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1.  Effect of Age and Biological Subtype on the Risk and Timing of Brain Metastasis in Breast Cancer Patients 
PLoS ONE  2014;9(2):e89389.
Brain metastasis is a major complication of breast cancer. This study aimed to analyze the effect of age and biological subtype on the risk and timing of brain metastasis in breast cancer patients.
Patients and Methods
We identified subtypes of invasive ductal carcinoma of the breast by determining estrogen receptor, progesterone receptor and HER2 status. Time to brain metastasis according to age and cancer subtype was analyzed by Cox proportional hazard analysis.
Of the 2248 eligible patients, 164 (7.3%) developed brain metastasis over a median follow-up of 54.2 months. Age 35 or younger, HER2-enriched subtype, and triple-negative breast cancer were significant risk factors of brain metastasis. Among patients aged 35 or younger, the risk of brain metastasis was independent of biological subtype (P = 0.507). Among patients aged 36–59 or >60 years, those with triple-negative or HER2-enriched subtypes had consistently increased risk of brain metastasis, as compared with those with luminal A tumors. Patients with luminal B tumors had higher risk of brain metastasis than luminal A only in patients >60 years.
Breast cancer subtypes are associated with differing risks of brain metastasis among different age groups. Patients age 35 or younger are particularly at risk of brain metastasis independent of biological subtype.
PMCID: PMC3933537  PMID: 24586742
2.  Production of shikimic acid from Escherichia coli through chemically inducible chromosomal evolution and cofactor metabolic engineering 
Shikimic acid (SA) produced from the seeds of Chinese star anise (Illicium verum) is a key intermediate for the synthesis of neuraminidase inhibitors such as oseltamivir (Tamiflu®), an anti-influenza drug. However, plants cannot deliver a stable supply of SA. To avoid the resulting shortages and price fluctuations, a stable source of affordable SA is required. Although recent achievements in metabolic engineering of Escherichia coli strains have significantly increased SA productivity, commonly-used plasmid-based expression systems are prone to genetic instability and require constant selective pressure to ensure plasmid maintenance. Cofactors also play an important role in the biosynthesis of different fermentation products. In this study, we first constructed an E. coli SA production strain that carries no plasmid or antibiotic marker. We then investigated the effect of endogenous NADPH availability on SA production.
The pps and csrB genes were first overexpressed by replacing their native promoter and integrating an additional copy of the genes in a double gene knockout (aroK and aroL) of E. coli. The aroG fbr , aroB, aroE and tktA gene cluster was integrated into the above E. coli chromosome by direct transformation. The gene copy number was then evolved to the desired value by triclosan induction. The resulting strain, E. coli SA110, produced 8.9-fold more SA than did the parental strain E. coli (ΔaroKΔaroL). Following qRT-PCR analysis, another copy of the tktA gene under the control of the 5Ptac promoter was inserted into the chromosome of E. coli SA110 to obtain the more productive strain E. coli SA110. Next, the NADPH availability was increased by overexpressing the pntAB or nadK genes, which further enhanced SA production. The final strain, E. coli SA116, produced 3.12 g/L of SA with a yield on glucose substrate of 0.33 mol/mol.
An SA-producing E. coli strain that carries neither a plasmid nor an antibiotic marker was constructed by triclosan-induced chromosomal evolution. We present the first demonstration that increasing NADPH availability by overexpressing the pntAB or nadK genes significantly enhances SA production.
PMCID: PMC3923554  PMID: 24512078
Shikimic acid; Escherichia coli; Chemically induced chromosomal evolution; NADPH; Transhydrogenase; NAD kinase
3.  Polyadenylated Messenger RNA in Paired Helical Filament-Immunoreactive Neurons in Alzheimer Disease 
An antibody raised against isolated paired helical filaments (PHF) was used to identify tangle-bearing (PHF+) neurons in autopsy brain tissue from six Alzheimer disease (AD) patients and six age-matched controls (AMC). A comparison of the levels of polyadenylated messenger RNA [poly(A)+ mRNA] in PHF+ and PHF− neurons of similar cross-sectional area in temporal and parietal lobe and cerebellum from four AD and four AMC brains was made by analysis of in situ hybridization of [3H] polyuridylate [poly(U)] to intracellular poly(A)+ mRNA. In PHF+ neurons, the level of poly(A)+. mRNA was approximately two-thirds that in similar-sized PHF− neurons in either AD or AMC. The level of poly(A)+ mRNA in PHF− neurons in regions of the brain that have more of the histopathologically defined effects in AD was similar to that in regions with less effects.
PMCID: PMC3886638  PMID: 1972628
Paired helical filament; Tangle-bearing neurons; Alzheimer disease; Polyadenylated messenger RNA
4.  Ursolic Acid Provides Kidney Protection in Diabetic Rats☆ 
Diabetic nephropathy (DN) is one of the most serious microvascular complications of diabetes and the leading cause of end-stage renal failure. However, the treatment of DN is still a problem in the world. Inflammatory process plays a critical role in the development of DN. Therefore, anti-inflammatory treatment of DN is worth exploring now and in the future.
The study aimed to evaluate the impact of ursolic acid (UA) on renal function in streptozotocin-induced diabetes.
Rats with streptozotocin-induced diabetes were treated with UA for 16 weeks. After 16 weeks, urine albumin excretion, serum creatinine, and blood urea nitrogen were measured. In addition, renal oxidative stress level, nuclear factor kappa-B (NF-κB) activity, P-selectin expression, and kidney histopathologic changes were evaluated.
Sixteen weeks following streptozotocin injection, the rats produced significant alteration in renal function and increased oxidative stress, NF-κB activity, and P-selectin expression in the kidneys. Interestingly, UA significantly prevented biochemical and histopathologic changes in the kidneys associated with diabetes. Compared with untreated diabetic rats, UA treatment lowered urine albumin excretion, renal oxidative stress level, NF-κB activity, and P-selectin expression. Moreover, UA treatment also improved renal histopathologic changes in rats with diabetes.
UA treatment exhibited a protective effect on kidneys in diabetic rats, implying that UA could be a potential treatment for diabetic nephropathy.
PMCID: PMC3898186  PMID: 24465045
diabetic nephropathy; nuclear factor kappa-B; oxidative stress; ursolic acid
5.  Evaluation of conservative treatment and timing of surgical intervention for mild forms of cervical spondylotic myelopathy 
The optimal management approach for patients with mild forms of cervical spondylotic myelopathy (MCSM) has not been well established. The aim of the present study was to investigate the outcome of conservative treatment, identify prognostic factors and provide evidence for the timing of surgical intervention. A total of 90 patients with MCSM attending hospital between February 2007 and January 2009 were prospectively enrolled. Initially, all patients received conservative treatment and were followed up periodically. When a deterioration in myelopathy was clearly identified, surgical treatment was conducted. Clinical and radiological factors correlating with the deterioration were examined, and final clinical outcomes were evaluated using the Japanese Orthopedic Association (JOA) score. At the end of January 2012, follow-ups of >3 years were completed. Seventy-eight patients were available for data analysis. Only 21 patients (26.9%) deteriorated and underwent surgery thereafter (group A), while the remaining 57 patients (73.1%) were treated conservatively throughout (group B). Statistical analysis revealed that segmental instability and cervical spinal stenosis were adverse factors for the prognosis of conservative treatment. Although the JOA scores of the patients in group A declined initially, following surgical intervention, no significant differences were identified in JOA scores between the two groups at the time of the final follow-up (P=0.46). In summary, conservative treatment is effective in MCSM patients. Patients with segmental instability and cervical spinal stenosis have a tendency to deteriorate, but conservative treatment remains the recommendation for the first action. If the myelopathy deteriorates during conservative treatment, timely surgical intervention is effective.
PMCID: PMC3786935  PMID: 24137278
cervical myelopathy; conservative treatment; surgical treatment; prognosis
6.  A ubiquitin-binding protein FAAP20 links RNF8-mediated ubiquitination to the Fanconi anemia DNA Repair network 
Molecular Cell  2012;47(1):61-75.
The Fanconi anemia (FA) protein network is necessary for repair of DNA interstrand crosslinks (ICLs), but its control mechanism remains unclear. Here we show that the network is regulated by a ubiquitin signaling cascade initiated by RNF8 and its partner, UBC13; and mediated by FAAP20, a component of the FA core complex. FAAP20 preferentially binds the ubiquitin product of RNF8-UBC13; and its recruitment to ICLs requires this ubiquitin-binding activity, RNF8 and UBC13. Both RNF8 and FAAP20 are required for recruitment of FA core complex and FANCD2 to ICLs, whereas RNF168 can modulate efficiency of the recruitment. RNF8 and FAAP20 are needed for efficient FANCD2 monoubiquitination, a key step of the FA network; RNF8 and FA core complex work in the same pathway to promote cellular resistance to ICLs. Thus, the RNF8-FAAP20 ubiquitin cascade is critical for recruiting FA core complex to ICLs and for normal function of the FA network.
PMCID: PMC3398238  PMID: 22705371
RNF8; FAAP20; RNF168; UBC13; Fanconi Anemia; ubiquitin
7.  High-efficiency Transduction of Human Monocyte-derived Dendritic Cells by Capsid-modified Recombinant AAV2 Vectors 
Vaccine  2012;30(26):3908-3917.
Phosphorylation of surface-exposed tyrosine residues negatively impacts the transduction efficiency of recombinant AAV2 vectors. Pre-treatment of cells with specific cellular serine/threonine kinase inhibitors also significantly increased the transduction efficiency of AAV2 vectors. We reasoned that site-directed mutagenesis of surface-exposed serine residues might allow the vectors to evade phosphorylation and thus lead to higher transduction efficiency. Each of the 15 surface-exposed serine (S) residues was substituted with valine (V) residues, and the transduction efficiency of three of these mutants, S458V, S492Vand S662V, was increased by up to∼20-fold in different cell types. The S662V mutant was efficient in transducing human monocyte-derived dendritic cells (moDCs), a cell type not readily amenable to transduction by the conventional AAV vectors, and did not induce any phenotypic changes in these cells. Recombinant S662V-AAV2 vectors encoding a truncated human telomerase (hTERT) gene were generated and used to stimulate cytotoxic T cells (CTLs) against target cells. S662V-AAV2-hTERT vector-transduced DCs resulted in rapid, specific T-cell clone proliferation and generation of robust CTLs, which led to specific cell lysis of K562 cells. These studies suggest that high-efficiency transduction of moDCs by serine-modified AAV2 vectors is feasible, which supports the potential utility of these vectors for future human DC vaccine studies.
PMCID: PMC3356484  PMID: 22497875
adeno-assosiated virus vectors; capsid proteins; serine-phosphorylation; serine/threonine kinase; dendritic cells; gene expression
8.  Optimization of the Capsid of Recombinant Adeno-Associated Virus 2 (AAV2) Vectors: The Final Threshold? 
PLoS ONE  2013;8(3):e59142.
The ubiquitin-proteasome pathway plays a critical role in the intracellular trafficking of AAV2 vectors, and phosphorylation of certain surface-exposed amino acid residues on the capsid provides the primary signal for ubiquitination. Removal of several critical tyrosine (Y) and serine (S) residues on the AAV2 capsid has been shown to significantly increase transduction efficiency compared with the wild-type (WT) vectors. In the present study, site-directed mutagenesis of each of the 17 surface-exposed threonine (T) residues was conducted, and the transduction efficiency of four of these mutants, T455V, T491V, T550V, and T659V, was observed to increase up to 4-fold in human HEK293 cells in vitro. The most critical Y, S, and T mutations were subsequently combined, and the quadruple-mutant (Y444+500+730F+T491V) AAV2 vector was identified as the most efficient. This vector increased the transduction efficiency ∼24-fold over the WT AAV2 vector, and ∼2–3-fold over the previously described triple-mutant (Y444+500+730F) vector in a murine hepatocyte cell line, H2.35, in vitro. Similar results were obtained in murine hepatocytes in vivo following tail vein injection of the Y444+500+730F+T491V scAAV2 vector, and whole-body bioluminescence imaging of C57BL/6 mice. The increase in the transduction efficiency of the Y-T quadruple-mutant over that of the Y triple-mutant correlated with an improved nuclear translocation of the vectors, which exceeded 90%. These observations suggest that further optimization of the AAV2 capsid by targeting amino acid residues involved in phosphorylation may not be possible. This study has thus led to the generation of a novel Y444+500+730F+T491V quadruple-mutant AAV2 vector with potential for use in liver-directed human gene therapy.
PMCID: PMC3602601  PMID: 23527116
9.  High-Efficiency Transduction of Primary Human Hematopoietic Stem Cells and Erythroid Lineage-Restricted Expression by Optimized AAV6 Serotype Vectors In Vitro and in a Murine Xenograft Model In Vivo 
PLoS ONE  2013;8(3):e58757.
We have observed that of the 10 AAV serotypes, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs), and that the transduction efficiency can be further increased by specifically mutating single surface-exposed tyrosine (Y) residues on AAV6 capsids. In the present studies, we combined the two mutations to generate a tyrosine double-mutant (Y705+731F) AAV6 vector, with which >70% of CD34+ cells could be transduced. With the long-term objective of developing recombinant AAV vectors for the potential gene therapy of human hemoglobinopathies, we generated the wild-type (WT) and tyrosine-mutant AAV6 vectors containing the following erythroid cell-specific promoters: β-globin promoter (βp) with the upstream hyper-sensitive site 2 (HS2) enhancer from the β-globin locus control region (HS2-βbp), and the human parvovirus B19 promoter at map unit 6 (B19p6). Transgene expression from the B19p6 was significantly higher than that from the HS2-βp, and increased up to 30-fold and up to 20-fold, respectively, following erythropoietin (Epo)-induced differentiation of CD34+ cells in vitro. Transgene expression from the B19p6 or the HS2-βp was also evaluated in an immuno-deficient xenograft mouse model in vivo. Whereas low levels of expression were detected from the B19p6 in the WT AAV6 capsid, and that from the HS2-βp in the Y705+731F AAV6 capsid, transgene expression from the B19p6 promoter in the Y705+731F AAV6 capsid was significantly higher than that from the HS2-βp, and was detectable up to 12 weeks post-transplantation in primary recipients, and up to 6 additional weeks in secondary transplanted animals. These data demonstrate the feasibility of the use of the novel Y705+731F AAV6-B19p6 vectors for high-efficiency transduction of HSCs as well as expression of the b-globin gene in erythroid progenitor cells for the potential gene therapy of human hemoglobinopathies such as β-thalassemia and sickle cell disease.
PMCID: PMC3597592  PMID: 23516552
10.  Development of optimized AAV3 serotype vectors: Mechanism of high-efficiency transduction of human liver cancer cells 
Gene therapy  2011;19(4):375-384.
Our recent studies have revealed that among the 10 different commonly used AAV serotypes, AAV3 vectors transduce human liver cancer cells extremely efficiently because these cells express high levels of human hepatocyte growth factor receptor (hHGFR), and AAV3 utilizes hHGFR as a cellular co-receptor for viral entry. In this report, we provide further evidence that both extracellular as well as intracellular kinase domains of hHGFR are involved in AAV3 vector entry and AAV3-mediated transgene expression. We also document that AAV3 vectors are targeted for degradation by the host cell proteasome machinery, and that site-directed mutagenesis of surface exposed tyrosine (Y) to phenylalanine (F) residues on AAV3 capsids significantly improves the transduction efficiency of Y701F, Y705F and Y731F mutant AAV3 vectors. The transduction efficiency of the Y705+731F double-mutant vector is significantly higher than each of the single-mutants in liver cancer cells in vitro. In immuno-deficient mouse xenograft models, direct intra-tumor injection of AAV3 vectors also led to high-efficiency transduction of human liver tumor cells in vivo. We also document here that the optimized tyrosine-mutant AAV3 vectors lead to increased transduction efficiency following both intra-tumor and tail-vein injections in vivo. The optimized tyrosine-mutant AAV3 serotype vectors containing pro-apoptotic genes should prove useful for the potential gene therapy of human liver cancers.
PMCID: PMC3519243  PMID: 21776025
AAV vectors; tyrosine mutants; human hepatocyte growth factor receptor; human liver cancer; gene therapy
11.  A Simple Method to Increase the Transduction Efficiency of Single-Stranded Adeno-Associated Virus Vectors In Vitro and In Vivo 
Human Gene Therapy  2011;22(5):633-640.
We have recently shown that co-administration of conventional single-stranded adeno-associated virus 2 (ssAAV2) vectors with self-complementary (sc) AAV2-protein phosphatase 5 (PP5) vectors leads to a significant increase in the transduction efficiency of ssAAV2 vectors in human cells in vitro as well as in murine hepatocytes in vivo. In the present study, this strategy has been further optimized by generating a mixed population of ssAAV2-EGFP and scAAV2-PP5 vectors at a 10:1 ratio to achieve enhanced green fluorescent protein (EGFP) transgene expression at approximately 5- to 10-fold higher efficiency, both in vitro and in vivo. This simple coproduction method should be adaptable to any ssAAV serotype vector containing transgene cassettes that are too large to be encapsidated in scAAV vectors.
Previous studies by this group have shown that coadministration of conventional single-stranded adenoassociated virus serotype 2 (ssAAV2) vectors with self-complementary (sc) AAV2–protein phosphatase-5 (PP5) vectors leads to a significant increase in the transduction efficiency of ssAAV2 vectors in human cells in vitro as well as in murine hepatocytes in vivo. Here, Ma and colleagues expand on those previous findings and describe a quadruple-plasmid transfection protocol that results in the production of ssAAV vectors that transduce cells up to 9-fold more efficiently than in the absence of PP5 coexpression.
PMCID: PMC3081437  PMID: 21219084
12.  Transplantation of insulin-producing cells from umbilical cord mesenchymal stem cells for the treatment of streptozotocin-induced diabetic rats 
Although diabetes mellitus (DM) can be treated with islet transplantation, a scarcity of donors limits the utility of this technique. This study investigated whether human mesenchymal stem cells (MSCs) from umbilical cord could be induced efficiently to differentiate into insulin-producing cells. Secondly, we evaluated the effect of portal vein transplantation of these differentiated cells in the treatment of streptozotocin-induced diabetes in rats.
MSCs from human umbilical cord were induced in three stages to differentiate into insulin-producing cells and evaluated by immunocytochemistry, reverse transcriptase, and real-time PCR, and ELISA. Differentiated cells were transplanted into the liver of diabetic rats using a Port-A catheter via the portal vein. Blood glucose levels were monitored weekly.
Human nuclei and C-peptide were detected in the rat liver by immunohistochemistry. Pancreatic β-cell development-related genes were expressed in the differentiated cells. C-peptide release was increased after glucose challenge in vitro. Furthermore, after transplantation of differentiated cells into the diabetic rats, blood sugar level decreased. Insulin-producing cells containing human C-peptide and human nuclei were located in the liver.
Thus, a Port-A catheter can be used to transplant differentiated insulin-producing cells from human MSCs into the portal vein to alleviate hyperglycemia among diabetic rats.
PMCID: PMC3404952  PMID: 22545626
Mesenchymal stem cell; Portal vein; Insulin-producing cells; Transplant
13.  Human Hepatocyte Growth Factor Receptor Is a Cellular Coreceptor for Adeno-Associated Virus Serotype 3 
Human Gene Therapy  2010;21(12):1741-1747.
Ling et al. identify human hepatocyte growth factor (hHGFR) as a novel receptor/coreceptor necessary for AAV3 entry into liver-specific cells. Using a variety of methods, the authors show that interference with the cell surface expression of HGFR significantly reduces the transduction efficiency of AAV3 vectors. Subsequent in vitro and in vivo experiments revealed that AAV3 specifically utilizes human HGFR (hHGFR), and not mouse HGFR (mHGFR).
Adeno-associated viruses (AAVs) use a variety of cellular receptors/coreceptors to gain entry into cells. A number of AAV serotypes are now available, and the cognate receptors/coreceptors for only a handful of those have been identified thus far. Of the 10 commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells in general. However, in our more recent studies, we observed that AAV3 vectors transduced human liver cancer cells remarkably well, which led to the hypothesis that AAV3 uses hepatocyte growth factor receptor (HGFR) as a cellular coreceptor for viral entry. AAV3 infection of human liver cancer cell lines was strongly inhibited by hepatocyte growth factor, HGFR-specific small interfering RNA, and anti-HGFR antibody, which corroborated this hypothesis. However, AAV3 vectors failed to transduce murine hepatocytes, both in vitro and in vivo, suggesting that AAV3 specifically uses human HGFR, but not murine HGFR, as a cellular coreceptor for transduction. AAV3 may prove to be a useful vector for targeting human liver cancers for the potential gene therapy.
PMCID: PMC2999576  PMID: 20545554
14.  High-Efficiency Transduction of Fibroblasts and Mesenchymal Stem Cells by Tyrosine-Mutant AAV2 Vectors for Their Potential Use in Cellular Therapy 
Human Gene Therapy  2010;21(11):1527-1543.
Previous reports have shown that site-directed mutagenesis of surface-exposed tyrosine residues on the capsid of AAV2 leads to enhanced transduction efficiency. In this report, Li and colleagues use these AAV2 tyrosine mutants and demonstrate that they are capable of transducing up to 90% of mouse and human bone marrow derived mesenchymal stem cells.
Adeno-associated virus 2 (AAV2) vectors transduce fibroblasts and mesenchymal stem cells (MSCs) inefficiently, which limits their potential widespread applicability in combinatorial gene and cell therapy. We have reported that AAV2 vectors fail to traffic efficiently to the nucleus in murine fibroblasts. We have also reported that site-directed mutagenesis of surface-exposed tyrosine residues on viral capsids leads to improved intracellular trafficking of the mutant vectors, and the transduction efficiency of the single tyrosine-mutant vectors is ∼10-fold higher in human cells. In the current studies, we evaluated the transduction efficiency of single as well as multiple tyrosine-mutant AAV2 vectors in murine fibroblasts. Our results indicate that the Y444F mutant vectors transduce these cells most efficiently among the seven single-mutant vectors, with >30-fold increase in transgene expression compared with the wild-type vectors. When the Y444F mutation is combined with additional mutations (Y500F and Y730F), the transduction efficiency of the triple-mutant vectors is increased by ∼130-fold and the viral intracellular trafficking is also significant improved. Similarly, the triple-mutant vectors are capable of transducing up to 80–90% of bone marrow-derived primary murine as well as human MSCs. Thus, high-efficiency transduction of fibroblasts with reprogramming genes to generate induced pluripotent stem cells, and the MSCs for delivering therapeutic genes, should now be feasible with the tyrosine-mutant AAV vectors.
Overview Summary
Fibroblasts and mesenchymal stem cells (MSCs) are promising targets for gene and cell therapy. Recombinant adeno-associated virus 2 (AAV2) vectors are currently in use in several Phase I/II clinical trials for gene therapy. However, the existing data show that AAV2 vector-mediated transduction of fibroblasts and MSCs is inefficient. We observed that AAV2 vectors containing mutations in three surface-exposed tyrosine residues significantly increase transduction efficiency in fibroblasts, as well as in both human and murine primary MSCs. The increased transduction efficiency of these triple-mutant AAV2 vectors correlates well with improved viral intracellular trafficking in fibroblasts. These data provide strong evidence that high-efficiency gene delivery and transgene expression by AAV vectors are indeed feasible, which should facilitate the generation of induced pluripotent stem cells, as well as the use of MSCs in combinatorial gene and cell therapy.
PMCID: PMC2987376  PMID: 20507237
15.  A histone-fold complex and FANCM form a conserved DNA remodeling complex to maintain genome stability 
Molecular cell  2010;37(6):865-878.
FANCM remodels branched DNA structures and plays essential roles in the cellular response to DNA replication stress. Here we show that FANCM forms a conserved DNA remodeling complex with a histone-fold heterodimer, MHF. We find that MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA remodeling complex that protects replication forks from yeast to human.
PMCID: PMC2847587  PMID: 20347428
FANCM; Fanconi Anemia; DNA repair; Histone fold; CENP-S; CENP-X
16.  High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors  
Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.
PMCID: PMC3197307  PMID: 21445055
17.  Mirror-Image Arachnoid Cysts in a Pair of Monozygotic Twins: A Case Report and Review of the Literature 
Mirror-imaging of arachnoid cysts (ACs) in monozygotic twins (MZ) is extremely rare. We describe a pair of MZ who developed mirror-imaging of ACs in the temporal fossas, and we also review the literature. Brain computer tomography (CT) and Magnetic Resonance Imaging (MRI) of the MZ revealed mirror-imaging of vast lesions of cerebrospinal fluid intensity in their temporal fossas. This is the second ever report of such a case according to the available literature. Unlike the prior case, our patients were 14 months, which is a much younger age than the patients of the previous report. Consequently, our case is better in supporting a genetic origin in the pathogenesis of AC. The findings in our case indicate that early neuroimaging is mandatory in the counterpart of the symptomatic patient with AC, irrespective of the absence of symptoms.
PMCID: PMC3133845  PMID: 21750644
Mirror image; Arachnoid cyst; Monozygotic twins
18.  14-3-3ζ Cooperates with ErbB2 to Promote Progression of Ductal Carcinoma in Situ to Invasive Breast Cancer by Inducing Epithelial-Mesenchymal Transition 
Cancer cell  2009;16(3):195-207.
ErbB2, a metastasis-promoting oncoprotein, is overexpressed in ~25% of invasive/metastatic breast cancers, but in 50–60% of non-invasive ductal carcinomas in situ (DCIS). It has been puzzling how a subset of ErbB2-overexpressing DCIS develops into invasive breast cancer (IBC). We found that co-overexpression of 14-3-3ζ in ErbB2-overexpressing DCIS conferred a higher risk of progression to IBC. ErbB2 and 14-3-3ζ overexpression, respectively, increased cell migration and decreased cell adhesion, two prerequisites of tumor cell invasion. 14-3-3ζ overexpression reduced cell adhesion by activating the TGFβ/Smads pathway that led to ZFHX1B/SIP-1 upregulation, E-cadherin loss, and epithelial-mesenchymal transition (EMT). Importantly, patients whose breast tumors overexpressed both ErbB2 and 14-3-3ζ had higher rates of metastatic recurrence and death than those whose tumors overexpressed only one.
PMCID: PMC2754239  PMID: 19732720
19.  Negative Autoregulation of Epstein-Barr Virus (EBV) Replicative Gene Expression by EBV SM Protein▿  
Journal of Virology  2009;83(16):8041-8050.
The Epstein-Barr virus (EBV) SM protein is essential for lytic EBV DNA replication and virion production. When EBV replication is induced in cells infected with an SM-deleted recombinant EBV, approximately 50% of EBV genes are expressed inefficiently. When EBV replication is rescued by transfection of SM, SM enhances expression of these genes by direct and indirect mechanisms. While expression of most EBV genes is either unaffected or enhanced by SM, expression of several genes is decreased in the presence of SM. Expression of BHRF1, a homolog of cellular bcl-2, is particularly decreased in the presence of SM. Investigation of the mechanism of BHRF1 downregulation revealed that SM downregulates expression of the immediate-early EBV transactivator R. In EBV-infected cells, R-responsive promoters, including the BHRF1 and SM promoters, were less active in the presence of SM, consistent with SM inhibition of R expression. SM decreased spliced R mRNA levels, supporting a posttranscriptional mechanism of R inhibition. R and BHRF1 expression were also found to decrease during later stages of EBV lytic replication in EBV-infected lymphoma cells. These data indicate that feedback regulation of immediate-early and early genes occurs during the lytic cycle of EBV regulation.
PMCID: PMC2715772  PMID: 19515786
20.  Silencing of Tumor Necrosis Factor Receptor 1 by siRNA in EC109 Cells Affects Cell Proliferation and Apoptosis 
Tumor necrosis factor receptor 1 (TNFR1) is a membrane receptor able to bind TNF-α or TNF-β. TNFR1 can suppress apoptosis by activating the NF-κB or JNK/SAPK signal transduction pathway, or it can induce apoptosis through a series of caspase cascade reactions; the particular effect may depend on the cell line. In the present study, we first showed that TNFR1 is expressed at both the gene and protein levels in the esophageal carcinoma cell line EC109. Then, by applying a specific siRNA, we silenced the expression of TNFR1; this resulted in a significant time-dependent promotion of cell proliferation and downregulation of the apoptotic rate. These results suggest that TNFR1 is strongly expressed in the EC109 cell line and that it may play an apoptosis-mediating role, which may be suppressed by highly activated NF-κB.
PMCID: PMC2760352  PMID: 19826638
21.  A Human Orthologue of Archaeal DNA Repair Protein Hef is Defective in Fanconi Anemia Complementation Group M 
Nature genetics  2005;37(9):958-963.
Fanconi anemia (FA) is a genetic disease featuring genomic instability and cancer predisposition1. Nine FA genes have been identified, and their products participate in a DNA damage response network involving BRCA1 and BRCA22,3. We have previously purified a FA core complex containing the FANCL ubiquitin ligase and 6 other FA proteins4–6. Each protein in this complex is essential for monoubiquitination of FANCD2, a key reaction in the FA DNA damage response pathway2,7. Here we show that another component of this complex, FAAP250, is mutated in FA patients of a new complementation group (FA-M). FAAP250, renamed FANCM, has sequence similarity to known DNA repair proteins, including archaeal Hef, yeast Mph1 and human ERCC4/XPF. FANCM can dissociate DNA triplex, possibly due to its ability to translocate on duplex DNA. FANCM is essential for FANCD2 monoubiquitination and becomes hyperphosphorylated in response to DNA damage. Our data suggest an evolutionary link between FA proteins and DNA repair; FANCM may act as an engine that translocates the FA core complex along DNA.
PMCID: PMC2704909  PMID: 16116422
Fanconi anemia; FANCM; Hef; MPH1; XPF/ERCC4; FANCD2
22.  FANCM of the Fanconi anemia core complex is required for both monoubiquitination and DNA repair 
Human Molecular Genetics  2008;17(11):1641-1652.
In response to DNA damage, the Fanconi anemia (FA) core complex functions as a signaling machine for monoubiquitination of FANCD2 and FANCI. It remains unclear whether this complex can also participate in subsequent DNA repair. We have shown previously that the FANCM constituent of the complex contains a highly conserved helicase domain and an associated ATP-dependent DNA translocase activity. Here we show that FANCM also possesses an ATP-independent binding activity and an ATP-dependent bi-directional branch-point translocation activity on a synthetic four-way junction DNA, which mimics intermediates generated during homologous recombination or at stalled replication forks. Using an siRNA-based complementation system, we found that the ATP-dependent activities of FANCM are required for cellular resistance to a DNA-crosslinking drug, mitomycin C, but not for the monoubiquitination of FANCD2 and FANCI. In contrast, monoubiquitination requires the entire helicase domain of FANCM, which has both ATP dependent and independent activities. These data are consistent with participation of FANCM and its associated FA core complex in the FA pathway at both signaling through monoubiquitination and the ensuing DNA repair.
PMCID: PMC2902294  PMID: 18285517
23.  BAF250B-associated SWI/SNF chromatin-remodeling complex is required to maintain undifferentiated mouse embryonic stem cells 
Stem cells (Dayton, Ohio)  2008;26(5):1155-1165.
Whether SWI/SNF chromatin remodeling complexes play roles in embryonic stem (ES) cells remains unknown. Here we show that SWI/SNF complexes are present in mouse ES cells, and their composition is dynamically regulated upon induction of ES cell differentiation. For example, the SWI/SNF purified from undifferentiated ES cells contains a high level of BAF155 and a low level of BAF170 (both of which are homologs of yeast SWI3 protein), whereas that from differentiated cells contains near equal amounts of both. Moreover, the levels of BAF250A and BAF250B decrease, whereas that of BRM increases, during the differentiation of ES cells. The altered expression of SWI/SNF components hinted that these complexes could play roles in ES cell maintenance or differentiation. We therefore generated ES cells with biallelic inactivation of BAF250B, and found that these cells display a reduced proliferation rate and an abnormal cell cycle. Importantly, these cells are deficient in self-renewal capacity of undifferentiated ES cells, and exhibit certain phenotypes of differentiated cells, including reduced expression of several pluripotency-related genes, and increased expression of some differentiation-related genes. These data suggest that the BAF250B-associated SWI/SNF is essential for mouse ES cells to maintain its normal proliferation and pluripotency. The work presented here underscores the importance of SWI/SNF chromatin remodeling complexes in pluripotent stem cells.
PMCID: PMC2409195  PMID: 18323406
chromatin remodeling; SWI/SNF; BAF250B; ARID1B; embryonic stem cells; ES cells; gene targeting; DNA microarray; gene expression profiling; embryonal carcinoma cells; EC cells; Pou5f1; Oct4; Oct3/4; trophoblast; primitive endoderm; BRG1; SMARCA4; BRM; SMARCA2; BAF250A; ARID1A; BAF180; PBRM1; BAF170; SMARCC2; BAF155; SMARCC1; BAF60A; SMARCD1; BAF57; SMARCE1; BAF53A; ACTL6A; ACTIN; hSNF5/INI1; SMARCB1
24.  An EGR2/CITED1 Transcription Factor Complex and the 14-3-3σ Tumor Suppressor Are Involved in Regulating ErbB2 Expression in a Transgenic-Mouse Model of Human Breast Cancer▿  
Molecular and Cellular Biology  2007;27(24):8648-8657.
Amplification and elevated expression of the ErbB2 receptor tyrosine kinase occurs in 20% of human breast cancers and is associated with a poor prognosis. We have previously demonstrated that mammary tissue-specific expression of activated ErbB2 under the control of its endogenous promoter results in mammary tumor formation. Tumor development was associated with amplification and overexpression of ErbB2 at both the transcript and protein levels. Here we demonstrate that the EGR2/Krox20 transcription factor and its coactivator CITED1 are coordinately upregulated during ErbB2 tumor induction. We have identified an EGR2 binding site in the erbB2 promoter and demonstrated by chromatin immunoprecipitation assays that EGR2 and CITED1 associate specifically with this region of the promoter. EGR2 and CITED1 were shown to associate, and expression from an erbB2 promoter-reporter construct was stimulated by EGR2 and was further enhanced by CITED1 coexpression. Furthermore, expression of the 14-3-3σ tumor suppressor led to downregulation of ErbB2 protein levels and relocalization of EGR2 from the nucleus to the cytoplasm. Taken together, these observations suggest that, in addition to an increased gene copy number and upregulation of EGR2 and CITED1, an elevated erbB2 transcript level involves the loss of 14-3-3σ, which sequesters a key transcriptional regulator of the erbB2 promoter.
PMCID: PMC2169423  PMID: 17938205
25.  Two distinct domains of the β subunit of Aquifex aeolicus leucyl-tRNA synthetase are involved in tRNA binding as revealed by a three-hybrid selection 
Nucleic Acids Research  2004;32(11):3294-3303.
The Aquifex aeolicus αβ-LeuRS is the only known heterodimeric class Ia aminoacyl-tRNA synthetase. In this study, we investigated the function of the β subunit which is believed to bind tRNALeu. A yeast three-hybrid system was constructed on the basis of the interaction of the β subunit with its cognate tRNALeu. Then, seven mutated β subunits exhibiting impaired tRNA binding capacities were selected out from a randomly mutated library. Two mutations were identified in the class Ia-helix-bundle-domain, which might interact with the D-hairpin of the tRNA analogous to other class Ia tRNA:synthetases complexes. The five other mutations were found in the LeuRS-specific C-terminal domain of which the folding is still unknown. tRNA affinity measurements and kinetic analyses performed on the isolated β subunits and on the co-expressed αβ-heterodimers showed for all the mutants an effect in tRNA affinity in the ground state. In addition, an effect on the transition state of the aminoacylation reaction was observed for a 21-residues deletion mutant of the C-terminal end. These results show that the genetic approach of the three hybrid system is widely applicable and is a powerful tool for the investigation of tRNA:synthetase interactions.
PMCID: PMC443541  PMID: 15208367

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