Proteasome inhibitors induced caspase-dependent apoptosis and accumulation of p21WAF1/Cip1 in human immature leukemic cells

The 26S proteasome is a non-lysosomal multicatalytic protease complex for degrading intracellular proteins by ATP/ubiquitin-dependent proteolysis. Tightly ordered proteasomal degradation of proteins critical for cell cycle control implies a role of the proteasome in maintaining cell proliferation and cell survival. In this study, we demonstrate that cell-permeable proteasome inhibitors, lactacystin, benzyloxycarbonyl(Z)-leucyl-leucyl-leucinal (ZLLLal; MG-132) and 4-hydroxy-5-iodo-3-nitrophenylacetyl-leucyl-leucyl-leucine vinyl sulfone (NLVS), induce apoptosis abundantly in p53-defective leukemic cell lines CCRF-CEM, U937 and K562 as well as in myelogenic and lymphatic leukemic cells obtained from adult individuals with relapsed acute leukemias. Leukemic cell apoptosis induced by the proteasome inhibitors was dependent on activation of caspase-3 and related caspase family proteases, because caspase-3 inhibitor N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartal (Ac-DEVD-cho) and, more effectively, the general caspase-inhibitor N-benzyloxycarbonyl-L-valyl-L-alanyl-L-aspartate fluoromethylketone (Z-VAD-fmk) were capable of blocking apoptosis induced by lactacystin, ZLLLal or NLVS. Induction of apoptosis by lactacystin or ZLLLal was accompanied by cell cycle arrest at G2/M phase and by accumulation and stabilization of cyclin-dependent kinase inhibitor p21WAF1/Cip and tumor suppressor protein p53. A role of p53 in mediating apoptosis or induction of p21WAF1/Cip1 was ruled out since CCRF-CEM and U937 cells express non-functional mutant p53, and K562 cells lack expression of p53. Viability and hematopoietic outgrowth of human CD34+ progenitor cells treated with lactacystin were slightly reduced, whereas treatment of CD34 + cells with ZLLLal or the cytostatic drugs doxorubicin and gemcitabine resulted in markedly reduced viability and hematopoietic outgrowth. These results demonstrate a basic role of the proteasome in maintaining survival of human leukemic cells, and may define cell-permeable proteasome inhibitors as potently anti-leukemic agents which exhibit a moderate hematopoietic toxicity in vitro.     

Direct binding of antithymoctye globulin to haemopoietic progenitor cells in aplastic anaemia

Antithymocyte globulin (ATG) is widely used in the treatment of aplastic anaemia (AA) and a response occurs in 60-80% of patients. However, its exact mechanism of action in the treatment of AA has yet to be determined. Previously, we have shown that ATG increases colony growth from purified bone marrow CD34+ cells of AA patients in vitro, and decreases stem cell apoptosis and the expression of soluble Fas receptor after ATG therapy in vivo. The aim of this study was to further examine the association of ATG with AA haemopoietic progenitor cells. We describe here that ATG bound directly to CD34+ cells. Forty-six patients and 20 normal control subjects were studied. ATG bound to CD34+ cells in normal control subjects (mean 90.38%) as determined by flow cytometry. The mean percentage of CD34+ cells binding to ATG was 59.90% in untreated aplastic patients, 83.24% in partial responders, 58.3% in non-responders and 62.73% in relapsed patients. In completely recovered patients, ATG binding was indistinguishable from control subjects. The functionality of AA patients' haemopoietic progenitor cells was assessed using colony assays. These results demonstrate the direct binding of ATG to CD34+ cells and suggest that differences in its binding to AA CD34+ cells could reflect functional differences in the haemopoietic stem cell compartment throughout the disease process.     

Efficient retroviral transduction of human B-lymphoid and myeloid progenitors: marked inhibition of their growth by the <Emphasis Type="Italic">Pax5</Emphasis> transgene

We applied a coculture system for the genetic manipulation of human B-lymphoid and myeloid progenitor cells using murine bone marrow stromal cell support, and investigated the effects of forced Pax5 expression in both cell types. Cytokine-stimulated cord blood CD34⁺ cells could be transduced at 85% efficiency and 95% cell viability by a single 24-h infection with RD114-pseudotyped retroviral vectors, produced by the packaging cell line Plat-F and bicistronic vector plasmids pMXs-Ig, pMYs-Ig, or pMCs-Ig, encoding EGFP. Infected CD34⁺ cells were seeded onto HESS-5 cells in the presence of stem cell factor and granulocyte colony-stimulating factor, allowing the extensive production of B progenitors and granulocytic cells. We examined the cell number and CD34, CD33, CD19, and CD20 lambda and kappa expressions by flow cytometry. Ectopic expression of Pax5 in CD34⁺ cells resulted in small myeloid progenitors coexpressing CD33 and CD19 and inhibited myeloid differentiation. After 6 weeks, the number of Pax5-transduced CD19⁺ cells was 40-fold lower than that of control cells. However, the expression of CD20 and the κ/λ chain on Pax5-transduced CD19⁺ cells suggests that the Pax5 transgene may not interfere with their differentiation. This report is the first to describe the effects of forced Pax5 expression in human hematopoietic progenitors.     

不同来源的CD34^＋细胞归巢相关分子的表达

目的 研究不同来源CD34^＋细胞归巢相关分子（HRM）的表达情况。方法采用免疫磁珠法（MACS）分选不同来源的CD34^＋细胞，免疫荧光标记流式细胞仪测定HRM的表达。结果骨髓（BM）、动员后的外周血（mPB）及脐血（UCB）来源的CD34^＋细胞均高表达细胞黏附分子CD49d、CD49e、CD54、CD11a、CD62L、CD44、CD31。UCB来源的CD34^＋细胞表面表达的细胞黏附分子中，CD49e表达显著低于BM和mPB来源的CD34^＋细胞（P〈0．05），CD54表达显著低于mPB来源者（P〈0．05），CXCR4表达显著低于BM来源者（P〈0．05）。结论UCB来源的造血干／祖细胞归巢能力低可能是UCB移植造血重建延迟的原因之一。     

P27基因在原发性肝癌中的表达及其与预后的关系

目的　探讨 P2 7在原发性肝癌中表达 ,揭示其与原发性肝癌临床病理指标和预后的关系。方法　采用免疫组化二步法 ,检测 4 3例原发性肝癌标本、2 1例肝硬化标本和 16例正常肝脏组织中的 P2 7表达情况 ,并对2 9例肝癌患者进行了随访。结果　 P2 7在正常肝脏组织中少有表达 ,肝硬化组阳性表达有所增加 ,但两组之间差异无显著性 (P>0 .0 5 ) ;P2 7在原发性肝癌组中阳性表达率为 86 .0 4 % ,与肝硬化组和正常对照组相比差异有显著性(P<0 .0 1)。肿瘤直径 >5 cm、多个瘤灶、低分化和有血管侵犯的原发性肝癌组织中 P2 7呈低表达 ;P2 7高表达的原发性肝癌患者较低表达者生存期明显延长 (P<0 .0 1)。结论　 P2 7基因在原发性肝癌组织中的表达有组织特异性 ;P2 7与原发性肝癌的浸润和转移有密切关系 ,可以作为原发性肝癌的预后判断指标之一     

Adenovirus expressing p27kip1 suppresses growth of established esophageal carcinoma xenograffs

AIM: To investigate the growth suppression of adenovirus expressing p27kip1 on established esophageal tumors in nude mice.METHODS: Esophageal carcinoma xenografts in nude mice were established by tumor tissue mass transplantation. The successfully constructed recombinant adenoviral vectors carrying p27kip1 gene (Adp27kip1) were directly injected into the esophageal tumors in nude mice. Compared to control group, the growth curve of tumor was drawn and the growth inhibition rate of tumor was calculated. The histology of tumors was examined by hematoxylin and eosin (H&E) staining. The expression of p27kip1 and survivin was detected in tumors by immunohistochemical technique.RESULTS: The growth of tumors in gene therapy group with Ad-p27kip1 was obviously suppressed compared to control group (0.42±0.08 g vs 1.17±0.30 g, t=6.39,P＜0.01), the inhibition rate of tumor growth reached 64.1%. Pathological detection showed that the tumors in nude mice were poorly differentiated esophageal squamous carcinoma. In addition, the expression of p27kip1 was increased, while the expression of survivin was decreased in tumors after being transfected with Ad-p27kip1.CONCLUSION: p27kip1 gene therapy mediated by adenovirus vector has a significant inhibitory effect on esophageal carcinoma in vivo. Up-regulated p27kip1expression and down-regulated survivin expression may be its important mechanisms.     

Angiogenesis in chronic myeloproliferative diseases detected by CD34 expression

Increased bone marrow angiogenesis estimated as bone marrow microvessel density (MVD), or as serum angiogenic factor levels and/or immunohistochemical expression of these factors in bone marrow biopsy has been demonstrated in a variety of hematological disorders including chronic myeloproliferative diseases (MPDs). The aim of this study was to investigate the MVD in 25 cases of myelofibrosis with myeloid metaplasia (MMM). MVD was estimated by CD34 immunohistochemical expression in bone marrow biopsies. A control group of 27 patients without bone marrow disease, eight cases of polycythemia vera (PV), 41 cases of essential thrombocythemia (ET) and nine cases of chronic myeloid leukemia (CML) were also studied. Moreover, in cases with MMM, MVD was correlated with clinical, laboratory, histological parameters and the outcome of the patients. Our study confirmed a significantly higher degree of angiogenesis in MMM, PV, ET and CML compared with controls (P < 0.001, P = 0.0007, P < 0.001 and P = 0.0008, respectively). Angiogenesis was higher in MMM than PV, ET and CML cases (P < 0.001, P < 0.001 and P = 0.008). Increased angiogenesis was correlated with hypercatabolic symptoms in MMM patients (P = 0.009). No correlation with other clinicopathological parameters or clinical outcome was found. However, definitive conclusions regarding the prognostic value of increased angiogenesis may require additional follow-up and a larger group of patients.     

Alcohol Ingestion Impairs Host Defenses Predisposing Otherwise Healthy Mice to Pneumocystis carinii Infection

Pulmonary infection with Pneumocystis carinii, an opportunistic pathogen, is associated with a variety of immunosuppressive states, Including human immunodeficiency virus infection. We hypothesized that alcohol ingestion might compromise host defenses against this pathogen and, in an immunocompromised host, increase the severity of infection. This hypothesis was tested in both acute and chronic ethanol-treated normal and CD4⁺ T-cell-depleted mice challenged with P. carinii organisms. Normal and CD4⁺ T-cell-depleted mice were given an intraperitoneal injection of ethanol or saline 0.5 hr before P. carinii challenge and killed 3 hr later for bronchoalveolar lavage. Acute alcohol treatment decreased significantly tumor necrosis factor (TNF) activity and the number of polymorphonuclear leukocytes (PMNLs) recovered in the lavage in response to the pathogen. Depletion of CD4⁺ T-cells did not potentiate the effect of alcohol on the early inflammatory response to the pathogen any further. In normal animals, in vivo interferon (IFN)-γ pretreatment augmented significantly the P. carinii- stimulated lung TNF response and PMNL recruitment. However, IFN-γ pretreatment prevented the alcohol-induced suppression of TNF secretion without affecting the PMNL recruitment. The effect of chronic alcohol consumption on the severity of infection was studied in long-term, alcohol-fed normal and CD4⁺-depleted mice challenged with P. carinii organisms. Lung histopathology showed that P. carinii infection was present in >60% of the alcohol-fed mice and in none of the controls. Also, a significantly higher number of PMNLs were recovered in the lavage fluid of alcohol-fed mice with persistent infection. There was no difference in lavage fluid and cell culture supernatant TNF and reactive nitrogen intermediates release between the two groups. CD4⁺ T-cell depletion did not alter significantly the effect of alcohol on any of the aforementioned parameters. We conclude that chronic alcohol ingestion alone induces immunosuppression sufficient to permit pulmonary infection with P. carinii. One possible mechanism for the adverse effects of alcohol on host defenses against P. carinii may be through suppressed host release of cytokines including TNF-α and IFN-γ.     

CD147分子和基质金属蛋白酶在类风湿关节炎滑膜中的表达

目的 探讨类风湿关节炎(RA)滑膜组织CD147的表达及其与基质金属蛋白酶(MMP)-01和MMP-2表达的相关性。方法 采用免疫组织化学SP(streptavidin／peroxidase)染色方法检测11例RA患者受损关节软骨-血管翳接合部（CPJ)滑膜组织中CD147和MMP-1及MMP-2的表达，并与3例骨关节炎（OA)患者滑膜组织CD147和MMP-1及MMP-2的检测相对照。结果 3例OA滑膜组织CD147和MMP-1及MMP-2的表达均为阴性，而11例RA滑膜组织中均有CD147和MMP-1及MMP-2的表达。其中，表达CD147的细胞为单核-巨噬细胞、淋巴细胞和滑膜成纤维样细胞，表达MMP-1及MMP-2的细胞为滑膜成纤维样细胞。统计学分析表明RA滑膜细胞CD147的表达和MMP-1及MMP-2的表达间存在显著相关性。结论 CD147在RA滑膜组织中表达增高，可能是导敏RA受损关节软骨、骨基质降解的重要因素之一。