Nuclear factor-Y and Epstein Barr virus in nasopharyngeal cancer.

PURPOSE: The Epstein Barr virus (EBV) is intimately associated with nasopharyngeal cancer (NPC) in a latent state expressing a limited number of genes. The process of switching from latency to replication is not well understood, particularly in response to DNA stress; hence, the focus of this study is on an EBV-positive NPC model. EXPERIMENTAL DESIGN: C666-1 cells were exposed to radiation (2-15 Gy) or cisplatin (0.1-50 microg/mL) assayed subsequently for relative EBV copy number (BamHI) and lytic gene expression (BRLF1 and BZLF1) using quantitative real-time PCR. Chromatin immunoprecipitation was conducted to assess the interaction of the transcription factor nuclear factor-Y (NF-Y) with promoter sequences. RESULTS: Radiation-induced and cisplatin-induced BamHI expression, along with increased levels of BRLF1 and BZLF1 in a dose-dependent and time-dependent manner, associated with the immediate nuclear transactivation of the transcription factor NF-Y and its own increased transcription of NF-Y subunits 8 h posttreatment. In silico analysis revealed three putative NF-Y consensus-binding sequences in the promoter region of BRLF1, which all interacted with NF-Y in response to radiation and cisplatin, confirmed using chromatin immunoprecipitation. Introduction of dominant-negative NF-YA reduced BRLF1 expression after radiation and cisplatin by 2.8-fold; in turn, overexpression of NF-YA resulted in a 2-fold increase in both BRLF1 and BZLF1 expression. CONCLUSIONS: These results show that NF-Y is an important mediator of EBV stress response in switching from a latent to lytic state. This novel insight could provide a potential therapeutic strategy to enhance NPC response to radiation and cisplatin.

Inhibition of mechanosensitive cation channels inhibits myogenic differentiation by suppressing the expression of myogenic regulatory factors and caspase-3 activity.

Mechanosensitive cation channels (MSC) are ubiquitous in eukaryotic cell types. However, the physiological functions of MSC in several tissues remain in question. In this study we have investigated the role of MSC in skeletal myogenesis. Treatment of C2C12 myoblasts with gadolinium ions (MSC blocker) inhibited myotube formation and the myogenic index in differentiation medium (DM). The enzymatic activity of creatine kinase (CK) and the expression of myosin heavy chain-fast twitch (MyHCf) in C2C12 cultures were also blocked in response to gadolinium. Treatment of C2C12 myoblasts with gadolinium ions did not affect the expression of either cyclin A or cyclin D1 in DM. Other inhibitors of MSC such as streptomycin and GsTMx-4 also suppressed the expression of CK and MyHCf in C2C12 cultures. The inhibitory effect of gadolinium ions on myogenic differentiation was reversible and independent of myogenic cell type. Real-time-polymerase chain reaction analysis revealed that inhibition of MSC decreases the expression of myogenic transcription factors MyoD, myogenin, and Myf-5. Furthermore, the activity of skeletal alpha-actin promoter was suppressed on MSC blockade. Treatment of C2C12 myoblasts with gadolinium ions prevented differentiation-associated cell death and inhibited the cleavage of poly (ADP-ribose) polymerase and activation of caspase-3. On the other hand, delivery of active caspase-3 protein to C2C12 myoblasts reversed the inhibitory effect of gadolinium ions on myogenesis. Our data suggest that inhibition of MSC suppresses myogenic differentiation by inhibiting the caspase-3 activity and the expression of myogenic regulatory factors.

Integrin-linked kinase mediates bone morphogenetic protein 7-dependent renal epithelial cell morphogenesis.

Bone morphogenetic protein 7 (BMP7) stimulates renal branching morphogenesis via p38 mitogen-activated protein kinase (p38(MAPK)) and activating transcription factor 2 (ATF-2) (M. C. Hu, D. Wasserman, S. Hartwig, and N. D. Rosenblum, J. Biol. Chem. 279:12051-12059, 2004). Here, we demonstrate a novel role for integrin-linked kinase (ILK) in mediating renal epithelial cell morphogenesis in embryonic kidney explants and identify p38(MAPK) as a target of ILK signaling in a cell culture model of renal epithelial morphogenesis. The spatial and temporal expression of ILK in embryonic mouse kidney cells suggested a role in branching morphogenesis. Adenovirus-mediated expression of ILK stimulated and expression of a dominant negative ILK mutant inhibited ureteric bud branching in embryonic mouse kidney explants. BMP7 increased ILK kinase activity in inner medullary collecting duct 3 (IMCD-3) cells, and adenovirus-mediated expression of ILK increased IMCD-3 cell morphogenesis in a three-dimensional culture model. In contrast, treatment with a small molecule ILK inhibitor or expression of a dominant negative-acting ILK (ILK(E359K)) inhibited epithelial cell morphogenesis. Further, expression of ILK(E359K) abrogated BMP7-dependent stimulation. To investigate the role of ILK in BMP7 signaling, we showed that ILK overexpression increased basal and BMP7-induced levels of phospho-p38(MAPK) and phospho-ATF-2. Consistent with its inhibitory effects on IMCD-3 cell morphogenesis, expression of ILK(E359K) blocked BMP7-dependent increases in phospho-p38(MAPK) and phospho-ATF-2. Inhibition of p38(MAPK) activity with the specific inhibitor, SB203580, failed to inhibit BMP7-dependent stimulation of ILK activity, suggesting that ILK functions upstream of p38(MAPK) during BMP7 signaling. We conclude that ILK functions in a BMP7/p38(MAPK)/ATF-2 signaling pathway and stimulates epithelial cell morphogenesis.

A conditionally replicating adenovirus for nasopharyngeal carcinoma gene therapy.

Successful attainment of tumor-specific gene expression was achieved in nasopharyngeal carcinoma (NPC) by exploiting the exclusive presence of the Epstein-Barr virus (EBV) genome in the cancer cells. In the current study, we have utilized an EBV-dependent transcriptional targeting strategy to construct a novel conditionally replicating adenovirus, adv.oriP.E1A. After treatment with adv.oriP.E1A, we observed extensive cell death in the EBV-positive NPC cell line C666-1. In contrast, no cytotoxicity was observed in a panel of other human EBV-negative cell lines, including fibroblasts from the nasopharynx. In vitro adenoviral replication was confirmed by the time-dependent increase in the expression of adenoviral capsid fiber protein and adenoviral DNA after C666-1 cells were infected with adv.oriP.E1A. Tumor formation was inhibited for more than 100 days after ex vivo infection of C666-1 cells with adv.oriP.E1A. Combination of local tumor radiation and adv.oriP.E1A caused complete disappearance of established tumors for at least 2 weeks in two distinct EBV-positive NPC xenograft models. Safety of this treatment was determined through the systemic delivery of adv.oriP.E1A in vivo, whereby minimal temporary perturbation of liver function was observed. We have successfully established a conditionally replicating adenovirus for EBV-positive NPC, which is both safe and efficacious, indicating a strategy that may be therapeutically applicable.

Progress toward skeletal gene therapy.

Skeletal gene therapy is an attractive new approach to the treatment of bone disorders. Impressive advances in our knowledge of the molecular genetic basis of skeletal disorders and fracture healing have led to the development of novel therapeutics based on ectopic expression of one or more genes in patient cells that can influence repair or regenerative processes in bone. Although still a relatively immature field, proof-of-principle for enhanced bone formation through skeletal gene therapy has already been established. The challenge now is to more precisely define optimal cellular targets and therapeutic genes, and to develop safe and efficient ways to deliver therapeutic genes to target cells. In this review, we will highlight some of the exciting advances that have been made in skeletal gene therapy in recent years, with a focus on treatment of localized skeletal lesions. Strengths and weaknesses of current approaches will be discussed, as will strategies for improved safety and therapeutic outcome in the future. Skeletal gene therapy can have an enormous impact on patient care. The next 5 years will present us with unparalleled opportunities to develop more effective therapeutic strategies and overcome obstacles presented by current gene transfer technologies.

p16 gene therapy: a potentially efficacious modality for nasopharyngeal carcinoma.

p16 is an important regulator of the cell cycle at the G(1) phase. Frequent aberration of p16 in nasopharyngeal carcinoma (NPC) suggests a role for this tumor suppressor gene in disease development. p16 gene transfer has been demonstrated to be effective in various human cancer models, including breast, lung, and prostate, causing cell cycle arrest, apoptosis, and tumor growth delay. We investigated the potential of adenoviral-mediated p16 therapy, in combination with ionizing radiation (RT), in two distinct NPC models. Two deltaE1 adenoviral vectors were employed: one carrying the human p16 gene (adv.p16), and the other a beta-galactosidase reporter gene (adv.beta-gal), both driven by the cytomegalovirus (CMV) promoter. Two NPC cell lines with differential endogenous p16 expression, CNE-1 (low) and CNE-2Z (high), were evaluated for protein expression, cytotoxicity, cell cycle analysis, apoptosis, and senescence. The CNE-1 cells were exquisitely sensitive to adv.p16, with 0.1% survival level after gene therapy [25 plaque-forming unit (pfu)/cell], which further decreased to 0.01% with the addition of RT (2 Gy). This reduction in survival was effected through necrosis, G1 arrest, and senescence. In contrast, CNE-2Z cells were resistant to adv.p16 gene transfer, with 75% surviving at an equivalent viral dose. This differential sensitivity was recapitulated in vivo in that adv.p16-treated CNE-1 cells formed no tumors in severe-combined-immunodeficiency (SCID) mice, followed for over 100 days. In contrast, tumor formation was detected 40 days after implantation of adv.p16-treated CNE-2Z cells. In conclusion, adv.p16 gene transfer appears to be highly effective against NPC that lack functional p16, which is the situation in the majority of NPC patients.

Heat-directed tumor cell fusion.

In previous studies we demonstrated that a modified human HSP70b promoter (HSE.70b) directs high levels of gene expression to tumor cells after mild hyperthermia treatment in the range of 41.5-44 degrees C. This transcriptional targeting system exhibits low basal activity at 37 degrees C, is highly induced (950-fold) after mild heat treatment (43 degrees C/30 min), and returns to basal activity levels within 12-24 hours of activation. Here we describe heat-directed targeting of an activated form of the Gibbon ape leukemia virus env protein (GALV FMG) to tumor cells. GALV FMG mediates cell-cell fusion, and when expressed in tumor cells can produce bystander effects of up to 1:200. Transient transfection of a HSE70b.GALV FMG minigene caused extensive syncytia formation in HeLa and HT-1080 cells following mild heat treatment (44 degrees C/30 min). Stable transfection into HT-1080 cells produced a cell line (HG5) that exhibits massive syncytia formation and a 60% reduction in viability relative to a vector-only control (CI1) following heat treatment in vitro. Mild hyperthermia also resulted in syncytia formation, necrosis, and complete macroscopic regression of HG5 xenograft tumors grown in the footpads of mice with severe combined immunodeficiency disorders (SCID). Median survival increased from 12.5 (in heated CI1 controls) to 52 days after a single heat treatment. Heat-directed tumor cell fusion may prove to be a highly beneficial adjunct to existing cancer treatment strategies that take advantage of the synergistic interaction between mild hyperthermia and radiation or chemotherapeutic drugs.

Heat-directed suicide gene therapy for breast cancer.

Adjuvant hyperthermia can improve treatment outcome for locally recurrent breast cancer (LRBC). Previously, we demonstrated that infection of human breast cancer cells with a recombinant adenovirus expressing beta-galactosidase from the human hsp70b gene promoter (Ad.70b.betagal) results in 50- to 800-fold increases in reporter gene expression following heat treatment (30 minutes at 43 degrees C). Here, we describe a heat-directed suicide gene therapy strategy based on an adenoviral vector (Ad.70b.CDTK) in which expression of the dual prodrug-activating E. coli cytosine deaminase/herpes simplex virus thymidine kinase (CDTK) fusion gene is under the control of the hsp70b promoter. Treatment of T47D and MCF-7 breast cancer cells with mild hyperthermia (43 degrees C/30 minutes) and prodrugs (100 microg/ml 5-fluorocytosine and 10 microg/ml ganciclovir) following infection with Ad.70b.CDTK (10-100 PFU/cell) resulted in 30- to 60-fold decreases in clonogenic survival relative to control cultures treated with heat or prodrugs alone. Clonogenic survival declined even further (up to 240-fold) following heat treatment at 41.5 degrees C for 120 minutes. A decreased clonogenic survival was accompanied by tumor cell apoptosis. These results demonstrate that this combined treatment strategy can be highly effective against heat- and radiation-resistant breast tumor cells and supports the continued development of heat-directed CDTK suicide gene therapy strategies for LRBC.

Overexpression of the 78-kDa glucose-regulated protein/immunoglobulin-binding protein (GRP78/BiP) inhibits tissue factor procoagulant activity.

Previous studies have demonstrated that overexpression of GRP78/BiP, an endoplasmic reticulum (ER)-resident molecular chaperone, in mammalian cells inhibits the secretion of specific coagulation factors. However, the effects of GRP78/BiP on activation of the coagulation cascade leading to thrombin generation are not known. In this study, we examined whether GRP78/BiP overexpression mediates cell surface thrombin generation in a human bladder cancer cell line T24/83 having prothrombotic characteristics. We report here that cells overexpressing GRP78/BiP exhibited significant decreases in cell surface-mediated thrombin generation, prothrombin consumption and the formation of thrombin-inhibitor complexes, compared with wild-type or vector-transfected cells. This effect was attributed to the ability of GRP78/BiP to inhibit cell surface tissue factor (TF) procoagulant activity (PCA) because conversion of factor X to Xa and factor VII to VIIa were significantly lower on the surface of GRP78/BiP-overexpressing cells. The additional findings that (i) cell surface factor Xa generation was inhibited in the absence of factor VIIa and (ii) TF PCA was inhibited by a neutralizing antibody to human TF suggests that thrombin generation is mediated exclusively by TF. GRP78/BiP overexpression did not decrease cell surface levels of TF, suggesting that the inhibition in TF PCA does not result from retention of TF in the ER by GRP78/BiP. The additional observations that both adenovirus-mediated and stable GRP78/BiP overexpression attenuated TF PCA stimulated by ionomycin or hydrogen peroxide suggest that GRP78/BiP indirectly alters TF PCA through a mechanism involving cellular Ca(2+) and/or oxidative stress. Similar results were also observed in human aortic smooth muscle cells transfected with the GRP78/BiP adenovirus. Taken together, these findings demonstrate that overexpression of GRP78/BiP decreases thrombin generation by inhibiting cell surface TF PCA, thereby suppressing the prothrombotic potential of cells.

A novel muscle-specific enhancer identified within the deletion overlap region of two XLDC patients lacking muscle exon 1 of the human dystrophin gene.

Previous studies point to the involvement of several discrete transcriptional enhancers in the modulation of dystrophin gene expression in skeletal and cardiac muscle. Analysis of deletion breakpoints in two X-linked dilated cardiomyopathy patients with mutations that remove muscle exon 1 identified a 3.2-kb deletion overlap region (XLDC3.2) between -1199 and +2057 bp predicted to contain regulatory elements essential for dystrophin gene expression in cardiac muscle. A novel-sequence-based search strategy was used to identify a 543-bp region downstream of muscle exon 1 rich in cardiac-specific transcriptional elements. Designated dystrophin muscle enhancer 2 (DME2), this candidate enhancer was seen to function in a position- and orientation-independent manner in muscle cell lines but not in fibroblasts. As only modest activity was observed in primary neonatal rat cardiomyocytes, DME2 is thought to play a role in dystrophin gene regulation at later stages of cardiac muscle development.

Duchenne muscular dystrophy: current knowledge, treatment, and future prospects.

The cloning of the dystrophin gene has led to major advances in the understanding of the molecular genetic basis of Duchenne, Becker, and other muscular dystrophies associated with mutations in genes encoding members of the dystrophin-associated glycoprotein complex. The recent introduction of pharmaceutical agents such as prednisone has shown great promise in delaying the progression of Duchenne muscular dystrophy but there remains a need to develop more long-term therapeutic interventions. Knowledge of the nature of the dystrophin gene and the glycoprotein complex has led many researchers to think that somatic gene replacement represents the most promising approach to treatment. The potential use of this strategy has been shown in the mdx mouse model of Duchenne muscular dystrophy, where germ line gene transfer of either a full-length or a smaller Becker-type dystrophin minigene prevents necrosis and restores normal muscle function.

A novel p53 transcriptional repressor element (p53TRE) and the asymmetrical contribution of two p53 binding sites modulate the response of the placental transforming growth factor-beta promoter to p53.

Previous studies in our laboratory and others identified placental transforming growth factor-beta (PTGF-beta) as an important downstream mediator of DNA damage signaling and a transcriptional target of p53. Here we show that accumulation of PTGF-beta mRNA in response to p53 overexpression is delayed relative to p21(WAF1), whereas the promoters of these genes respond to p53 with similar kinetics. Mutational analyses of two p53 binding sites within the PTGF-beta promoter revealed that site p53-1 (+29 bp) is responsible for as much as 80% of the transcriptional response to p53. This is consistent with electrophoretic mobility shift assays showing that site p53-1 binds p53 with a much higher affinity than site p53-2 (-850 bp). We also describe for the first time a novel 21-bp element (-222 to -242 bp) that acts to down-regulate the PTGF-beta promoter response to p53. Termed the p53 transcriptional repressor element (p53TRE), this sequence was shown to suppress p53 transactivation in a position- and promoter-independent fashion and to associate with a 28-kDa protein expressed in several tumor cell lines. A p53 suppressor element and asymmetric p53 binding sites may participate determining the activation thresholds of p53-responsive promoters in a cell- and context-specific manner.

Expression of the prodrug-activating enzyme DT-diaphorase via Ad5 delivery to human colon carcinoma cells in vitro.

Intratumoral injection of recombinant adenoviral type 5 (Ad5) vectors that carry prodrug-activating enzymes like DT-diaphorase (DTD) could be used to selectively target tumor cells for chemotherapy. To demonstrate the feasibility of this approach, Ad5 vectors were constructed, which express human DTD minigenes for both wild-type and mutant (C-to-T change in nucleotide 609 in DTD cDNA) DTD under the control of the cytomegalovirus (CMV) promoter. HT29 human colon carcinoma cells express wild-type DTD, whereas BE human colon carcinoma cells express mutant DTD, have low to undetectable DTD activity, and are 4- to 6-fold more resistant to mitomycin C (MMC) than HT29 cells. A test of the ability of Ad5 to infect these cells (using a beta-galactosidase CMV-driven minigene) indicated that 90-100% of BE cells were infected at a multiplicity of infection (MOI) of 100, whereas only 15-40% of HT29 cells were infected at this MOI. Infection of BE cells in vitro with recombinant Ad5 carrying a minigene for wild-type DTD at MOIs of 3-100 resulted in a progressive increase in DTD activity and a maximal 8-fold increase in sensitivity to MMC as measured by a colony-forming assay. HT29 cells were sensitized 2- to 3-fold following treatment with Ad5.DTD at an MOI of 100. These results indicate that adenovirus-mediated gene transfer and expression of wild-type DTD can sensitize resistant tumor cells to MMC and that this therapeutic strategy may exert a significant bystander effect.