Phyllanthus emblica Linn. fruit extract potentiates the anticancer efficacy of mitomycin C and cisplatin and reduces their genotoxicity to normal cells in vitro.

OBJECTIVE: Fruit of Phyllanthus emblica Linn. (PE) is widely consumed as a functional food and used as a folk medicine due to its remarkable nutritional and pharmacological effects. Mitomycin C (MMC) and cisplatin (cDDP) are the most widely used forms of chemotherapeutic drug, but their clinical use is limited by their genotoxicity to normal cells. We aimed to determine whether PE has potential to reduce the genotoxicity, while improving the anticancer effect, of MMC and cDDP. METHODS: Cell proliferation was evaluated using the trypan blue exclusion assay and colony-forming assay. Genomic instability (GIN) was measured using the cytokinesis-block micronucleus assay. RESULTS: Co-treatment (72 h) with PE at 20-320 µg/ml significantly enhanced the efficacy of MMC (0.05 µg/ml) and cDDP (1 µg/ml) against Colo205 colorectal cancer cells (P<0.05), and at 80-320 µg/ml significantly decreased MMC- and cDDP-induced GIN and multinucleation in normal colonic NCM460 cells (P<0.05). PE significantly decreased the mitotic index (P<0.01), blocked mitotic progression (P<0.05), and promoted apoptosis (P<0.01) in MMC- and cDDP-treated NCM460 cells, suggesting that PE-mediated inhibition of mitosis and induction of apoptosis may limit the division and survival of highly damaged cells. Also, PE was found to inhibit the clonal expansion of MMC- and cDDP-treated NCM460 cells (P<0.05) and decrease the heterogeneity of the surviving clones. CONCLUSIONS: PE potentiates the anticancer efficacy of MMC and cDDP, while preventing their genotoxicity and inhibiting clonal expansions of unstable genomes in normal cells. These data suggest that PE has the potential to reduce the risk of secondary cancers induced by chemotherapeutics.

Association between the MTHFR C677T polymorphism, blood folate and vitamin B12 deficiency, and elevated serum total homocysteine in healthy individuals in Yunnan Province, China.

BACKGROUND: An increased serum total homocysteine (tHcy) concentration is typically associated with genetic defects involved in Hcy metabolism or related nutritional deficiencies. In this study, the combined effects of methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and folate and vitamin B12 deficiency on serum total Hcy (tHcy) levels were evaluated in a healthy Chinese population in Yunnan Province, China. METHODS: The MTHFR C677T polymorphism was genotyped in 330 volunteers (164 men and 166 women) using polymerase chain reaction-restriction fragment length polymorphism analysis. Folate, vitamin B12, and tHcy concentrations were determined by corpuscle immune chemiluminescence assays. The tHcy concentration was determined using an enzymatic assay. RESULTS: Significant negative correlations (p<0.001) were observed between the serum levels of tHcy and folate (r=-0.252) and vitamin B12 (r=-0.243). Men had significantly higher serum tHcy concentrations than women (p<0.001). Individuals with the MTHFR TT genotype had significantly higher serum tHcy concentrations than individuals with the CC and CT genotypes (p<0.001). The folate level of red blood cells was significantly increased in individuals with the TT genotype than in individuals with the CC genotype (p<0.05). Moreover, in the low vitamin group, the serum tHcy level was significantly correlated with the levels of folate (r=-0.334, p=0.001) and vitamin B12 (r=-0.212, p=0.046). CONCLUSION: The MTHFR C677T polymorphism, folate deficiency, and B12 deficiency were significantly associated with elevated serum tHcy levels. Among these three factors, folate deficiency had the greatest contribution to the serum tHcy concentration, followed by (in order of decreasing effect) MTHFR C677T and vitamin B12 deficiency. Thus, folic acid and vitamin B12 supplementation could help prevent diseases associated with tHcy accumulation, especially in individuals with the MTHFR 677TT genotype.

Four microRNAs promote prostate cell proliferation with regulation of PTEN and its downstream signals in vitro.

BACKGROUND: Phosphatase and tensin homologue (PTEN), as a tumor suppressor, plays vital roles in tumorigenesis and progression of prostate cancer. However, the mechanisms of PTEN regulation still need further investigation. We here report that a combination of four microRNAs (miR-19b, miR-23b, miR-26a and miR-92a) promotes prostate cell proliferation by regulating PTEN and its downstream signals in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We found that the four microRNAs (miRNAs) could effectively suppress PTEN expression by directly interacting with its 3' UTR in prostate epithelial and cancer cells. Under-expression of the four miRNAs by antisense neutralization up-regulates PTEN expression, while overexpression of the four miRNAs accelerates epithelial and prostate cancer cell proliferation. Furthermore, the expression of the four miRNAs could, singly or jointly, alter the expression of the key components in the phosphoinositide 3-kinase (PI3K)/Akt pathway, including PIK3CA, PIK3CD, PIK3R1 and Akt, along with their downstream signal, cyclin D1. CONCLUSIONS: These results suggested that the four miRNAs could promote prostate cancer cell proliferation by co-regulating the expression of PTEN, PI3K/Akt pathway and cyclin D1 in vitro. These findings increase understanding of the molecular mechanisms of prostate carcinogenesis and progression, even provide valuable insights into the diagnosis, prognosis, and rational design of novel therapeutics for prostate cancer.

Role and fate of SP100 protein in response to Rep-dependent nonviral integration system.

Previously, we studied an AAVS1 site-specific non-viral integration system with a Rep-donor plasmid and a plasmid containing adeno-associated virus integration element. Our earlier study focused on the plasmid vector itself, but the cellular response to the system was still unknown. SP100 is a member of the promyelocytic leukemia nuclear bodies. It is involved in many cellular processes such as transcriptional regulation and the cellular intrinsic immune response against viral infection. In this study, we revealed that SP100 inhibited the Rep-dependent nonviral integration. Conversely, transient expression of Rep78 increased the degradation of SP100. This degradation was inhibited by treatment with MG132, an inhibitor of the ubiquitin proteasome. SP100 and Rep78 are both located in the nucleolus, which provides the spatial possibility for their interaction. Rep78 was coimmunoprecipitated with the enhanced green fluorescent protein (EGFP)-SP100 fusion protein but not EGFP, which verified the interaction between Rep78 and SP100. These results have enriched our knowledge about the cellular protein SP100 and Rep-dependent nonviral integration. It may lead to an improvement in the application of Rep-related transgene integration method and in the selection of target cells.

Different effects of telmisartan and valsartan on human aortic vascular smooth muscle cell proliferation.

BACKGROUND: Vascular smooth muscle cell proliferation is an important process in the development of atherosclerosis and is associated with other cellular processes in atherogenesis. Telmisartan is reported to have partial peroxisome proliferator-activated receptor (PPAR)-γ activating properties and has been referred to as selective PPAR modulators, but valsartan just blocks angiotensin II (AngII) type 1 (AT1) receptors. This study aimed to compare the different effects of telmisartan and valsartan on human aortic smooth muscle cells (HASMCs) proliferation. METHODS: Ability of telmisartan and valsartan to inhibit proliferation of HASMCs was evaluated by the Cell Counting Kit-8 (CCK-8) in continuous cell culture. Whether the antiproliferative effects of telmisartan and valsartan depend on their effects on AngII receptors or activating the peroxisome PPAR-γ was also investigated in this study. RESULTS: Telmisartan inhibited proliferation of HASMCs by 52.4% (P < 0.01) at the concentration of 25 µmol/L and the effect depended on the dose of telmisartan, but valsartan had little effect on HASMCs proliferation (P > 0.05) and no dose response. When tested in cells stimulated with AngII, telmisartan had the same inhibition of HASMCs by 59.2% (P < 0.05) and valsartan also inhibited it by 41.6% (P < 0.05). Telmisartan and valsartan had the same effect on down-regulating AT1 receptor expression and telmisartan was superior to valsartan up-regulating AngII type 2 (AT2) receptor expression. Antiproliferative effects of telmisartan were observed when HASMCs were treated with the PPAR-γ antagonist GW9662 but antiproliferative effects of the PPAR-γ activator pioglitazone were not observed. CONCLUSIONS: Telmisartan, but not valsartan, inhibits HASMCs proliferation and has dose-dependent response without stimulation of AngII. AT2 receptor up-regulation of telmisartan contributes to its greater antiproliferative effects than valsartan. Its PPAR-γ activation does not play a critical role in inhibiting HASMCs proliferation.

MicroRNA-7 inhibits tumor growth and metastasis by targeting the phosphoinositide 3-kinase/Akt pathway in hepatocellular carcinoma.

UNLABELLED: MicroRNAs (miRNAs) are known to be involved in carcinogenesis and tumor progression in hepatocellular carcinoma (HCC). Recently, microRNA-7 (miR-7) has been proven to play a substantial role in glioblastoma and breast cancer, but its functions in the context of HCC remain unknown. Here, we demonstrate that miR-7 inhibits HCC cell growth and metastasis in vitro and in vivo. We first screened and identified a novel miR-7 target, phosphoinositide 3-kinase catalytic subunit delta (PIK3CD). Overexpression of miR-7 would specifically and markedly down-regulate its expression. miR-7-overexpressing subclones showed significant cell growth inhibition by G(0) /G(1) -phase cell-cycle arrest and significant impairment of cell migration in vitro. To identify the mechanisms, we investigated the phosphoinositide 3-kinase (PI3K)/Akt pathway and found that Akt, mammalian target of rapamycin (mTOR), and p70S6K were down-regulated, whereas 4EBP1 was up-regulated in miR-7-overexpressing subclones. We also identified two novel, putative miR-7 target genes, mTOR and p70S6K, which further suggests that miR-7 may be a key regulator of the PI3K/Akt pathway. In xenograft animal experiments, we found that overexpressed miR-7 effectively repressed tumor growth (3.5-fold decrease in mean tumor volume; n = 5) and abolished extrahepatic migration from liver to lung in a nude mouse model of metastasis (n = 5). The number of visible nodules on the lung surface was reduced by 32-fold. A correlation between miR-7 and PIK3CD expression was also confirmed in clinical samples of HCC. CONCLUSION: These findings indicate that miR-7 functions as a tumor suppressor and plays a substantial role in inhibiting the tumorigenesis and reversing the metastasis of HCC through the PI3K/Akt/mTOR-signaling pathway in vitro and in vivo. By targeting PIK3CD, mTOR, and p70S6K, miR-7 efficiently regulates the PI3K/Akt pathway. Given these results, miR-7 may be a potential therapeutic or diagnostic/prognostic target for treating HCC.

The 156KELK159 tetrapeptide of HIV-1 integrase is critical for lentiviral gene integration.

HIV-1 integrase (HIV-1 IN), a key element of HIV-1-derived lentiviral vectors, is crucial for the stable maintenance of the vector gene by inserting them into host genome. HIV-1 IN has been found to have functions other than integration, such as involving in virion morphology, viral DNA synthesis and viral DNA nuclear import. In our study, the yeast two-hybrid assay identified a tetrapeptide 156KELK159 in HIV-1 IN that was crucial for HIV-1 IN and Daxx interaction. To investigate the functions of the tetrapeptide 156KELK159 of the HIV-1 IN, both the wild type HIV-1 IN and a mutant without 156KELK159 were used to package the EGFP reporter gene contained lentivirus. p24 based titer assay revealed that deleting the tetrapeptide did not affect virus packaging. The result was verified by quantitative real time PCR with viral specific primers. But the 156KELK159 was crucial for lentiviral gene integration. Deleting the tetrapeptide made the percentage of cells expressing the reporter gene significantly decreased and did not affect the level of DNA entered into the cells or nucleus. Real time reverse transcription PCR and FACS were used to detect the lentiviral report gene expression in infection maintaining cells and revealed 156KELK159 did not affect lentiviral vector gene expression. Our results may shed light on the regulatory mechanism of gene integration of lentivirus.

Cdc20 mediates D-box-dependent degradation of Sp100.

Cdc20 is a co-activator of the anaphase-promoting complex/cyclosome (APC/C complex), which recruits substrates at particular phases of the cell cycle and mediates their degradation. Sp100 is a PML-NB scaffold protein, which localizes to nuclear particles during interphase and disperses from them during mitosis, participates in viral resistance, transcriptional regulation, and apoptosis. However, its metabolism during the cell cycle has not yet been fully characterized. We found a putative D-box in Sp100 using the Eukaryotic Linear Motif (ELM) predictor database. The putative D-box of Sp100 was verified by mutational analysis. Overexpression of Cdc20 resulted in decreased levels of both endogenous Sp100 protein and overexpressed Sp100 mRNA in HEK 293 cells. Only an overexpressed D-box deletion mutant of Sp100 accumulated in HEK293 cells that also overexpressed Cdc20. Cdc20 knockdown by cdc20 specific siRNA resulted in increased Sp100 protein levels in cells. Furthermore, we discovered that the Cdc20 mediated degradation of Sp100 is diminished by the proteasome inhibitor MG132, which suggests that the ubiquitination pathway is involved in this process. However, unlike the other Cdc20 substrates, which display oscillating protein levels, the level of Sp100 protein remains constant throughout the cell cycle. Additionally, both overexpression and knockdown of endogenous Sp100 had no effect on the cell cycle. Our results suggested that sp100 is a novel substrate of Cdc20 and it is degraded by the ubiquitination pathway. The intact D-box of Sp100 was necessary for this process. These findings expand our knowledge of both Sp100 and Cdc20 as well as their role in ubiquitination.

Development of chimeric gene regulators for cancer-specific gene therapy with both transcriptional and translational targeting.

Cancer gene therapy has been of great challenge in achieving maximal high levels of specificity and more rational efficiency in target cancer cell. We herein developed a novel approach for cancer-specific gene therapy using both transcriptional and translational targeting regulation. We integrated the tumor-specific gene promoter of hTERT, the 5'UTR of bFGF-2, the enhancer of woodchuck hepatitis virus post-transcriptional regulatory element (WRE), and/or the 3'UTR of the human EGFR into two major chimeric gene regulators. We found that chimeric gene regulator I (hTERT_5'UTR...WRE_BGHpolyA) enhanced the specificity of expression in hepatocellular carcinoma (HCC) cells up to 300% in total due to increases at both the transcriptional and translational levels but only 120-200% enhancement at the transcriptional level and 120-180% enhancement at the translational level. In addition, chimeric gene regulator II (hTERT_5'UTR...WRE_3'UTR_BGHpolyA) improved the specificity to 550% and also highly strengthened the stability of the mRNA. In vitro cytotoxicity assays demonstrated that HCC cell growth was inhibited by HSV-1 TK expression under the control of both chimeric regulators, with a relative cell viability of approximately 80% for 2 days and approximately 85% for 4 days after transfection, respectively. These observations represent a new approach for highly tumor-specific gene expression and also provide insights into application to cancer gene therapy.

Functional differentiation between Rep-mediated site-specific integration and transcriptional repression of the adeno-associated viral p5 promoter.

The adeno-associated virus (AAV) p5 promoter controls expression of Rep68 and Rep78, which are responsible for specific integration of the viral genome into the AAVS1 site of the human genome. The p5 promoter contains a Rep-binding element (RBE) sequence that acts as a substrate of the Rep proteins for both site-specific integration of p5 itself and transcriptional suppression of the p5 promoter. To differentiate these two Rep-mediated functions, we dissected the p5 core structure TATA/RBE/YY1+1 through a series of mutations. Mutations in the TATA box or YY1+1 region of p5IEE significantly reduced Rep-mediated site-specific integration (RMSSI) and p5 promoter transcriptional activity, but only the TATA box is involved in Rep-mediated transcriptional suppression (RMTS). Point mutations at nucleotides 266, 267, 268, 270, and 273 of the GAGTGAGC motif in p5 RBE significantly reduced RMSSI efficiency. However, only p5G270T lost the affinity of Rep binding and had significant reduction of RMTS. It appears that RMTS is determined by the affinity of p5RBE for Rep whereas RMSSI requires more stringent conditions. Thus, RMTS and RMSSI can be differentiated by point mutations in the p5 promoter, which is useful in gene therapy in a helper vector to drive Rep expression, as the mutant promoters seldom integrate themselves but remain the RMTS feature for reduced cytotoxicity caused by a high level of Rep protein.

PhiC31 integrase interacts with TTRAP and inhibits NFkappaB activation.

Phage PhiC31 integrase-mediated gene delivery is believed to be safer than using retroviral vectors since the protein confines its insertion of the target gene to a limited number of sites in mammalian genomes. To evaluate its safety in human cells, it is important to understand the interactions between this integrase and cellular proteins. Here we show that PhiC31 integrase interacts with TTRAP as presented by yeast two-hybrid and co-immunoprecipitation assays. Reducing the expression of endogenous TTRAP can increase the efficiency of PhiC31 integrase-mediated integration. A possible effect of interaction between PhiC31 integrase and TTRAP was highlighted by the fact that PhiC31 integrase inhibited the NFkappaB activation mediated by IL-1 in a dose-dependent manner. Because low dose of PhiC31 integrase can mediate considerable recombination events, we suggest that low dose of PhiC31 integrase be used when this integrase is applied in human cells.

Cellular protein TTRAP interacts with HIV-1 integrase to facilitate viral integration.

TTRAP is a PML-NB protein that is involved in the NF-kappaB signaling pathway. TTRAP was recently identified by yeast two-hybrid analysis as a HIV-1 integrase (HIV-1 IN) interacting protein. This interaction was verified by co-immunoprecipitation, GST pull-down, and intracellular imaging, and deletion assays suggested that the N-terminal 180 residues of TTRAP are responsible for the interaction. In stable TTRAP knock-down cell lines, the integration of viral vectors decreased significantly compared with non-silenced cell lines. Conversely, overexpression of TTRAP by transient transfection increased the percentage of integration events. This is the first time that TTRAP has been shown to interact with HIV-1 IN and facilitate lentiviral vector integration. These findings reveal a new function of TTRAP and expand our understanding of the cellular response to HIV infection. The interaction between TTRAP and HIV-1 IN may be useful in designing new anti-viral strategies as well as for improving the efficiency of lentiviral-vector-mediated gene delivery.

TAT-phiC31 integrase mediates DNA recombination in mammalian cells.

Streptomyces phage integrase phiC31 is capable of mediating site-specific insertions in mammalian genomes. To avoid potential toxicity of long-term expression of phiC31 in host cells, we developed a method employing a cell-permeable TAT-phiC31 integrase. His6-tagged phiC31 proteins with or without an HIV TAT intercellular transducing peptide were generated and purified. Both of them retained integrase activity in vitro. However, TAT-phiC31 but not phiC31 was able to mediate a specific integration between two att sites in the genome of 293-PB [EGFP] report cell line. Transduced TAT-phiC31 was mainly localized in the cytoplasm that is similar to the localization of phiC31 when expressed through cDNA transfection. Adding a nuclear localization signal (NLS) peptide to the C-terminus of TAT-phiC31 facilitated nuclear localization of the integrase with an increased efficiency of recombination in the reporter cell line. These results demonstrated that TAT can mediate a cell membrane entry of phiC31 protein to perform a site-specific integration in mammalian cells. This is a simple and possibly safer method of site-specific recombination for gene delivery.

Vector retargeting for cancer gene therapy.

Vector tropism is a research hot spot in cancer gene therapy, and targeted viral vectors play a key role in the enhancement of safety and efficiency in cancer gene therapy. Vector retargeting is one of the important strategies on viral vector targeting. This review mainly focused on the progresses of vector retargeting in cancer gene therapy, and summarized relevant pathways and strategies.

Eliminating bacteria backbone of naked DNA enhanced hFIX expression and reduced inflammatory response in mice.

To test the hypothesis that the persistent high level of transgene expression of linear DNA eliminating bacterial backbone (LDEBB) results from less cytokine induction in vivo. We systematically investigated the effect of circular DNA (C DNA), linear DNA (L DNA) and LDEBB on gene expression in mice by hydrodynamics-based plasmid administration, and then determined serum cytokine levels in mice by enzyme linked immunosorbent assay (ELISA). The expression of human clotting factor IX (hFIX) gene in mice treated with LDEBB, L DNA or C DNA reached a maximum 1-day after injection (9809, 6447, 2368 ng/mL), respectively. Thirty days after injection, hFIX concentrations dropped to baseline in mice treated with C DNA group, while L DNA group and LDEBB group decreased to 207 and 377 ng/mL, respectively, at the same time-point. Mice receiving LDEBB encoding hFIX expressed approximately 1.5 to 20-fold more serum hFIX than mice injected with L DNA and C DNA for a period of 8 months, respectively. However, mice receiving LDEBB are much less inflammatory than L DNA and C DNA as shown by a 4-fold reduction in serum levels of both TNF-alpha and IL-12. These results demonstrate that LDEBB is not silenced and is capable of expressing persistently high levels of transgene in vivo, which result from less cytokine induction in vivo. LDEBB provides a promising approach and useful therapeutic strategy to improve naked DNA delivery.

TTRAP is a novel PML nuclear bodies-associated protein.

PML nuclear body (PML NB) is an important macromolecular nuclear structure that is involved in many essential aspects of cellular function. Tens of proteins have been found in PML NBs, and promyelocytic leukemia protein (PML) has been proven to be essential for the formation of this structure. Here, we showed that TRAF and TNF receptor-associated protein (TTRAP) was a novel PML NBs-associated protein. TTRAP colocalized with three important PML NBs-associated proteins, PML, DAXX and Sp100 in the typical fashion of PML NBs. By yeast mating assay, TTRAP was identified to interact with these PML NBs-associated proteins. The transcription and expression of TTRAP could be induced by IFN-gamma, representing another common feature of PML NBs-associated proteins. These results would not only be important for understanding PML NBs but also be helpful in studying the TTRAP function in the future.

Daxx interacts with HIV-1 integrase and inhibits lentiviral gene expression.

The death-associated protein Daxx is a ubiquitously expressed gene in mammals and is widely involved in transcriptional regulation and cellular intrinsic immune response against incoming virus. We found here that knocking down endogenous Daxx with specific siRNA increased HIV-1-derived lentiviral reporter gene expression in 293T cells. This repressive effect of Daxx is not due to its inhibition on viral gene integration into the cellular genome and is independent of the ubiquitin promoter on the vFUGW lentiviral vector. Instead, this inhibition is dependent on Daxx's interaction with HIV-1 integrase. A histone deacetylases (HDACs) inhibitor increased reporter gene expression to the level similar to Daxx knockdown in vFUGW infected cells but there was no additive effect in combination of HDACs inhibitor and Daxx-specific siRNA. Our results suggest that Daxx may associate with HIV-1-derived lentiviral DNA via interacting with HIV-1 integrase and recruit HDACs to viral DNA to repress lentiviral gene expression.

[Reconstruction and expression of recombinant lentiviral vector for human amelogenin in 293T cell line].

PURPOSE: To construct a recombinant lentiviral vector for human amelogenin and to investigate the amelogenin expression of recombinant lentivirus FUAmW in 293T cell line. METHODS: A lentiviral expression vector for human amelogenin was constructed by recombinant DNA technique. Recombinant plasmid FUAmW was confirmed by restriction endonuclease and DAN sequence analysis respectively. High tites recombinant lentivirus were prepared from 293T cells by polytheylenimine(PEI) mediated transient cotransfection. The generated recombinant FUGW viruses and recombinant FUAmW viruses were used to infect 293T cells, respectively. The expression of human amelogenin in 293T cells was detected by RT-RCR and Western-blot. RESULTS: The sequence analysis of recombinant plasmid FUAmW showed that the human amelogenin encoding mature protein was inserted into lentiviral vector FUW accurately. Human amelogenin expressions were observed in 293T cells 72 hours after infecting with recombinant FUAmW viruses. CONCLUSIONS: The recombinant lentiviral vector FUAmW can be constructed correctly and transfected into 293T cells. Human amelogenin can be expressed in 293T cells infected with recombinant FUAmw virus. Supported by National Natural Science Foundation of China (Grant No.30672315).

Cloning and expression of translation elongation factor 2 (EF-2) in zebrafish.

We have identified a developmentally regulated gene translation elongation factor 2 (EF-2) in zebrafish (GenBank Accession No. AAQ91234). Analysis of DNA sequence of zebrafish EF-2 shows that the 2826 bp cDNA spans an open reading frame from nucleotide 55 to 2631 and encodes a protein of 858 amino acids. It shares an identity of 92, 93, 93, 92, 79 and 80% in amino acid sequence to human, mouse, Chinese hamster, Gallus gullus, C. elegans and Drosophila EF-2, respectively. Zebrafish EF-2 protein has 16 conserved domains, GTP-binding domain is found in the NH2 terminus, and the ADP-ribosylation domain locates at the COOH terminus. Whole mount in situ hybridization on zebrafish embryos shows that the transcripts of EF-2 gene are detected during the early development of zebrafish embryo and constantly change from 5-somite stage to protruding-mouth stage. It expresses strongly throughout envelope at 5-somite stage. Then the stained cells concentrate strongly in the eyes, brain and muscle tissue. From prim-25 stage the stained cells only appear strongly in the lens and the anterior portion of the cerebellum.

DAXX interacts with phage PhiC31 integrase and inhibits recombination.

Phage PhiC31 integrase has potential as a means of inserting therapeutic genes into specific sites in the human genome. However, the possible interactions between PhiC31 integrase and cellular proteins have never been investigated. Using pLexA-PhiC31 integrase as bait, we screened a pB42AD-human fetal brain cDNA library for potential interacting cellular proteins. Among 61 positives isolated from 10(6) independent clones, 51 contained DAXX C-terminal fragments. The strong interaction between DAXX and PhiC31 was further confirmed by co-immunoprecipitation. Deletion analysis revealed that the fas-binding domain of DAXX is also the region for PhiC31 binding. Hybridization between a PhiC31 integrase peptide array and an HEK293 cell extract revealed that a tetramer, 451RFGK454, in the C-terminus of PhiC31 is responsible for the interaction with DAXX. This tetramer is also necessary for PhiC31 integrase activity as removal of this tetramer resulted in a complete loss of integrase activity. Co-expression of DAXX with PhiC31 integrase in a HEK293-derived PhiC31 integrase activity reporter cell line significantly reduced the PhiC31-mediated recombination rate. Knocking down DAXX with a DAXX-specific duplex RNA resulted in increased recombination efficiency. Therefore, endogenous DAXX may interact with PhiC31 causing a mild inhibition in the integration efficiency. This is the first time that PhiC31 was shown to interact with an important cellular protein and the potential effect of this interaction should be further studied.