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Linzhao Cheng

Publications and source records attributed to Linzhao Cheng.

At least 19 recordsLinked to original sources
PubMed2007

A stem cell-like chromatin pattern may predispose tumor suppressor genes to DNA hypermethylation and heritable silencing.

Adult cancers may derive from stem or early progenitor cells. Epigenetic modulation of gene expression is essential for normal function of these early cells but is highly abnormal in cancers, which often show aberrant promoter CpG island hypermethylation and transcriptional silencing of tumor suppressor genes and pro-differentiation factors. We find that for such genes, both normal and malignant embryonic cells generally lack the hypermethylation of DNA found in adult cancers. In embryonic stem cells, these genes are held in a 'transcription-ready' state mediated by a 'bivalent' promoter chromatin pattern consisting of the repressive mark, histone H3 methylated at Lys27 (H3K27) by Polycomb group proteins, plus the active mark, methylated H3K4. However, embryonic carcinoma cells add two key repressive marks, dimethylated H3K9 and trimethylated H3K9, both associated with DNA hypermethylation in adult cancers. We hypothesize that cell chromatin patterns and transient silencing of these important regulatory genes in stem or progenitor cells may leave these genes vulnerable to aberrant DNA hypermethylation and heritable gene silencing during tumor initiation and progression.

Adult↗
PubMed2007

Promoting human embryonic stem cell renewal or differentiation by modulating Wnt signal and culture conditions.

We previously showed that Wnt3a could stimulate human embryonic stem (hES) cell proliferation and affect cell fate determination. In the absence of feeder cell-derived factors, hES cells cultured under a feeder-free condition survived and proliferated poorly. Adding recombinant Wnt3a in the absence of feeder cell derived-factors stimulated hES cell proliferation but also differentiation. In the present study, we further extended our analysis to other Wnt ligands such as Wnt1 and Wnt5a. While Wnt1 displayed a similar effect on hES cells as Wnt3a, Wnt5a had little effect in this system. Wnt3a and Wnt1 enhanced proliferation of undifferentiated hES cells when feeder-derived self-renewal factors and bFGF are also present. To explore the possibility to promote the proliferation of undifferentiated hES cells by activating the Wnt signaling, we overexpressed Wnt3a or Wnt1 gene in immortalized human adult fibroblast (HAFi) cells that are superior in supporting long-term growth of undifferentiated hES cells than primary mouse embryonic fibroblasts. HAFi cells with or without a Wnt transgene can be propagated indefinitely. Over-expression of the Wnt3a gene significantly enhanced the ability of HAFi feeder cells to support the undifferentiated growth of 3 different hES cell lines we tested. Co-expression of three commonly-used drug selection genes in Wnt3a-overpressing HAFi cells further enabled us to select rare hES clones after stable transfection or transduction. These immortalized engineered feeder cells (W3R) that co-express growth-promoting genes such as Wnt3a and three drug selection genes should empower us to efficiently make genetic modified hES cell lines for basic and translational research.

Cell Culture Techniques↗
PubMed2006

Inducible and reversible transgene expression in human stem cells after efficient and stable gene transfer.

We report here a lentiviral vector system for regulated transgene expression. We used the tetracycline repressor fused with a transcriptional suppression domain (tTS) to specifically suppress transgene expression. Human cells were first transduced with a tTS-expressing vector and subsequently transduced with a second lentiviral vector-containing transgene controlled by a regular promoter adjacent to a high-affinity tTS-binding site (tetO). After optimizing the location of the tetO site in the latter vector, we achieved a better inducible transgene expression than the previous lentiviral vectors using the tetracycline repressor systems. In this new system, the transgene transcription from a cellular promoter such as EF1alpha or ubiquitin-C promoter is suppressed by the tTS bound to the nearby tetO site. In the presence of the tetracycline analog doxycycline (Dox), however, the tTS binding is released from the transgene vector and transcription from the promoter is restored. Thus, this system simply adds an extra level of regulation, suitable for any types of promoters (ubiquitous or cell-specific). We tested this tTS-suppressive, Dox-inducible system in 293T cells, human multipotent hematopoietic progenitor cells, and three human embryonic stem cell lines, using a dual-gene vector containing the green fluorescent protein reporter or a cellular gene. We observed a tight suppression in the uninduced state. However, the suppression is reversible, and transgene expression was restored at 5 ng/ml Dox. The lentiviral vectors containing the tTS-suppressive, Dox-inducible system offer a universal, inducible, and reversible transgene expression system in essentially any mammalian cell types, including human embryonic stem cells.

Cell Line, Tumor↗
PubMed2006

PC3, but not DU145, human prostate cancer cells retain the coregulators required for tumor suppressor ability of androgen receptor.

BACKGROUND: Androgen receptor (AR) functions in normal prostate epithelium as a tumor suppressor to inhibit continuous proliferation of these cells. Such tumor suppressor function of AR is lost in androgen depletion independent (ADI) prostate cancers. In type-I ADI cancers AR is not expressed, while in type-II ADI cancers AR is recaptured as an oncogene. The PC3 and DU145 human prostate cancer cell lines are representative of the earlier type-I ADI prostate cancers. While these cells do not express AR, it is unclear whether they retained the coactivators necessary for AR-dependent tumor suppression. To answer this question the response to AR protein expression by PC3 and DU145 cells was evaluated. METHODS: To do this, a lentiviral AR (Lenti-AR) expression system was engineered to encode an AR transcript which includes appropriate 5' and 3' untranslated regions (UTRs) containing all previously identified post-transcriptional regulatory sequences. AR expression and transcriptional activity were evaluated in Lenti-AR transduced cells by Western blot and luciferase assay, respectively. Cell growth in culture and in mouse xenografts was evaluated in correlation to expression changes in p21, p27, and p45(SKP2) proteins. RESULTS: Lenti-AR transduced PC3 and DU145 lines expressed transcriptionally functional AR protein at appropriate physiological levels. Expression and engagement of AR protein in PC3-Lenti-AR cells resulted in transactivation of p21 and subsequent growth inhibition of these cells in culture and in mouse xenografts. Such inhibition was due to induced G1 arrest of these cells as documented by expression changes in p27 and p45(SKP2) proteins. Such growth inhibition was not observed in DU145-Lenti-AR cells. CONCLUSIONS: These results document that PC3, but not DU145 cells retain the coregulators needed for AR tumor suppressor ability.

Animals↗
PubMed2006

HES1 inhibits cycling of hematopoietic progenitor cells via DNA binding.

Notch signaling is implicated in stem cell self-renewal, differentiation, and other developmental processes, and the Drosophila hairy and enhancer of split (HES) 1 basic helix-loop-helix protein is a major downstream effector in the Notch pathway. We found that HES1 was expressed at high levels in the hematopoietic stem cell (HSC)-enriched CD34+/[CD38/Lin](- /low) subpopulation but at low levels in more mature progenitor cell populations. When CD34+ cells were cultured for 1 week, the level of HES1 remained high in the CD34+ subset that had remained quiescent during ex vivo culture but was reduced in CD34+ cells that had divided. To investigate the effects of HES1 in human and mouse hematopoietic stem-progenitor cells (HSPCs), we constructed conditional lentiviral vectors (lentivectors) to introduce transgenes encoding either wild-type HES1 or a mutant lacking the DNA-binding domain (BHES1). We found that lentivector-mediated HES1 expression in CD34+ cells inhibited cell cycling in vitro and cell expansion in vivo, associated with upregulation of the cell cycle inhibitor p21(cip1/Waf1) (p21). The HES1 DNA-binding domain was required for these actions. HES1 did not induce programmed cell death or alter differentiation in HSPCs, and while short-term repopulating activity was reduced in HES1-transduced mouse and human cells, long-term reconstituting HSC function was preserved. Our data characterize the complex, cell context-dependent actions of HES1 as a major downstream Notch signaling regulator of HSPC function.

Animals↗
PubMed2006

Culture of human embryonic stem cells on human and mouse feeder cells.

This chapter describes the methods we use to maintain and expand undifferentiated human embryonic stem (hES) cells on human and mouse feeder cells. All of the available hES cells have been derived and propagated on primary mouse embryonic fibroblasts as feeder cells that have been mitotically inactivated. We found that hES cells can be successfully cultured on selected human feeder cells, such as marrow stromal cells derived from adult bone marrow and breast skin fibroblasts. Detailed protocols to use human and mouse feeder cells are described here, together with our method to split hES cells by trypsin/ethylenediaminetetraacetic acid-mediated dissociation. We also describe methods we use to characterize hES cells expanded on either human or mouse feeder cells, including alkaline phosphatase staining, immunostaining for cell-surface markers associated with undifferentiated hES cells, and teratoma formation in mice.

Animals↗
PubMed2005

Electrophysiological properties of pluripotent human and mouse embryonic stem cells.

Pluripotent embryonic stem cells (ESCs) possess promising potential for cell-based therapies, but their electrophysiological properties have not been characterized. Here we describe the presence of ionic currents in mouse (m) and human (h) ESCs and their physiological function. In mESCs, tetraethylammonium (TEA)-sensitive depolarization-activated delayed rectifier K+ currents (IK(DR)) (8.6 +/- 0.9 pA/pF at +40 mV; IC50 = 1.2 +/- 0.3 mM), which contained components sensitive to 4-aminopyridine (4-AP) (IC50 = 0.5 +/- 0.1 mM) and 100 nM Ca2+-activated K+ current (IK(Ca)) blocker iberiotoxin (IBTX),were detected in 52.3% of undifferentiated cells.IK(DR) was similarly present in hESCs (approximately 100%) but with an approximately sixfold higher current density (47.5 +/- 7.9 pA/pF at +40 mV). When assayed by bromodeoxyurindine incorporation, application of TEA, 4-AP, or IBTX significantly reduced the proliferation of mESCs and hESCs in a dose-dependent manner (p < .05). A hyperpolarization-activated inward current (I(h)) (-2.2 +/- 0.4 pA/pF at -120 mV) was detected in 23% of mESCs but not hESCs. Neither Na(v) nor Ca(v) currents were detected in mESCs and hESCs. Microarray and reverse transcription-polymerase chain reaction analyses identified several candidate genes for the ionic currents discovered. Collectively, our results indicate that pluripotent ESCs functionally express several specialized ion channels and further highlight similarities and differences between the two species. Practical considerations for the therapeutic use of ESCs are discussed.

4-Aminopyridine↗
PubMed2005

Radiofrequency-enhanced vascular gene transduction and expression for intravascular MR imaging-guided therapy: feasibility study in pigs.

PURPOSE: To evaluate the feasibility of radiofrequency (RF)-enhanced vascular gene transduction and expression by using a magnetic resonance (MR) imaging-heating guidewire as an intravascular heating vehicle during MR imaging-guided therapy. MATERIALS AND METHODS: The institutional committee for animal care and use approved the experimental protocol. The study included in vitro evaluation of the use of RF energy to enhance gene transduction and expression in vascular cells, as well as in vivo validation of the feasibility of intravascular MR imaging-guided RF-enhanced vascular gene transduction and expression in pig arteries. For in vitro experiments, approximately 10(4) vascular smooth muscle cells were seeded in each of four chambers of a cell culture plate. Next, 1 mL of a green fluorescent protein gene (gfp)-bearing lentivirus was added to each chamber. Chamber 4 was heated at approximately 41 degrees C for 15 minutes by using an MR imaging-heating guidewire connected to a custom RF generator. At day 6 after transduction, the four chambers were examined and compared at confocal microscopy to determine the efficiency of gfp transduction and expression. For the in vivo experiments, a lentivirus vector bearing a therapeutic gene, vascular endothelial growth factor 165 (VEGF-165), was transferred by using a gene delivery balloon catheter in 18 femoral-iliac arteries (nine artery pairs) in domestic pigs and Yucatan pigs with atherosclerosis. During gene infusion, one femoral-iliac artery in each pig was heated to approximately 41 degrees C with RF energy transferred via the intravascular MR imaging-heating guidewire, while the contralateral artery was not heated (control condition). At day 6, the 18 arteries were harvested for quantitative Western blot analysis to compare VEGF-165 transduction and expression efficiency between RF-heated and nonheated arterial groups. RESULTS: Confocal microscopy showed gfp expression in chamber 4 that was 293% the level of expression in chamber 1 (49.6% +/- 25.8 vs 16.8% +/- 8.0). Results of Western blot analysis showed VEGF-165 expression for normal arteries in the RF-heated group that was 300% the level of expression in the nonheated group (70.4 arbitrary units [au] +/- 107.1 vs 23.5 au +/- 29.8), and, for atherosclerotic arteries in the RF-heated group, 986% the level in the nonheated group (129.2 au +/- 100.3 vs 13.1 au +/- 4.9). CONCLUSION: Simultaneous monitoring and enhancement of vascular gene delivery and expression is feasible with the MR imaging-heating guidewire.

Animals↗
PubMed2005

Defining the role of Wnt/beta-catenin signaling in the survival, proliferation, and self-renewal of human embryonic stem cells.

We used a panel of human and mouse fibroblasts with various abilities for supporting the prolonged growth of human embryonic stem cells (hESCs) to elucidate growth factors required for hESC survival, proliferation, and maintenance of the undifferentiated and pluripotent state (self-renewal). We found that supportive feeder cells secrete growth factors required for both hESC survival/proliferation and blocking hESC spontaneous differentiation to achieve self-renewal. The antidifferentiation soluble factor is neither leukemia inhibitory factor nor Wnt, based on blocking experiments using their antagonists. Because Wnt/beta-catenin signaling has been implicated in cell-fate determination and stem cell expansion, we further examined the effects of blocking or adding recombinant Wnt proteins on undifferentiated hESCs. In the absence of feeder cell-derived factors, hESCs cultured under a feeder-free condition survived/proliferated poorly and gradually differentiated. Adding recombinant Wnt3a stimulated hESC proliferation but also differentiation. After 4-5 days of Wnt3a treatment, hESCs that survived maintained the undifferentiated phenotype but few could form undifferentiated hESC colonies subsequently. Using a functional reporter assay, we found that the beta-catenin-mediated transcriptional activation in the canonical Wnt pathway was minimal in undifferentiated hESCs, but greatly upregulated during differentiation induced by the Wnt treatment and several other methods. Thus, Wnt/beta-catenin activation does not suffice to maintain the undifferentiated and pluripotent state of hESCs. We propose a new model for the role of Wnt/beta-catenin signaling in undifferentiated hESCs.

Animals↗
PubMed2005

Arsenic suppresses gene expression in promyelocytic leukemia cells partly through Sp1 oxidation.

The mechanism by which arsenic dramatically affects gene expression remains poorly understood. Here we report that prolonged exposure of acute promyelocytic leukemia NB4 cells to low levels of arsenic trioxide increased the expression of a set of genes responsible for reactive oxygen species (ROS) production. We hypothesize that arsenic-induced ROS in turn contribute partially to altered gene expression. To identify genes responsive to arsenic-induced ROS, we used microarray gene expression analysis and identified genes that responded to arsenic and hydrogen peroxide but whose response to arsenic was reversed by an ROS scavenger, N-acetyl-L-cysteine. We found that 26% of the genes significantly responsive to arsenic might have been directly altered by ROS. We further explored the mechanisms by which ROS affects gene regulation and found that the Sp1 transcription factor was oxidized by arsenic treatment, with a corresponding decrease in its in situ binding on the promoters of 3 genes, hTERT, C17, and c-Myc, whose expressions were significantly suppressed. We conclude that ROS contributed partly to arsenic-mediated gene regulation and that Sp1 oxidation contributed to gene suppression by arsenic-induced ROS.

Antineoplastic Agents↗
PubMed2004

Myocyte enhancer factor 2 mediates calcium-dependent transcription of the interleukin-2 gene in T lymphocytes: a calcium signaling module that is distinct from but collaborates with the nuclear factor of activated T cells (NFAT).

The second messenger calcium plays an essential role in the T cell receptor-mediated signal transduction pathways leading to transcription of the interleukin-2 gene. A key mechanism of calcium signaling has been shown to be mediated by calcineurin and NFAT. We report herein that the transcription factor myocyte enhancer factor (MEF)-2 is another calcium signal transducer involved in the regulation of the interleukin (IL)-2 promoter. A MEF2-binding site was identified in proximity to the TATA box of the IL-2 promoter. This site was shown to be bound by MEF2 in both resting and activated T cells. Overexpression of MEF2 enhanced, while overexpression of a dominant negative form of MEF2 or the MEF2-specific transcriptional corepressors Cabin1 and histone deacetylase 4 inhibited, the T cell receptor-dependent activation of an IL-2 reporter gene. Down-regulation of MEF2 by RNA interference in primary human T cells led to the inhibition of endogenous IL-2 transcription. These results suggest that MEF2 is required for the transcriptional activation of IL-2 and likely other cytokine genes in response to calcium signaling and may serve as a novel target for development of immunosuppressants.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Lentivirus-mediated gene transfer and expression in established human tumor antigen-specific cytotoxic T cells and primary unstimulated T cells.

In this report, we evaluated the efficiency of stable gene transfer into established CD8(+) human tumor antigen-specific cytotoxic T cell (CTL) lines and peripheral blood lymphocytes (PBL) by oncoretroviral and lentiviral vectors. In the oncoretroviral vector, the green fluorescent protein (GFP) reporter gene was regulated by the murine stem cell virus (MSCV) promoter. In three human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors, the GFP transgene was regulated by either a chimeric MSCV/HIV-1 promoter, or cellular promoters from human housekeeping genes PGK and EF1 alpha. We found that several lines of proliferating tumor-specific CTL were poorly (=2%) transduced by the oncoretroviral vector that transduced Jurkat T cell line efficiently (=80%). In contrast, three lentiviral vectors transduced 38-63% of these proliferating CTL. More interestingly, all lentiviral vectors packaged without the HIV-1 accessory proteins transduced human bulk PBL and purified CD4(+) and CD8(+) lymphocyte subsets without prior stimulation. Detailed analysis indicated that the lentiviral vectors containing the EF1 alpha or PGK ubiquitous promoter can transduce unstimulated PBL and achieve low-level transgene expression in the absence of any T-cell activation. However, T-cell activation subsequent to the transduction of unstimulated PBL is required for high-level transgene expression. Transduced PBL expressing transgene delivered by the lentiviral vectors still preserved resting and naïve cell phenotypes. Taken together, prior T cell stimulation and HIV-1 accessory proteins are dispensable for lentivirus-mediated gene transfer into resting naïve and memory T lymphocytes. These results will have significant implications for the study of T-cell biology and for the improvement of clinical gene therapies of acquired immune deficiency syndrome (AIDS) and cancer.

Animals↗
PubMed2003

In vivo retroviral gene transfer by direct intrafemoral injection results in correction of the SCID phenotype in Jak3 knock-out animals.

Efficient retroviral gene transfer to pluripotential hematopoietic stem cells (PHSCs) requires ex vivo culture in multiple hematopoietic growth factors (HGFs) to promote cell division. While treatment of PHSCs with HGF can render stem cells viable targets for retroviral infection, HGFs can promote differentiation, loss of self-renewal potential, and affect the homing/engraftment capacity of PHSCs. To avoid the negative impacts observed with ex vivo transduction protocols, we developed a murine model for in vivo retroviral infection by direct intrafemoral injection (DII), thus abolishing the need for removal of cells from their native microenvironment and the signals necessary to maintain their unique physiology. Using this approach we have demonstrated in vivo retroviral gene transfer to colony-forming units-c (CFU-c), short-term reconstituting cells, and PHSCs. Moreover, direct intrafemoral injection of Jak3 knock-out mice with retroviral particles encoding the Jak3 gene resulted in reconstitution of normally deficient lymphocyte populations concomitant with improved immune function. In addition, DII can be used to target the delivery of other gene therapy vectors including adenoviral vectors to bone marrow cells in vivo. Taken together, these results demonstrate that in vivo retroviral gene transfer by direct intrafemoral injection may be a viable alternative to current ex vivo gene transfer approaches.

Animals↗
PubMed2003

Cotransplantation of human mesenchymal stem cells enhances human myelopoiesis and megakaryocytopoiesis in NOD/SCID mice.

OBJECTIVE: For approximately 5% of autologous transplant recipients and a higher proportion of allogeneic transplant recipients, low level and delayed platelet engraftment is an ongoing problem. Mesenchymal stem cells (MSC), which can be derived from bone marrow as well as other organs, are capable of differentiation into multiple cell types and also support hematopoiesis in vitro. Because cotransplantation of marrow-derived stromal cells has been shown to enhance engraftment of human hematopoietic stem cells, we hypothesized that cotransplantation of MSC could enhance platelet and myeloid cell development. MATERIALS AND METHODS: We tested this hypothesis by transplantation of CD34-selected mobilized human peripheral blood stem cells (PBSC) into sublethally irradiated NOD/SCID mice with or without culture-expanded human MSC and evaluated human myeloid, lymphoid, and megakaryocytic engraftment with flow cytometry and in vitro cultures. RESULTS: We find that MSC cotransplantation enhances human cell engraftment when a limiting dose (<1 x 10(6)) of CD34 cells is administered. This enhancement is characterized by a shift in the differentiation of human cells from predominantly B lymphocytes to predominantly CD13(+), CD14(+), and CD33(+) myeloid cells with a corresponding increase in myeloid CFU in the marrow. Megakaryocytopoiesis is enhanced by MSC cotransplantation as assessed by an increase in both marrow CFU-MK and circulating human platelets. In contrast, MSC do not affect the percentage of human bone marrow cells that expresses CD34(+). CONCLUSIONS: Cotransplantation of human mesenchymal stem cells with CD34(+)-selected hematopoietic stem cells enhances myelopoiesis and megakaryocytopoiesis.

Animals↗
PubMed2003

Lentiviral vectors with two independent internal promoters transfer high-level expression of multiple transgenes to human hematopoietic stem-progenitor cells.

Lentiviral vectors (LVs) offer several advantages over traditional oncoretroviral vectors. LVs efficiently transduce slowly dividing cells, including hematopoietic stem-progenitor cells (HSCs), resulting in stable gene transfer and expression. Additionally, recently developed self-inactivating (SIN) LVs allow promoter-specific transgene expression. For many gene transfer applications, transduction of more than one gene is needed. We obtained inconsistent results in our attempts to coexpress two transgenes linked by an internal ribosomal entry site (IRES) element in a single bicistronic LV transcript. In more than six bicistronic LVs we constructed containing a gene of interest followed by an IRES and the GFP reporter gene, GFP fluorescence was undetectable in transduced cells. We therefore investigated how to achieve consistent and efficient coexpression of two transgenes by LVs. In a SIN LV containing the elongation factor 1alpha promoter, we included a second promoter from cytomegalovirus, the phosphoglycerate kinase gene, or the HLA-DRalpha gene. Using a single LV containing two constitutive promoters, we achieved strong and sustained expression of both transgenes in transduced engrafting CD34(+) HSCs and their progeny, as well as in other human cell types. Thus, such dual-promoter LVs can coexpress multiple transgenes efficiently in a single target cell and will enable many gene transfer applications.

Animals↗
PubMed2003

Immunotherapy of established tumors using bone marrow transplantation with antigen gene--modified hematopoietic stem cells.

A major focus of cancer immunotherapy is to develop strategies to induce T-cell responses through presentation of tumor antigens by dendritic cells (DCs). Current vaccines are limited in their ability to efficiently transfer antigens to DCs in vivo. Ex vivo-generated DCs can be efficiently loaded with antigen but after reinjection, few DCs traffic to secondary lymphoid organs, the critical sites for antigen presentation. To enhance efficiency and durability of antigen presentation by DCs, we transduced hematopoietic stem-progenitor cells (HSCs) with a model tumor antigen and then transplanted the gene-modified cells into irradiated recipient mice, which resulted in efficient expression of the transgene in a large proportion of donor derived DCs in lymphoid organs. The combination of bone marrow transplantation (BMT) using transduced HSCs, systemic agents that generate and activate DCs, and mature T-cell infusion resulted in substantial expansion and activation of antigen-specific T cells. This tripartite strategy provided potent antigen-specific immunotherapy for an aggressive established tumor.

Animals↗