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author:("Zheng, xinjin")
1.  New Insights into FAK Phosphorylation Based on a FAT Domain-Defective Mutation 
PLoS ONE  2014;9(9):e107134.
Mounting evidence suggests that the FAK N-terminal (FERM) domain controls FAK phosphorylation and function; however, little is known regarding the role of the C terminal (FAT) domain in FAK regulation. We identified a patient-derived FAK mutant, in which a 27-amino acid segment was deleted from the C-terminal FAT domain (named FAK-Del33). When FAK-Del33 was overexpressed in specific tumor cell lines, Y397 phosphorylation increased compared with that observed in cells expressing FAK-WT. Here, we attempt to unveil the mechanism of this increased phosphorylation. Using cell biology experiments, we show that FAK-Del33 is incapable of co-localizing with paxillin, and has constitutively high Y397 phosphorylation. With a kinase-dead mutation, it showed phosphorylation of FAK-Del33 has enhanced through auto-phosphorylation. It was also demonstrated that phosphorylation of FAK-Del33 is not Src dependent or enhanced intermolecular interactions, and that the hyperphosphorylation can be lowered using increasing amounts of transfected FERM domain. This result suggests that Del33 mutation disrupting of FAT's structural integrity and paxillin binding capacity leads to incapable of targeting Focal adhesions, but has gained the capacity for auto-phosphorylation in cis.
PMCID: PMC4166415  PMID: 25226367
2.  An aberrant spliced transcript of focal adhesion kinase is exclusively expressed in human breast cancer 
To clarify the roles of a new aberrantly spliced transcript of FAK that lacks exon 26 (denoted -26-exon FAK) in human breast cancers.
Transcripts of FAK expressed in 102 human breast tumor tissues and 52 corresponding normal tissues were analyzed by RT-PCR and DNA sequencing, as well as agarose gel electrophoresis. The cDNA of -26-exon FAK was cloned and expressed in MCF-10A cells, and then the kinase activity, cellular localization and migration capability of FAK were examined by western blotting, immunofluorescent staining and migration assays, respectively. The expression levels of FAK were analyzed by western blotting in MCF-7 cells treated with TNF-α or in MCF-10A cells upon serum deprivation. The MCF-10A cells transfected with a plasmid expressing -26-exon FAK were cultured in serum-free medium and cell apoptosis was analyzed by flow cytometry.
The -26-exon FAK transcript was exclusively present in human breast tumor tissues and the encoded protein possessed the same kinase activity, cellular localization and cell migration-promoting ability as wild-type FAK. In MCF-7 cells treated with TNF-α, and in MCF-10A cells upon serum deprivation, the -26-exon FAK was resistant to proteolysis while wild-type FAK was largely cleaved. In addition, the -26-exon FAK, but not wild-type FAK, inhibited cell apoptosis.
The -26-exon FAK transcript, which is exclusively expressed in human breast tumor tissues, encodes a protein that possesses the same kinase activity and biological function as the wild-type FAK, but because it is resistant to the caspase-mediated cleavage that induces the proteolysis of the wild-type form, it ultimately prevents apoptosis.
PMCID: PMC4040474  PMID: 24885534
Exon 26; FAK; Breast cancer; Caspase
3.  Pseudo attP sites in favor of transgene integration and expression in cultured porcine cells identified by streptomyces phage phiC31 integrase 
BMC Molecular Biology  2013;14:20.
Phage PhiC31 integrase integrates attB-containing plasmid into pseudo attP site in eukaryotic genomes in a unidirectional site-specific manner and maintains robust transgene expression. Few studies, however, explore its potential in livestock. This study aims to discover the molecular basis of PhiC31 integrase-mediated site-specific recombination in pig cells. We show that PhiC31 integrase can mediate site-specific transgene integration into the genome of pig kidney PK15 cells. Intramolecular recombination in pig PK15 cell line occurred at maximum frequency of 82% with transiently transfected attB- and attP-containing plasmids. An optimal molar ratio of pCMV-Int to pEGFP-N1-attB at 5:1 was observed for maximum number of cell clones under drug selection. Four candidate pseudo attP sites were identified by TAIL-PCR from those cell clones with single-copy transgene integration. Two of them gave rise to higher integration frequency occurred at 33%. 5′ and 3′ junction PCR showed that transgene integration mediated by PhiC31 integrase was mono-allelic. Micro- deletion and insertion were observed by sequencing the integration border, indicating that double strand break was induced by the recombination. We then constructed rescue reporter plasmids by ABI-REC cloning of the four pseudo attP sites into pBCPB + plasmid. Transfection of these rescue plasmids and pCMV-Int resulted in expected intramolecular recombination between attB and pseudo attP sites. This proved that the endogenous pseudo attP sites were functional substrates for PhiC31 integrase-mediated site-specific recombination. Two pseudo attP sites maintained robust extracellular and intracellular EGFP expression. Alamar blue assay showed that transgene integration into these specific sites had little effect on cell proliferation. This is the first report to document the potential use of PhiC31 integrase to mediate site-specific recombination in pig cells. Our work established an ideal model to study the position effect of identical transgene located in diverse chromosomal contexts. These findings also form the basis for targeted pig genome engineering and may be used to produce genetically modified pigs for agricultural and biomedical uses.
PMCID: PMC3844521  PMID: 24010979
PhiC31 integrase; Pig; Pseudo attP site; TAIL-PCR
4.  Effect of Chronic Restraint Stress on Human Colorectal Carcinoma Growth in Mice 
PLoS ONE  2013;8(4):e61435.
Stress alters immunological and neuroendocrinological functions. An increasing number of studies indicate that chronic stress can accelerate tumor growth, but its role in colorectal carcinoma (CRC) progression is not well understood. The aim of this study is to investigate the effects of chronic restraint stress (CRS) on CRC cell growth in nude mice and the possible underlying mechanisms. In this study, we showed that CRS increased the levels of plasma catecholamines including epinephrine (E) and norepinephrine (NE), and stimulated the growth of CRC cell-derived tumors in vivo. Treatment with the adrenoceptor (AR) antagonists phentolamine (PHE, α-AR antagonist) and propranolol (PRO, β-AR antagonist) significantly inhibited the CRS-enhanced CRC cell growth in nude mice. In addition, the stress hormones E and NE remarkably enhanced CRC cell proliferation and viability in culture, as well as tumor growth in vivo. These effects were antagonized by the AR antagonists PHE and PRO, indicating that the stress hormone-induced CRC cell proliferation is AR dependent. We also observed that the β-AR antagonists atenolol (ATE, β1- AR antagonist) and ICI 118,551 (ICI, β2- AR antagonist) inhibited tumor cell proliferation and decreased the stress hormone-induced phosphorylation of extracellular signal-regulated kinases-1/2 (ERK1/2) in vitro and in vivo. The ERK1/2 inhibitor U0126 also blocked the function of the stress hormone, suggesting the involvement of ERK1/2 in the tumor-promoting effect of CRS. We conclude that CRS promotes CRC xenograft tumor growth in nude mice by stimulating CRC cell proliferation through the AR signaling-dependent activation of ERK1/2.
PMCID: PMC3621827  PMID: 23585898
5.  Enrichment of Prostate Cancer Stem-Like Cells from Human Prostate Cancer Cell Lines by Culture in Serum-Free Medium and Chemoradiotherapy 
The discovery of rare subpopulations of cancer stem cells (CSCs) has created a new focus in cancer research. As CSCs demonstrate resistance to chemoradiation therapy relative to other cancer cells, this allows the enrichment of CSC populations by killing apoptosis-susceptible cancer cells. In this study, three commonly used human prostate cancer (PCa) cell lines (DU145, PC-3 and LNCaP) were examined for their expression of the putative stem cell markers CD133 and CD44 via flow cytometric analysis. Under normal culture conditions, CD133+/CD44+ cells were only present in the DU145 cell line, and comprised only a minor percentage (0.1% ± 0.01%) of the total population. However, the proportion of these CD133+/CD44+ prostate CSCs could be increased in these cell lines via culture in serum-free medium (SFM), or through chemotherapy or radiotherapy. Indeed, after culture in SFM, the proportion of CD133+/CD44+ cells in DU145 and PC-3 had increased to 10.3% and 3.0%, respectively. Moreover, the proportion had increased to 9.8% enriched by chemotherapy and 3.5% by radiotherapy in DU145. Colony-formation tests, cell invasion assays, and tumor xenografts in BALB/c nude mice were used to evaluate the stem cell properties of CD133+/CD44+ PCa cells that were isolated via fluorescence-activated cell sorting (FACS). CD133+/CD44+ cells had an enhanced colony-formation capability and invasive ability in vitro, and displayed greater tumorigenic properties in vivo. These results demonstrate the presence of CD133+/CD44+ prostate CSCs in established PCa cell lines and that populations of these cells can be enriched by culture in SFM or chemoradiotherapy. Finding novel therapies to override chemoradiation resistance in the prostate CSCs is the key to improve long-term results in PCa management.
PMCID: PMC3677682  PMID: 23781140
Cancer stem cells; Prostate cancer; Serum-free medium culture; Chemotherapy; Radiotherapy.
6.  An asymmetric PCR-based, reliable and rapid single-tube native DNA engineering strategy 
BMC Biotechnology  2012;12:39.
Widely used restriction-dependent cloning methods are labour-intensive and time-consuming, while several types of ligase-independent cloning approaches have inherent limitations. A rapid and reliable method of cloning native DNA sequences into desired plasmids are highly desired.
This paper introduces ABI-REC, a novel strategy combining asymmetric bridge PCR with intramolecular homologous recombination in bacteria for native DNA cloning. ABI-REC was developed to precisely clone inserts into defined location in a directional manner within recipient plasmids. It featured an asymmetric 3-primer PCR performed in a single tube that could robustly amplify a chimeric insert-plasmid DNA sequence with homologous arms at both ends. Intramolecular homologous recombination occurred to the chimera when it was transformed into E.coli and produced the desired recombinant plasmids with high efficiency and fidelity. It is rapid, and does not involve any operational nucleotides. We proved the reliability of ABI-REC using a double-resistance reporter assay, and investigated the effects of homology and insert length upon its efficiency. We found that 15 bp homology was sufficient to initiate recombination, while 25 bp homology had the highest cloning efficiency. Inserts up to 4 kb in size could be cloned by this method. The utility and advantages of ABI-REC were demonstrated through a series of pig myostatin (MSTN) promoter and terminator reporter plasmids, whose transcriptional activity was assessed in mammalian cells. We finally used ABI-REC to construct a pig MSTN promoter-terminator cassette reporter and showed that it could work coordinately to express EGFP.
ABI-REC has the following advantages: (i) rapid and highly efficient; (ii) native DNA cloning without introduction of extra bases; (iii) restriction-free; (iv) easy positioning of directional and site-specific recombination owing to formulated primer design. ABI-REC is a novel approach to DNA engineering and gene functional analysis.
PMCID: PMC3408372  PMID: 22768962
Asymmetric; Bridge PCR; Intramolecular homologous recombination; Myostatin

Results 1-6 (6)