喉癌组织中MKRN1和HSP90基因表达的研究

【摘要】目的 正常细胞中MKRN1和HSP90基因的平衡决定了端粒酶活性和端粒长度,使细胞保持正常的状态。二者的表达异常可能与肿瘤的发生发展有关。为探讨MKRN1和HSP90基因的表达与喉癌发生发展之间的关系，我们研究了喉癌组织中MKRN1和HSP90基因mRNA的表达情况。方法 提取35例喉癌组织的总mRNA，用半定量RT-PCR的方法检测MKRN1和HSP90基因mRNA的表达情况。结果 35例喉鳞癌中有18例MKRN1基因mRNA表达下调，占51.4%（18/35）；16例HSP90基因mRNA表达上调，占45.7%（16/35）。 MKRN1基因在肿瘤组织中的表达水平为0.74±0.63,在癌旁正常对照组织中为1.21±0.76，（P＜0.01）。HSP90基因在肿瘤组织中表达水平为1.82±1.25,而在癌旁正常对照组织中为1.23±1.04,（P＜0.05）,统计学上具有显著差异。MKRN1与HSP90基因的表达存在负相关(r=-0.458, P＜0.05)。HSP90基因表达情况与肿瘤的病理分期和患者年龄相关 (P＜0.05) ，MKRN1的表达与肿瘤的临床特征均无相关性(P＞0.05)。结论 喉癌组织中MKRN1和HSP90基因mRNA的表达异常存在相关性，二者表达平衡的破坏可能在喉癌的发生发展中起着重要的作用。     

Members of the heat-shock protein 70 family promote cancer cell growth by distinct mechanisms

Whereas the stress-inducible heat-shock protein 70 (Hsp70) has gained plenty of attention as a putative target for tumor therapy, little is known about the role of other Hsp70 proteins in cancer. Here we present the first thorough analysis of the expression and function of the cytosolic Hsp70 proteins in human cancer cells and identify Hsp70-2, a protein essential for spermatogenesis, as an important regulator of cancer cell growth. Targeted knock-down of the individual family members by RNA interference revealed that both Hsp70 and Hsp70-2 were required for cancer cell growth, whereas the survival of tumorigenic as well as nontumorigenic cells depended on Hsc70. Cancer cells depleted for Hsp70 and Hsp70-2 displayed strikingly different morphologies (detached and round vs. flat senescent-like), cell cycle distributions (G2/M vs. G1 arrest) and gene expression profiles. Only Hsp70-2 depletion induced the expression of macrophage inhibitory cytokine-1 that was identified as a target of P53 tumor-suppressor protein and a mediator of the G1 arrest and the senescent phenotype. Importantly, concomitant depletion of Hsp70 and Hsp70-2 had a synergistic antiproliferative effect on cancer cells. Thus, highly homologous Hsp70 proteins bring about nonoverlapping functions essential for cell growth and survival.     

Heat shock proteins HSP25, HSP60, HSP72, HSP73 in isoosmotic cortex and hyperosmotic medulla of rat kidney

The distribution of heat shock proteins (HSP) HSP60, HSP73, HSP72 and HSP25 in the isoosmotic cortex and the hyperosmotic medulla of the rat kidney was investigated using Western blot analysis and immunohistochemistry. HSP73 was homogeneously distributed throughout the whole kidney. The level of HSP60 was high in the renal cortex and low in the medulla. HSP25 and HSP72 were present in large amounts in the medulla. Only low levels of HSP25 and almost undetectable amounts of HSP72 were found in the cortex. HSP25 exists in one nonphosphorylated and several phosphorylated isoforms. Western blot analysis preceded by isoelectric focussing showed that HSP25 predominates in its nonphosphorylated form in the outer medulla but in its phosphorylated form in cortex and inner medulla. Although this intrarenal distribution pattern was not changed during prolonged anaesthesia (thiobutabarbital sodium), a shift from the nonphosphorylated to the phosphorylated isoforms of HSP25 occurred in the medulla. The characteristic intrarenal distribution of the constitutively expressed HSPs (HSP73, HSP60, HSP25) may reflect different states of metabolic activity in the isoosmotic (cortex) and hyperosmotic (medulla) zones of the kidney. The high content of inducible HSP72 in the medulla most likely is a consequence of the osmotic stress imposed upon the cells by the high urea and salt concentrations in the hyperosmotic medullary environment.     

Monocyte-derived RANTES is intrinsically elevated in periodontal disease while MCP-1 levels are related to inflammation and are inversely correlated with IL-12 levels

Bacteria colonizing tooth surfaces are essential in the induction of an inflammatory response in the periodontal tissues, but do not cause periodontitis in everyone, implicating differences in the host immune response. These possible differences were studied using lipopolysaccharide (LPS)-stimulated whole blood cell cultures (WBCC), which revealed a down regulation of monocyte derived interleukin-12 (IL-12p70) in untreated periodontitis patients and an up regulation after therapy. IL-12p70 is a crucial factor in the differentiation of Th1 cell responses. Since CC chemokines are able to influence the T cell differentiation via cytokine secretion in antigen-presenting cells, the production of CC chemokines in periodontitis was evaluated. Therefore WBCC were stimulated with LPS from Escherichia coli for 18 h and the levels of IL-12p70 and CC chemokines were measured in the supernatants by ELISA. Untreated periodontitis patients released 2 fold more RANTES (regulated on activation normal T cell expressed and secreted) (P = 0.01) and lower levels of IL-12p70 in comparison to controls (P < 0.05). A trend towards higher levels of macrophage chemoattractant protein-1 (MCP-1) (P = 0.07) was also seen in untreated periodontitis patients; while similar levels of monocyte derived chemokine (MDC) and macrophage inflammatory proteins-1 alpha and -1 beta (MIP-1 alpha and -1 beta) were found. After periodontal therapy no changes were seen with regard to MDC, MIP-1 alpha, MIP-1 beta and RANTES, whereas the MCP-1 levels decreased (P < 0.05) and the IL-12p70 levels strongly increased (P < 0.01). The data showed a consistent inverse correlation between the levels of MCP-1 and IL-12p70, and their proportional changes after therapy correlated with the clinical inflammatory response after therapy. This indicates that the disease state regulates the release of IL-12p70 and MCP-1 in E. coli LPS-stimulated WBCC. In contrast, the persistent augmented levels of RANTES after therapy are suggestive for an intrinsic behaviour.     

Phenotypic characterization and analysis of allogeneic T cell responses in ZAP-70 dominant negative transgenic mice

Antigen stimulation of T cells results in a series of biochemical events including the interaction of both SH2 domains of ZAP-70 with phosphorylated ITAMS on the T cell receptor. In order to study the physiological relevance of decreasing native ZAP-70–SH2 interaction in vivo, we generated transgenic mice expressing a T cell-specific, dominant negative form of ZAP-70 consisting of only the tandem SH2 domains (ZAP-NC). Phenotypically, these animals had a comparable distribution of lymphocyte subsets in the thymus and spleen compared with the wild-type (WT) controls. However, examination of peripheral blood revealed a slow but progressive decrease in the number of lymphocytes, particularly CD4⁺ cells, with age (17% reduction by 3 months, 58% reduction by 6 months). Allogeneic responses were then evaluated in vitro as well as in vivo using a subcutaneous sponge matrix implant. Although spleen cells cultured for 4 days in vitro with alloantigen developed normal functional responses, allogeneic responses generated in vivo within a subcutaneous sponge matrix were impaired. This was characterized by a depression in cytotoxic T lymphocyte (CTL) activity, a 82% reduction in the frequency of helper T cells, and a 78% reduction in the capacity of sponge-infiltrating lymphocytes to produce IL-2 in response to secondary antigen stimulation. These results indicate that although overt lymphocyte development and in vitro function were unremarkable, expression of a truncated ZAP-70 affected the in vivo survival of peripheral lymphocytes and altered the in vivo generation of functional activity to alloantigen.     

HSP70在米非司酮引产胎儿肝、肾、脑组织中的表达

目的对米非司酮引产胎儿肝、肾、脑组织进行热休克蛋白70的检测,探讨米非司酮对胎儿损伤的可能性.方法米非司酮引产胎儿17例,水囊引产胎儿5例, 采用免疫组化方法,检测HSP70在胎儿肝肾脑组织中的表达.结果实验组中HSP70均呈阳性表达,且有不同程度的表达,对照组均呈阴性表达.结论米非司酮可不同程度造成胎儿肝、肾、脑组织损伤.     

Heat shock protein 70 gene polymorphisms in rheumatoid arthritis

A role for heat shock proteins (hsp) in rheumatoid arthritis has been suggested. In addition, the specific binding of human HSP70 protein to QKRAA and RRRAA motifs within the HV3 region of disease-associated DRB1*0401 and DRB1*1001 molecules, respectively, has been proposed as being relevant to rheumatoid arthritis. The purpose of this work was to analyze the influence of HSP70 gene polymorphism on the susceptibility to or severity of rheumatoid arthritis and to investigate the possible contribution of these HSP70 polymorphisms in determining HLA-DRB1*0401/*1001 disease association. The frequencies of the HSP70-1, HSP70-2 and HSP70-hom genotypes were analyzed by PCR-RFLP using BsrBI , Pst I and Nco I enzymes, respectively, in patients with heumatoid arthritis and in healthy controls. No significant differences were observed when HSP70 alleled istribution between the groups under study were compared. Moreover, we did not observe any significant difference in HSP70 allele frequencies between patients positive for HLA-DRB1*0401/*1001 alleles and matched controls. Our data indicate that HSP70 gene polymorphisms do not appear to be relevant in the susceptibility to or severity of rheumatoid arthritis.     

Expression and localization of heat shock proteins in rat basophilic leukemia cells: differential modulation by degranulation,thermal or oxidative stress

BACKGROUND: Rat basophilic leukemia (RBL-2H3) cells are well characterized in terms of morphological and biochemical changes upon activation, and have been extensively used as a model system for studying the mechanisms of the immediate hypersensitivity reaction. To investigate whether overexpression of heat shock/stress proteins (HSP) is involved in the mast cell-dependent reactivity, we examined the adaptive responses of RBL-2H3 cells to classical stress conditions such as heat shock or oxidative injury produced by an aqueous extract of tobacco smoke. METHODS: HSP were determined by flow cytometry and immunocytochemistry. Degranulation was confirmed as the release of beta-hexosaminidase, determined spectrophotometrically, and by electron microscopy experiments. RESULTS: We found that RBL-2H3 cells respond to heat shock or oxidative injury by the synthesis of both the inducible 72 kDa HSP (Hsp70), and the oxidation-specific 32 kDa heme oxygenase (HO)-1. Heat shock induced mainly Hsp70 in a cell growth-dependent manner, whereas oxidative stress induced mainly HO-1 in a cell growth-independent manner. However, heat shock or oxidative stress had no significant effects on degranulation. CONCLUSION: Stress-mediated synthesis of HSP was not associated with RBL-2H3 degranulation and likewise, degranulation did not induce HSP.     

CD27/CD70 interaction directly drives B cell IgG and IgM synthesis

CD27 is a T cell activation antigen expressed on a majority of peripheral blood T cells. CD27 is also expressed on a subpopulation of human B cells, and it is reported that CD27⁺ B cells secrete both IgG and IgM. CD70, a ligand for CD27, is expressed on activated T and B cells, suggesting an interaction between T and B cells via CD27/CD70 ligation. Here, we analyze B cell immunoglobulin synthesis using a CD70 transfectant and present functional data showing that B cells secrete large amounts of IgG and IgM as a result of the CD27/CD70 interaction. A flow cytometric analysis showed that CD27 expression was increased and CD70 was expressed on tonsillar and peripheral blood B cells after activation with Staphylococcus aureus Cowan strain (SAC) plus interleukin (IL-2). In addition, the proliferation of B cells was enhanced mildly by the addition of CD70 transfectant, and its proliferation was blocked by anti-CD70 mAb. More importantly, the CD70 transfectant enhanced IgG and IgM production by purified B cells greatly in the presence of SAC plus IL-2. The enhancement was completely blocked by the addition of either anti-CD70 mAb or anti-CD27 mAb. Strongly suggesting that the interaction of CD27 with its ligand, CD70, on B cells plays an important role in B cell growth and differentiation to produce IgG and IgM.