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To investigate the regulatory effects of Olaparib on natural killer cell activating receptor (NKG2D) ligands expression on human acute myeloid leukemia (AML) cell line HL-60, and to explore the molecular mechanism of Olaparib on HL-60 cells.After HL-60 cells in logarithmic growth phase were treated with Olaparib at different concentrations for different times (24, 48 h), the expression of NKG2D ligand on the surface of HL-60 cells was detected by flow cytometry. Western blot was used to dectect the expression of ERK expression in HL-60 cells. The killing effect of NK cells to HL-60 cells was detected by CFSE/PI method.10 μmol/L Olaparib could upregulate the expression of NKG2D ligand on the surface of HL-60 cell at 24 and 48 hours, while 5 μmol/L Olaparib could induce up-regulation of the expression of ULBP-2 and ULBP-3 at 48 hours. Western blot analysis showed that ERK phosphorylation of HL-60 cells was enhanced after treating with Olaparib. The killing effect of NK cells to HL-60 cells could be enhanced by Olaparib, however, ERK inhibitor could suppress the killing effect of NK cells to HL-60 cells.Olaparib can upregulate NKG2D ligands expression on the surface of HL-60 cells and enhance the cytotoxicity of NK cell to HL-60 cells. The mechanism may be related to Olaparib promoting ERK phosphorylation expression.Olaparib对HL-60细胞NKG2D配体表达的调节作用及相关机制研究.研究Olaparib对人急性髓系白血病细胞株HL-60细胞表面NKG2D配体表达的调节作用, 并初步探索其内在调控机制。.对数生长期的HL-60细胞经不同浓度Olaparib（1.25、2.5、5、10 μmol/L）作用24、48 h后, 采用流式细胞术检测细胞表面NKG2D配体表达情况；Western blot检测HL-60细胞内ERK蛋白表达变化情况；CFSE/PI法检测NK细胞对HL-60细胞的杀伤作用。.10 μmol/L Olaparib作用24和48 h均可上调HL-60细胞表面NKG2D配体的表达, 5 μmol/L Olaparib作用48 h可诱导ULBP-2、ULBP-3表达上调；Western blot检测结果显示, Olaparib处理后的HL-60细胞内ERK磷酸化水平增强。Olaparib可增强NK细胞对HL-60细胞的杀伤作用, 但ERK抑制剂可下调NK细胞对HL-60细胞的杀伤作用。.Olaparib可上调HL-60细胞表面NKG2D配体表达, 增强NK细胞对其的杀伤作用, 其机制可能与Olaparib促进ERK磷酸化表达有关。.

Breast cancer patients with positive epidermal growth factor receptor (EGFR) expression have significantly worse post-relapse prognosis than patients with negative EGFR expression. Vinorelbine (NVB) is usually reserved as a salvage therapy after anthracyclines and taxanes in patients with breast cancer. To see whether EGFR expression has a predictive value in NVB-mediated effect on human breast cancer cells, we examined 50 primary breast cancer samples. Of these, 42% were found to be NVB sensitive by ATP-tumour chemosensitivity assay. Sensitivity was correlated with EGFR expression level (p = 0.001). To dynamically examine EGFR's effect on NVB sensitivity in breast cancer cells, we used the real-time cell electronic sensing system with EGFR-positive and EGFR-negative breast cancer cell lines, MCF-7 and MDA-MB-435s, respectively. MCF-7 is NVB sensitive, while MDA-MB-435 is NVB resistant. NVB-induced cytotoxicity to MCF-7 can be partly reversed with inhibitory anti-EGFR antibody. NVB up-regulated EGFR expression in MCF-7 cells, which affects ERK1/2 phosphorylation. This cellular response mechanism may cause greater input to non-lethally damaged cells. These data suggest that EGFR expression can be used as a prognostic factor for breast cancer sensitivity to NVB, which could help identify appropriate treatments for breast cancer.

A multiple myeloma (MM) cell line, XG2, has high-level expression of CD40, a tumor necrosis factor receptor (TNFR) family member. CD40 is present on the surfaces of a large variety of cells, including B cells, endothelial cells, dendritic cells and some carcinoma cells, and delivers signals regulating diverse cellular responses, such as proliferation, differentiation, growth suppression, cell death. In this research, we study the effects of cross-linking of CD40 on myeloma cells using different concentrations of anti-CD40 monoclonal antibody (mAb), 5C11. We found that low concentrations of 5C11 induced proliferation of XG2, while high concentrations of 5C11 resulted in homotypic aggregation of XG2, and strongly suppression of its proliferation and apoptosis after 24 h of treatment. These dose-dependent effects of 5C11 were verified by flow cytometry, Western blotting and immunoprecipitation. Autocrine or paracrine induction of IL-6, and up-regulation of membrane TNF and phosphorylation of TNFR1 may partially explain the contradictory biological effects of CD40 cross-linking on XG2 by anti-CD40 mAb.

Current chemotherapy for esophageal cancer is conducted on the basis of empirical information from clinical trials, which fails to take into account the known heterogeneity of chemosensitivity between patients. This study was aimed to demonstrate the degree of heterogeneity of chemosensitivity in esophageal cancers.A total of 42 esophageal cancer specimens were collected. The heterogeneity of chemosensitivity in esophageal cancer specimens was examined using an ex vivo ATP-tumor chemosensitivity assay (ATP-TCA).Thirty eight specimens produced evaluable results (90.5%). The most active single agent tested was nedaplatin, to which 28.9% of samples were sensitive. Combinations of chemotherapy agents exhibited much higher sensitivity: cisplatin + paclitaxel was sensitive in 16 of 38 (42.1%) of samples, while nedaplatin+paclitaxel was more effective, which was sensitive in 20 of 38 cases (52.6%).There was a marked heterogeneity of chemosensitivity in esophageal cancer. Chemosensitivity testing may provide a practical method for testing new regimens before clinical trials in esophageal cancer patients.

Breast cancer patients with positive epidermal growth factor receptor (EGFR) expression have significantly worse post-relapse prognosis than patients with negative EGFR expression. Vinorelbine (NVB) is usually reserved as a salvage therapy after anthracyclines and taxanes in patients with breast cancer. To see whether EGFR expression has a predictive value in NVB-mediated effect on human breast cancer cells, we examined 50 primary breast cancer samples. Of these, 42% were found to be NVB sensitive by ATP-tumour chemosensitivity assay. Sensitivity was correlated with EGFR expression level (p = 0.001). To dynamically examine EGFR's effect on NVB sensitivity in breast cancer cells, we used the real-time cell electronic sensing system with EGFR-positive and EGFR-negative breast cancer cell lines, MCF-7 and MDA-MB-435s, respectively. MCF-7 is NVB sensitive, while MDA-MB-435 is NVB resistant. NVB-induced cytotoxicity to MCF-7 can be partly reversed with inhibitory anti-EGFR antibody. NVB up-regulated EGFR expression in MCF-7 cells, which affects ERK1/2 phosphorylation. This cellular response mechanism may cause greater input to non-lethally damaged cells. These data suggest that EGFR expression can be used as a prognostic factor for breast cancer sensitivity to NVB, which could help identify appropriate treatments for breast cancer.

CD40 is a costimulatory protein expressed on the surface of many different cells. It delivers signals regulating diverse cellular responses, including proliferation, differentiation, growth suppression, and cell death. In this study, we report a novel CD40 mutant (c.234C>A or p.H78Q) that is expressed in the U266 cell line and in freshly isolated tumor cells. Three-dimensional structural model and Scatchard analysis revealed that the mutated residue located in a region is important for binding to CD40L (CD154). Functional analysis indicated that the mutated CD40 was translocated to the CD40 signalosome and involved in CD40 signal transduction. In conclusion, the mutation in CD40 can lead to an alteration of function, including the change of antigen epitope and the binding affinity with CD40L.

To analyze the cloning result of CD40 mutant from RPMI8226 cells, a multiple myeloma (MM) cell line, and study the change of the expressions of costimulatory molecules and the apoptosis of RPMI8226 cells after activated with CD40.CD40 gene mutant in RPMI8226 cell was detected by RT-PCR and DNA sequencing. The cell lines were cultured with sCD40L, L929/CD40L, soluble 5C11 (an anti-CD40 mAb) plate-bound 5C11 and their respective controls. Their growth curves, change of phenotypes and cell cycles were detected. The signalosome of CD40 on RPMI8226 cells were analyzed with laser scanning confocal microscope.There was a single base substitution (TCA--TTA) in the open reading frame of CD40 from RPMI8226 cells, resulting in the conversion of a amino acid (Ser124Leu). Only plate-bound antibody could inhibit RPMI8226 cell proliferation [(2.5 +/- 0.6) x 10(5) vs (7.8 +/- 1.2) x 10(5), P0.05] and cause G1 arrested [(58.0 +/- 3.6)% vs (42.0 +/- 2.3)%, P0.05]. muCD40 was translocated to CD40 signalosome while CD40 activated.The mutated CD40 in RPMI8226 cell might decrease its affinity to CD40L, leading to the disorder of CD40 signal.

To prepare a novel functional mouse anti-human CD40 monoclonal mAb.Female BALB/c mice of 6-8 weeks old were immunized with CD40 transfectant (L929-CD40) as immunogen. The spleen B cells of the mice were fused with Sp2/0. The hybridoma cells were screened with CD40 transfectant (L929-CD40) by FCM. Fast-strip analysis was performed to identify Ig subclass of this mAb. The epitope recognized by this mAb was detected by Bio/5C11 competitive assay. The proliferation and cell cycle of tumor cells in vitro were studied by MTT assay and PI staining respectively.One hybridoma cell line named 2B6 was obtained, which had the property of secreting anti-human CD40 monoclonal antibody continuously and steadily. This mAb specifically recognized human CD40 molecule and promoted the proliferation of tumor cells in vitro.One hybridoma cell line which can secret a novel functional mouse anti-human CD40 mAb has been developed successfully. This mAb can specifically recognize human CD40 and influence the growth of tumor cells in vitro.

To measure the level of serum soluble CD40 (sCD40) in patients with acute hepatitis, hepatitis gravis and primary carcinoma of the liver, and to evaluate the relationship of sCD40 with biochemical marks and disease prognosis.Patients with acute hepatitis (n=49) hepatitis gravis (n=22) and primary carcinoma of the liver (n=13) were studied, and serum sCD40 was determined in these patients and compared with that of healthy controls (n=44) by enzyme linked immunosorbent assay (ELISA). The binding capacity of serum sCD40 to its ligand CD40L was detected by flow cytometry (FCM) in vitro.Concentration of sCD40 was significantly higher in patients with liver disease than that in healthy controls (P0.001), but no significant difference was found between the three types of liver disease (P=0.475). In the hepatitis gravis group, sCD40 concentration in dead patients was higher compared with that in the survivals (P0.05). Level of sCD40 in patients with acute hepatitis was correlated with serum alanine transaminase (ALT) and aspartic transaminase (AST). The serum sCD40 could bind CD40L in vitro.These data suggest that sCD40 is an important serological marker in liver disease to evaluate acute injury of hepatocytes, and it shows a relevance with the prognosis of hepatitis gravis. The highly elevated level of sCD40 suggest the involvement of CD40 and its ligand CD40L in liver disease.

To clone and express the recombinant human soluble CD40 ligand(CD40L) with biological activation.The isoleucine zipper (IZ) gene was fused at N-terminal of CD40L gene coding E107-L261 contributing to form trimer. The gene coding hexahistidine was fused at the N-terminal of IZ because the sCD40L-IZ fusion protein could be purified by affinity chromatography. Then the fusion gene was amplified and cloned into expression plasmid pET30a.The protein sCD40L-IZ with His-tag at the N-terminal was effectively expressed in E. coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was performed to acquire soluble sCD40L-IZ. The fusion protein sCD40L-IZ was conveniently purified using HiTrap affinity column with above 95% purity. The gel filtration chromatography and non-reduced SDS-PAGE identified the trimeric structure of the recombinant protein. The microscope analysis showed the sCD40L-IZ interacted with the membrane CD40 on XG2, a multiple myeloma cell line.The recombinant human trimeric CD40L-IZ was expressed in prokaryotic cells successfully, which has provided a basis for further study of the relationship between CD40L and apoptosis, CD40L and pathogenesis of illness. It is also useful to clinical treatment.

We examined the effects of CD40 activation with dexamethasone (Dex) or 60Co-gamma-irradiation on the growth of malignant B cells in vitro, using the human multiple myeloma (MM) cell line, XG2, and the B lymphoma Daudi cell line as models. Both lines are resistant to Dex and irradiation; 10(-7)M Dex or 10 Gy of gamma-irradiation induced only minimal growth arrest and apoptosis of the cells. Treatment of the cells with the agonistic anti-CD40 monoclonal antibody 5C11 partially inhibited the proliferation of the Daudi cells; XG2 underwent apoptosis. XG2 is an Interleukin-6 (IL-6)-dependent myeloma cell line and CD40 activation blocked XG2 in the G1 phase of the cell cycle, in a manner similar to the effect of IL-6 deprivation. Daudi was blocked in the G2/M phase after treatment with the agonistic CD40 mAb 5C11. Furthermore, the activation of CD40 on Daudi and XG2 enhanced their sensitivity to dexamethasone-and gamma-irradiation -induced growth arrest and apoptosis. CD40 activation stimulated both anti-apoptotic Bcl-XL and pro-apoptotic Bax mRNA synthesis in the Daudi cell line; CD40 activation increased the Bax mRNA level but had no effect on the Bcl-XL mRNA level in the XG2 cell line. Apoptosis in both cell lines was associated with an increasing ratio of Bax-to-Bcl-XL both in mRNA and in protein levels. It is concluded that use of the anti-CD40 mAb 5C11 either by itself or in combination with chemotherapy and/or radiotherapy may have significant therapeutic potential.

Ke-Qing Qian1, Heng Ye1, Yi-Wen Xiao1, Yong-Yi Bao2, Chun-Jian Qi11Department of Oncology; 2Department of Pathology, the Changzhou No. 2 People’s Hospital Affiliated to Nanjing Medical University, Changzhou, ChinaAbstract: Tumor lysis syndrome (TLS), a result of rapid cell lysis following tumor therapy, is a well recognized complication during the treatment of rapidly growing tumors. TLS rarely occurs in solid tumors. We present a case report of TLS in a patient with primary retroperitoneal soft tissue sarcoma. TLS occurred in the patient after four days’ combinational chemotherapy with cisplatin, adriamycin, and dacarbazine. These drugs were selected on the basis of an ex vivo ATP-based tumor sensitivity assay. TLS was properly controlled in the patient with concomitant remission of the sarcoma. Therefore, precautions should be taken to avoid this potentially fatal complication during treatment of solid tumors, especially with tumors highly sensitive to drugs.Keywords: tumor lysis syndrome, retroperitoneal soft tissue sarcoma, ATP-based tumor sensitivity assay (ATP-TCA)