Fei-Xiang Ding

Repression of the miR‐17‐92 cluster by p53 has an important function in hypoxia‐induced apoptosis

We here report that miR‐17‐92 cluster is a novel target for p53‐mediated transcriptional repression under hypoxia. We found the expression levels of miR‐17‐92 cluster were reduced in hypoxia‐treated cells containing wild‐type p53, but were unchanged in hypoxia‐treated p53‐deficient cells. The repression of miR‐17‐92 cluster under hypoxia is independent of c‐Myc. Luciferase reporter assays mapped the region responding to p53‐mediated repression to a p53‐binding site in the proximal region of the miR‐17‐92 promoter. Chromatin immunoprecipitation (ChIP), Re‐ChIP and gel retardation assays revealed that the binding sites for p53‐ and the TATA‐binding protein (TBP) overlap within the miR‐17‐92 promoter; these proteins were found to compete for binding. Finally, we show that pri‐miR‐17‐92 expression correlated well with p53 status in colorectal carcinomas. Over‐express miR‐17‐92 cluster markedly inhibits hypoxia‐induced apoptosis, whereas blocked miR‐17‐5p and miR‐20a sensitize the cells to hypoxia‐induced apoptosis. These data indicated that p53‐mediated repression of miR‐17‐92 expression likely has an important function in hypoxia‐induced apoptosis, and thus further our understanding of the tumour suppressive function of p53.

Multiepitope peptide‐loaded virus‐like particles as a vaccine against hepatitis B virus–related hepatocellular carcinoma

To develop a hepatitis B virus (HBV) therapeutic vaccine that can induce a broad but specific immune response and significant antitumor effects both in vivo and in vitro, we inserted HBV X protein (HBx)‐derived epitopes HBx(52‐60), HBx(92‐100), and HBx(115‐123); a novel subdominant cytolytic T lymphocyte (CTL) epitope HBx(140‐148); and the universal T helper epitope pan human leukocyte antigen DR‐binding epitope into HBV core protein to form multiepitope peptide‐loaded virus‐like particles (VLPs). CTL responses against epitope‐loaded VLPs were elicited by priming with VLP‐pulsed dendritic cells in both HLA‐A*0201 transgenic (Tg) mice and peripheral blood lymphocytes from HLA‐A2+/HBx+ HBV‐infected hepatocellular carcinoma (HCC) patients. The multiepitope peptide‐loaded VLPs demonstrated significantly higher immunogenicity in Tg mice than any single responsive epitope. Significant antitumor effects were demonstrated both with primary cultured autologous HCC cells in vitro and tumor‐bearing Tg mice in vivo in an HLA‐A2–restricted and epitope‐specific fashion. Conclusion: The significant antitumor effects both in vivo and in vitro demonstrate the potential of multiepitope peptide‐loaded VLPs as a vaccine against HCC. (HEPATOLOGY 2009.)

A novel, cheap and effective fusion expression system for the production of recombinant proteins

To develop faster, less expensive methods for expression and purification of proteins, the annexin B1-intein fusion expression system was constructed. The interest proteins fused to the annexin B1-intein tag were purified in a single-step method based on the Ca2+-binding activity of annexin B1, and the annexin B1-intein fusion tag was removed based on the self-cleaving activity of the intein. Moreover, we found that in some cases, fusion to annexin B1 can promote the solubility of heterologous proteins. The production of soluble and highly active of interleukin-2 and low-molecular single-chain urokinase in our results proved that the system was a novel, cheap and effective fusion expression system for the production of valuable soluble recombinant proteins in Escherichia coli.

Germline hMSH2 promoter mutation in a Chinese HNPCC kindred: evidence for dual role of LOH.

Hereditary non‐polyposis colorectal cancer (HNPCC) is a dominantly inherited cancer predisposition syndrome that is caused by germline mutations in mismatch repair genes. By screening the core promoters of hMSH2, hMLH1, and hMSH6 in 37 Chinese suspected HNPCC families, a novel germline mutation c.−78_−79delGT was found in the hMSH2 promoter. Its pathogenic effects were supported by the following findings: (a) it co‐segregated with HNPCC‐related cancers and was not present in the 220 control subjects, (b) tumors harboring the mutation lacked the expression of hMSH2 and showed high microsatellite instability, (c) it significantly decreased the promoter activity, and (d) it abolished the binding ability of the transcription factor E1A‐F. Loss of heterozygosity (LOH) was found in three of the tumors studied. Intriguingly, in the tumors from patients II:1 and III:1, LOH occurred in the wild‐type allele and agreed well with the traditional ‘two‐hit’ model. In contrast, in the tumor from patient III:3, LOH occurred in the mutant allele. A pathogenic somatic mutation (c.2210+1G>A) was also found in this tumor; therefore, we proposed that the ‘second hit’ was inactivated by somatic mutation, and the mutant allele was lost during tumor progression; this provided evidence for the new hypothesis for the dual role of LOH.

A preliminary study on the activation and antigen presentation of hepatitis B virus core protein virus-like particle-pulsed bone marrow-derived dendritic cells

Hepatitis B virus core protein virus-like particles (HBc-VLPs) act as a strong immunogen and are suitable for uptake by dendritic cells (DCs), in which they directly promote DC maturation and migration. To illustrate the utility of global proteomic analysis techniques in elucidating the molecular events that are altered in HBc-VLP-pulsed bone marrow-derived DCs (BMDCs) and to gain a better understanding of the molecular mechanisms of capture and processing of HBc-VLP-pulsed BMDCs, an antigen (Ag) delivery system based on HBc-VLP-pulsed BMDCs was developed. Two-dimensional electrophoresis (2-DE) and tandem mass spectrometry (MS/MS) analyses were utilized to analyze the differential protein expression patterns between HBc-VLP-pulsed and untreated BMDCs. Protein spots with significantly altered expression levels were detected, identified and validated. The results showed that exogenous HBc-VLPs were phagocytosed efficiently by BMDCs and enhanced the efficacy of BMDC maturation and Ag presentation, VLPs also induced high levels of Ag-specific CD8(+) T cells that displayed high cytotoxic T lymphocyte (CTL) activity in vivo. Several differentially expressed proteins, including growth factor receptor bound protein 2 (Grb2) and annexin A2 (AnxA2), were detected by proteomic analysis, identified by mass spectrometry and validated by western blot.

Annexin B1 from Taenia solium metacestodes is a newly characterized member of the annexin family.

Abstract We previously reported cloning of the Taenia solium annexin B1 gene from a metacestode cDNA expression library and demonstrated that it acts as a protective antigen for effective vaccine development against cysticercosis. In the present study we produced recombinant annexin B1 and antiserum against the protein to investigate its structural and functional properties. Western blotting of metacestode fractions indicated that T. solium annexin B1, similar to vertebrate annexins, associates with acid phospholipids in the presence of Ca2+. This property was confirmed by the recognition of apoptotic cells by labeled annexin B1. CD spectroscopy results demonstrated that α-helices are the main secondary structures of the protein. Ca2+ binding increases the α-helix content and causes significant thermal stabilization with a melting temperature increase of approximately 10°C. Functional Ca2+-dependent phospholipid binding sites of annexin B1 were investigated using mutant proteins. By changing a conserved acidic amino acid residue that putatively combines Ca2+ in each domain of annexin B1 singly or in combination, we found that Ca2+ binding in the first domain is more important than that at the other Ca2+ binding sites. Annexin B1 is a metacestode stage-specific antigen, with the protein being mainly localized in the teguments and surrounding cyst wall of T. solium metacestodes, suggesting a role in the parasite-host interaction.

Design and characterization of a platelet-targeted plasminogen activator with enhanced thrombolytic and antithrombotic potency.

To obtain a thrombus‐targeted plasminogen activator with high affinity for activated platelets and enhanced thrombolytic and antithrombotic potency, we engineered a sequence encoding RGDS (Arg‐Gly‐Asp‐Ser) peptide into the loop between domains II and III of the sequence‐deleted mutant of annexin B1 and then constructed a chimaeric plasminogen activator gene mAnxB1‐RGDS‐ScuPA by fusing ScuPA32k [low‐molecular‐mass single‐chain urokinase (32 kDa)] with the N‐terminus. The chimaeric protein was expressed in inclusion bodies in Escherichia coli at 25% of the total cellular protein content. Ion‐exchange and gel‐filtration chromatographies were applied to purify the chimaeric protein, achieving purity greater than 98%. We demonstrated that this chimaera can be expressed and purified in an active form; in vitro testing indicated that the chimaera fully retained the thrombolytic activity, platelet membrane‐binding activity and anti‐platelet aggregation activity of the parent molecules. The plasma clearance of the chimaera was similar to that of urokinase and ScuPA32k. In vivo experiments in a canine system indicated that animals administered the chimaera presented a decreased time to reperfusion, higher reperfusion ratio and less bleeding effects than treatment with urokinase. These results show that the chimaera is a platelet‐targeted plasminogen activator with enhanced thrombolytic and antithrombotic potency that may have advantages over currently available thrombolytic agents.

Translocation of annexin B1 in response to the stimulation of PMA and ionomycin in cervical cancer cells

Annexin B1 is a novel member of the annexin superfamily which was isolated from a Cysticercus cellulosae cDNA library. To investigate the physiological roles of annexin B1, we firstly performed immunohistochemical analysis on frozen Cysticercus cellulosae sections and found that annexin B1 was present not only in the tegument of the bladder wall, but also in the host‐derived inflammatory layer; In addition, ELISA analysis revealed that annexin B1 could be detected in the cystic fluid of Cysticercus cellulosae and the sera of pigs with cysticercosis. These findings indicated that annexin B1 might be a secretary protein. We further constructed a pEGFP—annexin B1 plasmid and transfected it into SiHa cells. We found that GFP—annexin B1 was stimulated to translocate to the plasma membrane by phorbol 12‐myristate 13‐acetate (PMA). By contrast, it was induced to distribute at the plasma and nuclear membranes by treatment with calcium ionophore ionomycin. PMA increased annexin B1 membrane binding, which might facilitate exocytosis. Moreover, translocation of the protein to the plasma and nuclear membranes after stimulated by ionomycin, was predicted to be related to an additional function.