Pi Wan Cheng

Activation of CMV promoter-controlled glycosyltransferase and β -galactosidase glycogenes by butyrate, tricostatin A, and 5-Aza-2′-deoxycytidine

Cytomegalovirus (CMV) immediate early promoter is a powerful promoter frequently used for driving the expression of transgenes in mammalian cells. However, this promoter gradually becomes silenced in stably transfected cells. We employed Chinese Hamster Ovary (CHO) and human pancreatic cancer (Panc 1) cells stably tansfected with three glycogenes driven by a CMV promoter to study the activation of silenced glycogenes. We found that butyrate, tricostatin A (TSA), and 5-aza-2′-deoxycytidine (5-Aza-dC) can activate these CMV-driven glycogenes. The increase in mRNA and protein of a glycogene occurred 8–10 h after butyrate treatment, suggesting an indirect effect of butyrate in the activation of the transgene. The enhanced expression of the trangenes by butyrate and TSA, known inhibitors of histone deacetylase, was independent of the transgene or cell type. However, the transgene can be activated by these two agents in only a fraction of the cells derived from a single clone, suggesting that inactivation of histone deacetylase can only partially explain silencing of the transgenes. Combination treatment of one or both agents with 5-Aza-dC, a known inhibitor of DNA methylase, resulted in a synergistic activation of the transgene, suggesting a cross-talk between histone acetylation and DNA demethylation. Understanding the mechanisms of the inactivation and reactivation of CMV promoter-controlled transgenes should help develop an effective strategy to fully activate the CMV promoter-controlled therapeutic genes silenced by the host cells. Published in 2005.

Expression of core 2 β-1,6-N-acetylglucosaminyltransferase in a human pancreatic cancer cell line results in altered expression of MUC1 tumor- associated epitopes

Many tumor-associated epitopes possess carbohydrate as a key component, and thus changes in the activity of glycosyltransferases could play a role in generating these epitopes. In this report we describe the stable transfection of a human pancreatic adenocarcinoma cell line, Panc1-MUC1, with the cDNA for mucin core 2 GlcNAc-transferase (C2GnT), which creates the core 2 β-1,6 branch in mucin-type glycans. These cells lack endogenous C2GnT activity but express a recombinant human MUC1 cDNA. C2GnT-transfected clones expressing different levels of C2GnT were characterized using monoclonal antibodies CC49, CSLEX-1, and SM-3, which recognize tumor-associated epitopes. Increased C2GnT expression led to greatly diminished expression of the CC49 epitope, which we identified as NeuAcα2,6(Galβ1,3)GalNAcα-Ser/Thr in the Panc1-MUC1 cells. This was accompanied by the emergence of the CSLEX-1 epitope, sialyl Lewis x (NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAc-R), an important selectin ligand. Despite this, however, the C2GnT transfectants could not bind to selectins. Increased C2GnT expression also led to masking of the SM-3 peptide epitope, which persisted after the removal of sialic acid, further suggesting greater complexity of the core 2-associated O-glycans on MUC1. The results of this study suggest that C2GnT could play a regulatory role in the expression of certain tumor-associated epitopes.

Transferrin-Facilitated Lipofection Gene Delivery Strategy: Characterization of the Transfection Complexes and Intracellular Trafficking

We previously showed that mixing transferrin with a cationic liposome prior to the addition of DNA, greatly enhanced the lipofection efficiency. Here, we report characterization of the transfection complexes in formulations prepared with transferrin, lipofectin, and DNA (pCMVlacZ) in various formulations. DNA in all the formulations that contain lipofectin was resistant to DNase I treatment. Transfection experiments performed in Panc 1 cells showed that the standard formulation, which was prepared by adding DNA to a mixture of transferrin and lipofectin, yielded highest transfection efficiency. There was no apparent difference in zeta potential among these formulations, but the most efficient formulation contained complexes with a mean diameter of three to four times that of liposome and the complexes in other gene delivery formulations. Transmission electron microscopic examination of the standard transfection complexes formulated using gold-labeled transferrin showed extended circular DNA decorated with transferrin as compared to extensively condensed DNA found in lipofectin-DNA complexes and heterogeneous structures in other formulations. By confocal microscopy, DNA and transferrin were found to colocalize at the perinuclear space and in the nucleus, suggesting cotransportation intracellularly, including nuclear transport. We propose that transferrin enhances the transfection efficiency of the standard lipofection formulation by preventing DNA condensation, and facilitating endocytosis and nuclear targeting.

p53 and PTEN/MMAC1/TEP1 Gene Therapy of Human Prostate PC-3 Carcinoma Xenograft, Using Transferrin- Facilitated Lipofection Gene Delivery Strategy

We previously reported that supplementation of a cationic liposome with transferrin (Tf) greatly enhanced lipofection efficiency (P.-W. Cheng, Hum. Gene Ther. 1996;7:275-282). In this study, we examined the efficacy of p53 and PTEN tumor suppressor gene therapy in a mouse xenograft model of human prostate PC-3 carcinoma cells, using a vector consisting of dimyristoyloxypropyl-3-dimethylhydroxyethyl ammonium bromide (DMRIE)-cholesterol (DC) and Tf. When the volume of the tumors grown subcutaneously in athymic nude mice reached 50-60 mm(3), three intratumoral injections of the following four formulations were performed during week 1 and then during week 3: (1) saline, (2) DC + Tf + pCMVlacZ, (3) DC + Tf + pCMVPTEN, and (4) DC + Tf + pCMVp53 (standard formulation). There was no significant difference in tumor volume and survival between group 1 and group 2 animals. As compared with group 1 controls, group 3 animals had slower tumor growth during the first 3 weeks but thereafter their tumor growth rate was similar to that of the controls. By day 2 posttreatment, group 4 animals had significantly lower tumor volume relative to initial tumor volume as well as controls at the comparable time point. Also, animals treated with p53 survived longer. Treatment with DC, Tf, pCMVp53, DC + pCMVp53, or Tf + pCMVp53 had no effect on tumor volume or survival. Expression of p53 protein and apoptosis were detected in tumors treated with the standard formulation, thus associating p53 protein expression and apoptosis with efficacy. However, p53 protein was expressed in only a fraction of the tumor cells, suggesting a role for bystander effects in the efficacy of p53 gene therapy. We conclude that intratumoral gene delivery by a nonviral vector consisting of a cationic liposome and Tf can achieve efficacious p53 gene therapy of prostate cancer.

Effects of epidermal growth factor, transferrin, and insulin on lipofection efficiency in human lung carcinoma cells.

Poor transfection efficiency is the major drawback of lipofection. We showed previously that addition of transferrin (TF) to Lipofectin enhanced the expression of a reporter gene in HeLa cells by 120-fold and achieved close to 100% transfection efficiency. The purpose of this study was to determine whether TF and other ligands could improve the efficiency of lipofection in lung carcinoma cells. Confluent A549, Calu3, and H292 cells were transfected for 18 hours with a plasmid DNA (pCMVlacZ) using Lipofectin plus TF, insulin, or epidermal growth factor as the vector. The transfected cells were assessed for transfection efficiency by β-galactosidase activity (light units/μg protein) and the percentage of blue cells following 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside staining. Lipofectin supplemented with epidermal growth factor yielded the largest enhancement of lipofection efficiency (≤23-fold over that by Lipofectin alone) in all three cell lines. Insulin significantly enhanced the lipofection efficiency in A549 and Calu3 cells but not in H292 cells, whereas TF showed significant lipofection efficiency-enhancing effect in Calu3 and H292 cells but not in A549 cells. The transfection efficiency correlated well with the amounts of DNA delivered to the nucleus as well as the amounts of the receptor. These results indicate that the gene delivery strategy employing ligand-facilitated lipofection can achieve high transfection efficiency in human lung carcinoma cells. In addition, enhancement of the expression of the receptor may be a possible strategy for increasing the efficiency of gene targeting.

TNFα enhances the motility and invasiveness of prostatic cancer cells by stimulating the expression of selective glycosyl- and sulfotransferase genes involved in the synthesis of selectin ligands

Sialyl Lewis x (sLe(x)) plays an important role in cancer metastasis. But, the mechanism for its production in metastatic cancers remains unclear. The objective of current study was to examine the effects of a proinflammatory cytokine on the expression of glycosyltransferase and sulfotransferase genes involved in the synthesis of selectin ligands in a prostate cancer cell line. Androgen-independent human lymph node-derived metastatic prostate cancer cells (C-81 LNCaP), which express functional androgen receptor and mimic the castration-resistant advanced prostate cancer, were used. TNFα treatment of these cells increased their binding to P-, E- and L-selectins, anti-sLe(x) antibody, and anti-6-sulfo-sialyl Lewis x antibody by 12%, 240%, 43%, 248% and 21%, respectively. Also, the expression of C2GnT-1, B4GalT1, GlcNAc6ST3, and ST3Gal3 genes was significantly upregulated. Further treatment of TNFα-treated cells with either anti-sLe(x) antibody or E-selectin significantly suppressed their in vitro migration (81% and 52%, respectively) and invasion (45% and 56%, respectively). Our data indicate that TNFα treatment enhances the motility and invasion properties of LNCaP C-81 cells by increasing the formation of selectin ligands through stimulation of the expression of selective glycosyl- and sulfotransferase genes. These results support the hypothesis that inflammation contributes to cancer metastasis.

Lectin enhancement of the lipofection efficiency in human lung carcinoma cells.

Poor transfection efficiency of human lung carcinoma cells by lipofection begs further development of more efficient gene delivery strategies. The purpose of this study was to determine whether lectins can improve the lipofection efficiency in lung carcinoma cells. A549, Calu3, and H292 cells grown to 90% confluence were transfected for 18 h with a plasmid DNA containing a beta-galactosidase reporter gene (pCMVlacZ) using lipofectin plus a lectin as the vector. Ten different lectins, which exhibit a wide range of carbohydrate-binding specificities, were examined for their abilities to enhance the efficiency of lipofection. The transfected cells were assessed for transfection efficiency by beta-galactosidase activity (units/microg protein) and % blue cells following X-Gal stain. Lipofectin supplemented with Griffonia simplicifolia-I (GS-I) yields largest enhancement of the lipofection efficiency in A549 and Calu3 cells (5.3- and 28-fold, respectively). Maackia amurensis gives the largest enhancement (6.5-fold) of lipofection efficiency in H292 cells. The transfection efficiency correlates with the amounts of DNA delivered to the nucleus. Binding of FITC-labeled GS-I and the enhancement of the lipofection efficiency by GS-I were inhibited by alpha-methyl-D-galactopyranoside, indicating an alpha-galactoside-mediated gene transfer to lung carcinoma cells. We conclude that lectin-facilitated lipofection is an efficient gene delivery strategy. Employment of cell type-specific lectins may allow for efficient cell type-specific gene targeting.

Restoration of Compact Golgi Morphology in Advanced Prostate Cancer Enhances Susceptibility to Galectin-1–Induced Apoptosis by Modifying Mucin O-Glycan Synthesis

Prostate cancer progression is associated with upregulation of sialyl-T antigen produced by β-galactoside α-2,3-sialyltransferase-1 (ST3Gal1) but not with core 2-associated polylactosamine despite expression of core 2 N-acetylglucosaminyltransferase-L (C2GnT-L/GCNT1). This property allows androgen-refractory prostate cancer cells to evade galectin-1 (LGALS1)–induced apoptosis, but the mechanism is not known. We have recently reported that Golgi targeting of glycosyltransferases is mediated by golgins: giantin (GOLGB1) for C2GnT-M (GCNT3) and GM130 (GOLGA2)-GRASP65 (GORASP1) or GM130-giantin for core 1 synthase. Here, we show that for Golgi targeting, C2GnT-L also uses giantin exclusively whereas ST3Gal1 uses either giantin or GM130-GRASP65. In addition, the compact Golgi morphology is detected in both androgen-sensitive prostate cancer and normal prostate cells, but fragmented Golgi and mislocalization of C2GnT-L are found in androgen-refractory cells as well as primary prostate tumors (Gleason grade 2–4). Furthermore, failure of giantin monomers to be phosphorylated and dimerized prevents Golgi from forming compact morphology and C2GnT-L from targeting the Golgi. On the other hand, ST3Gal1 reaches the Golgi by an alternate site, GM130-GRASP65. Interestingly, inhibition or knockdown of non-muscle myosin IIA (MYH9) motor protein frees up Rab6a GTPase to promote phosphorylation of giantin by polo-like kinase 3 (PLK3), which is followed by dimerization of giantin assisted by protein disulfide isomerase A3 (PDIA3), and restoration of compact Golgi morphology and targeting of C2GnT-L. Finally, the Golgi relocation of C2GnT-L in androgen-refractory cells results in their increased susceptibility to galectin-1–induced apoptosis by replacing sialyl-T antigen with polylactosamine. Implications: This study demonstrates the importance of Golgi morphology and regulation of glycosylation and provides insight into how the Golgi influences cancer progression and metastasis. Mol Cancer Res; 12(12); 1704–16. ©2014 AACR.

Glycosyltransferase-specific Golgi targeting mechanisms

Background: The Golgi docking mechanisms for transport vesicles carrying glycosyltransferases are largely unknown. Results: C1GalT1 utilizes GM130-GRASP65 when GRASP65 is available but GM130-Giantin without GRASP65, whereas C2GnT-M employs Giantin for Golgi targeting. Conclusion: The Golgi-targeting mechanism is glycosyltransferase-specific. Significance: Understanding the Golgi-targeting mechanisms of glycosyltransferases may help uncover altered glycosylation in some diseases. Glycosylation of secreted and membrane-bound mucins is carried out by glycosyltransferases localized to specific Golgi compartments according to the step in which each enzyme participates. However, the Golgi-targeting mechanisms of these enzymes are not clear. Herein, we investigate the Golgi-targeting mechanisms of core 1 β3 galactosyltransferase (C1GalT1) and core 2 β1,6-N-acetylglucosaminyltransferase-2 or mucus type (C2GnT-M), which participate in the early O-glycosylation steps. siRNAs, co-immunoprecipitation, and confocal fluorescence microscopy were employed to identify the golgins involved in the Golgi docking of vesicular complexes (VCs) that carry these two enzymes. We have found that these VCs use different golgins for docking: C2GnT-M-carrying VC (C2GnT-M-VC) utilizes Giantin, whereas C1GalT1-VC employs GM130-GRASP65 complex. However, in the absence of GRASP65, C1GalT1-VC utilizes GM130-Giantin complex. Also, we have found that these VCs are 1.1–1.2 μm in diameter, specific for each enzyme, and independent of coat protein complex II and I (COPII and COPI). These two fluorescently tagged enzymes exhibit different fluorescence recovery times in the Golgi after photobleaching. Thus, novel enzyme-specific Golgi-targeting mechanisms are employed by glycosyltransferases, and multiple Golgi docking strategies are utilized by C1GalT1.

Biosynthesis and Function of ß 1,6 Branched Mucin-Type Glycans

The contribution of carbohydrate structure to biomolecular, cellular, and organismal function is well-established, but has not yet received the attention it deserves, perhaps due to the complexity of the structures involved and to a lack of simple experimental methods for relating structure and function. In particular, β1,6 GlcNAc branching plays a key functional role in processes ranging from inflammation and immune system function to tumor cell metastasis. For instance, synthesis of the core 2 β1,6 branched structure in the mucin glycan chain by C2GnT enables the expression of functional structures at the termini of polylactosamine chains, such as blood group antigens and sialyl Lewis x. Also, IGnT can create multiple branches on the polylactosamine chain, which may serve as a mechanism for amplifying the functional potency of cell surface glycoproteins and glycolipids. The family of enzymes which creates β1,6 branched structure in mucin glycans is proving to be quite complex, since multiple isoforms appear to exist for these enzymes, and some of the enzymes are adept at forming more than one type of β1,6 branched structure, as in the case ofC2GnT-M. Furthermore, the enzymes do not appear to be restricted to acting on mucin-type acceptor structures, but are able to act on glycolipid structures as well. Much remains to be learned regarding the specific biological niche filled by each of these enzymes and how their activities complement one another, as well as the manner in which the activities of these enzymes are regulated in the cell.