈ－ＴｄＲ释放法测定ＣＢ－ＬＡＫ和ＰＢ－ＬＡＫ细胞的杀伤活性。结果：①ｒＩＬ－２（５００ｕ／ｍｌ）能明显提高ＣＢ－ＬＡＫ对Ｒａｊｉ细胞的杀伤活性，０ｈ的ＣＢ－ＬＡＫ活性为１３．６３％，ｒＩＬ－２（５００ｕ／ｍｌ）诱导４８、７２、９６、１２０ｈ后，ＣＢ－ＬＡＫ活性分别提高至２２．２８％、２６．２３％、２８．５５％和２５．７６％（Ｐ〈０．０１）；②ＤＣＰ（１００ｍｇ／Ｌ或４００ｍｇ／Ｌ）与ｒＩＬ－２（５００ｕ／ｍｌ）联合诱导ＣＢ－ＬＡＫ的活性，比单纯ｒＩＬ－２（５００ｕ／ｍｌ）的作用     

Cholesterol content and fluidity of normal human and chronic lymphocytic leukaemia lymphocytes in relation to serum cholesterol level

No significant difference was found between a group of healthy individuals and a group of patients with unequivocal chronic lymphocytic leukaemia (CLL) in either the cholesterol content or the fluidity of the circulating lymphocytes, there existing similar degrees of variation within each group. Serum cholesterol level (free and total) varied considerably among the donors, but there was no significant difference between the two groups. Moreover, lymphocyte cholesterol content did not correlate with serum cholesterol level. Furthermore, blood lymphocyte count varied independently of serum cholesterol level.

Cholesterol was quantitatively determined by a specific microanalytical procedure involving gas-liquid chromatography. The fluidity of living lymphocytes was measured as the degree of fluorescence depolarisation of the probe molecule diphenylhexatriene. The lymphocyte preparations were free from other cell types and could be considered representative of the populations in the whole bloods. Cryopreserved samples showed slight losses in cholesterol.

These findings on all three counts contradict the evidence on which Inbar and Shinitzky [21] advanced their hypothesis, invoking cholesterol as a bioregulator of CLL through its influence on the surface membrane fluidity of lymphocytes. They do not, however, exclude the possible influence of cholesterol on the clinical and haematological behaviour of individual patients.     

大连地区母血和脐血铊浓度及其相关的影响因素

目的：检测分析大连地区母血和脐血中铊浓度及其相关影响因素,为孕期合理保健提供资料。方法：收集125对母血及脐血,采用电感耦合等离子体_质谱（ICP_MS）法测定其血中铊的浓度,同时就孕妇家庭和社会环境等因素对血铊浓度的影响进行单因素分析和多元线性回归分析。结果：大连地区母血和脐血中铊的浓度分别为48.8±29.9ng/L和42.0±29.2ng/L,两者间的差异具有统计学意义（P〈0.05）,且母血和脐血的铊浓度之间呈正相关（r=0.731）。母血铊浓度因居住地和饮水来源、有无被动吸烟、有无妊娠并发症、是否近期家庭装修等因素的不同而存在差异;多元线性回归分析结果显示,其中饮水来源、有无被动吸烟和有无妊娠并发症等3个因素进入回归方程。结论：大连地区孕妇及其胎儿均受到铊暴露,居住地、妊娠并发证、被动吸烟、饮水来源、家庭装修等因素均影响母血铊浓度。     

脐血CIK细胞的体外扩增特性研究

目的动态观察脐血CIK细胞体外扩增特性及其表面抗原变化,并对其细胞毒活性进行检测。方法提取健康足月产妇的胎儿脐带血单个核细胞,第0天加入rh-IFN-γ,第1天加入rhIL-2、抗CD3单克隆抗体来诱导培养CIK细胞,以后每3d更换培养液1次,并补加rhIL-2及抗CD3McAb。用流式细胞仪动态检测培养物的免疫表型变化,同时进行细胞计数,并使用MTT法检测各阶段脐血CIK细胞的细胞毒活性。结果脐血CIK细胞在培养2￣3周后大量增生,扩增约(64.4±16)倍,其中CD3+CD5+6细胞扩增约900倍,是CIK的主要效应细胞,且CD+3CD+8的T细胞达(74.7±10.42)%,CD2+5细胞比例也明显增加,而CD3+4细胞比例却减少。另外CD+3CD1+6CD5+6细胞比例升高最显著;脐血CIK细胞对白血病细胞株K562,HL-60及原代白血病细胞有较高的杀伤活性。结论脐血单个核细胞在体外可以被培养为CIK细胞,且扩增潜能极大,扩增倍数极高,细胞毒活性强,脐血CIK细胞有望应用于恶性肿瘤的生物免疫治疗。     

肿瘤患者外周血淋巴细胞化疗药物敏感性检测的应用研究

目的：探讨肿瘤患者外周血淋巴细胞对临床常用化疗药物的敏感性,筛选个体敏感的化疗药物,指导临床用药。方法：采用MTT法检测154例肿瘤患者外周血淋巴细胞对常用15种化疗药物的敏感性。结果：本方法测定的结果与临床常规化疗的结果基本一致。结论：MTT法是一种简便、快速、可行的体外药敏实验方法,可为肿瘤患者化疗提供重要的实验依据。     

B-CLL cell surface markers and mitogen-induced thymidine uptake: a comparison between lymph node cells and peripheral blood lymphocytes in 25 patients

Lymphocytes from peripheral blood and lymph nodes from 25 patients with B-CLL were analysed by immunofluorescent staining of surface membrane immunoglobulin (SmIg), the B-cell marker B1, and the T-cell markers Leu 1/2/3/4. Tritium-thymidine uptake was measured in mitogen-stimulated 4-day cultures. Differences in these parameters between cells from the two sources in each patient were calculated with the paired T-test. All cell samples showed a clonal B-cell population. More blood lymphocytes than lymph node cells expressed the monoclonal SmIg (mu or gamma, p = 0.04; kappa or lambda, p less than 0.01), and T-cells were more frequent in lymph nodes (p = 0.01), where the T helper/suppressor ratio was higher. Furthermore, lymph node cells showed a higher thymidine uptake in response to the mitogens LPS, PWM, and Cowan (p = 0.01, p = 0.01, and p = 0.004, respectively), but there was no difference in the responses to EBV, DxS, TPA and PHA (p greater than 0.5). The higher response in lymph node cells to some mitogens might in part be explained by differences in the numbers of accessory cells, such as T-helper cells, but also by the existence of leukaemic B-cell subsets with different mitogen response patterns and different distributions within the lymphoid compartments. The characterization of subsets within a malignant cell clone might be of clinical importance.     

Blockade of MEK signaling potentiates 5‐Aza‐2′‐deoxycytidine‐induced apoptosis and upregulation of p21waf1 in acute myelogenous leukemia cells

We have recently reported that the mitogen‐activated protein kinase/ERK kinase (MEK) inhibitor AZD6244 (ARRY‐142886) strikingly potentiated the effects of histone deacetylase inhibitor to induce growth arrest and apoptosis of acute myelogeneous leukemia (AML) cells in association with enhanced upregulation of p21^waf1. This study examined the effects of the MEK inhibitor on the action of DNA methyltransferase inhibitor 5‐Aza‐2′‐deoxycytidine (5‐AzadC), another epigenetic agent in AML cells. AZD6244 significantly potentiated the ability of 5‐AzadC to induce growth arrest and apoptosis of NB4, and freshly isolated AML cells. In parallel, 5‐AzadC induced expression of p21^waf1 in AML cells, which was potently enhanced in the presence of AZD6244. Further studies explored the molecular mechanisms by which 5‐AzadC induced expression of p21^waf1 and found that a low dose of 5‐AzadC (1 μM) induced acetylation of histone H3 on the p21^waf1 gene promoter; however, higher dose of this compound (3 or 5 μM) potently induced DNA damage as assessed by expression of γH2AX, in NB4 cells. These effects were strikingly enhanced by concomitant blockade of MEK signaling. Furthermore, knock‐down of p21^waf1 by the siRNA rescued NB4 cells from 5‐AzadC‐mediated growth inhibition. Taken together, combination of 5‐AzadC and the MEK inhibitor may be useful for treatment of individuals with a subset of AML. © 2009 UICC     

5′nucleotidase,adenosine deaminase and purine nucleoside phosphorylase activities in acute leukaemia

Three enzymes concerned in purine degradation, 5′nucleotidase (5′NT), adenosine deaminase (ADA) and purine nucleoside phosphyorylase (PNP) have been measured biochemically in the bone marrow or peripheral blood blasts from 75 patients with acute leukaemia, from 18 patients with blast crisis of chronic granulocytic leukaemia and in the bone marrow and peripheral blood lymphocytes from 14 normal donors. Characteristic patterns among the different sub-types of acute leukaemia have been detected, with high ADA, low 5′NT and PNP in Thy-ALL, high 5′NT and ADA in c-ALL, high PNP and low ADA in AML. The cells in CGL blast transformation resembled the enzymatic pattern of either AML or c-ALL respectively. However, no significant correlation was found between any pair of enzymes in any group of leukaemia, normal bone marrow or peripheral blood lymphocytes studied here.     

The later administration of progesterone more rapidly activates dormant mouse mammary tumor cells initiated by 3′-methyl-4-dimethylaminoazobenzene

Implantation of progesterone at 1 month of age induced the development of mammary tumors in female C57BL/6 × DS-F₁ mice that had been treated with 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB) neonatally, and that had undergone ovariectomy and received implants of estradiol-17β (E₂) pellets at 1 month of age, and the incidence of mammary tumors became 100% at 15 months of age. On the other hand, no mammary tumors developed in these mice with implants of E₂ pellets alone. Implantation of progesterone alone also induced no mammary tumors in mice that had been treated with 3′-Me-DAB neonatally, and had undergone ovariectomy at 1 month of age. Implantation of progesterone at 4, 6, 8, and 10 months of age also caused the prompt development of mammary tumors as implantation of progesterone at 1 month of age. When ages at which the incidence became 50% were estimated on curves of the incidences, these ages on implantation of progesterone at 1, 4, 6, 8, and 10 were about 11, 13, 14, 14, and 14 months of age. These results suggest that progesterone together with estrogen promotes the development of mammary tumors induced by 3′-Me-DAB, and that the later progesterone is administered, the more rapidly it activates dormant mammary tumor cells initiated by 3′-Me-DAB.