Yiu-Loon Chui

Elucidation of the c-Jun N-Terminal Kinase Pathway Mediated by Epstein-Barr Virus-Encoded Latent Membrane Protein 1

ABSTRACT Epstein-Barr virus (EBV) is associated with several human diseases including infectious mononucleosis and nasopharyngeal carcinoma. EBV-encoded latent membrane protein 1 (LMP1) is oncogenic and indispensable for cellular transformation caused by EBV. Expression of LMP1 in host cells constitutively activates both the c-Jun N-terminal kinase (JNK) and NF-κB pathways, which contributes to the oncogenic effect of LMP1. However, the underlying signaling mechanisms are not very well understood. Based mainly on overexpression studies with various dominant-negative constructs, LMP1 was generally thought to functionally mimic members of the tumor necrosis factor (TNF) receptor superfamily in signaling. In contrast to the prevailing paradigm, using embryonic fibroblasts from different knockout mice and the small interfering RNA technique, we find that the LMP1-mediated JNK pathway is distinct from those mediated by either TNF-α or interleukin-1. Moreover, we have further elucidated the LMP1-mediated JNK pathway by demonstrating that LMP1 selectively utilizes TNF receptor-associated factor 6, TAK1/TAB1, and c-Jun N-terminal kinase kinases 1 and 2 to activate JNK.

Phenotypic alterations induced by the Hong Kong‐prevalent Epstein‐Barr virus‐encoded LMP1 variant (2117‐LMP1) in nasopharyngeal epithelial cells

Epstein‐Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC), a common cancer in Hong Kong. The EBV‐encoded LMP1 protein is believed to play an important role in cell transformation. We have previously identified a prevalent LMP1 variant (2117‐LMP1) that is expressed in 86% of primary NPC in Hong Kong. In this study, the biologic phenotypes induced by 2117‐LMP1 were compared with those of the prototypic B95.8‐LMP1 in an immortalized nasopharyngeal epithelial cell line, NP69. The 2117‐LMP1 could induce cell proliferation and resistance to apoptosis induced by growth factor deprivation. Expression of 2117‐LMP1 also suppressed expression of p16, p21 and Bax but induced expression of CDK2 and A20. Compared with B95.8‐LMP1, 2117‐LMP1 could induce a higher migration ability in NP69 cells but was less efficient in inducing morphologic changes, anchorage‐independent growth and cell invasion. Relatively weaker ability of 2117‐LMP1 than B95.8‐LMP1 in upregulation of vimentin, VEGF and MMP9 as well as in downregulation of E‐cadherin was observed. 2117‐LMP1 could activate higher level of NF‐κB activity in HEK 293 cells than B95.8‐LMP1. The present study supports a role of 2117‐LMP1 in NPC development by enhancing cell proliferation, cell death inhibition and migration in premalignant nasopharyngeal epithelial cells. Furthermore, our study reveals significant functional differences between 2117‐LMP1 and the prototypic B95.8‐LMP1. Our results provide insights into the pathologic significance of this prevalent LMP1 variant, 2117‐LMP1, in the development of NPC in the Hong Kong population.

BRE is an antiapoptotic protein in vivo and overexpressed in human hepatocellular carcinoma

BRE binds to the cytoplasmic domains of tumor necrosis factor receptor-1 and Fas, and in cell lines can attenuate death receptor-initiated apoptosis by inhibiting t-BID-induced activation of the mitochondrial apoptotic pathway. Overexpression of BRE by transfection can also attenuate intrinsic apoptosis and promote growth of the transfected Lewis lung carcinoma line in mice. There is, however, a complete lack of in vivo data about the protein. Here, we report that by using our BRE-specific monoclonal antibody on the immunohistochemistry of 123 specimens of human hepatocellular carcinoma (HCC), significant differences in BRE expression levels between the paired tumoral and non-tumoral regions (P<2.2e−16) were found. Marked overexpression of BRE was detected in majority of the tumors, whereas most non-tumoral regions expressed the same low level of the protein as in normal livers. To investigate whether BRE overexpression could promote cell survival in vivo, liver-specific transgenic BRE mice were generated and found to be significantly resistant to Fas-mediated lethal hepatic apoptosis. The transgenic model also revealed post-transcriptional regulation of Bre level in the liver, which was not observed in HCC and non-HCC cell lines. Indeed, all cell lines analysed express high levels of BRE. In conclusion, BRE is antiapoptotic in vivo, and may promote tumorigenesis when overexpressed.

Transgenic cyclooxygenase-2 expression and high salt enhanced susceptibility to chemical-induced gastric cancer development in mice

Cyclooxoygenase (COX)-2 overexpression is involved in gastric carcinogenesis. While high-salt intake is a known risk factor for gastric cancer development, we determined the effects of high salt on gastric chemical carcinogenesis in COX-2 transgenic (TG) mice. COX-2 TG mice were developed in C57/BL6 strain using the full-length human cox-2 complementary DNA construct. Six-week-old COX-2 TG and wild-type (WT) littermates were randomly allocated to receive alternate week of N-methyl-N-nitrosourea (MNU, 240 p.p.m.) in drinking water or control for 10 weeks. Two groups of mice were further treated with 10% NaCl during the initial 10 weeks. All mice were killed at the end of week 50. Both forced COX-2 overexpression and high-salt intake significantly increased the frequency of gastric cancer development in mice as compared with WT littermates treated with MNU alone. However, no additive effect was observed on the combination of high salt and COX-2 expression. We further showed that MNU and high-salt treatment increased chronic inflammatory infiltrates and induced prostaglandin E(2) (PGE(2)) production in the non-cancerous stomach. Whereas high-salt treatment markedly increased the expression of inflammatory cytokines (tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-1 beta and IL-6) in the gastric mucosa, COX-2 overexpression significantly altered the cell kinetics in the MNU-induced gastric cancer model. In conclusion, both high salt and COX-2 overexpression promote chemical-induced gastric carcinogenesis, possibly related to chronic inflammation, induction of PGE(2), disruption of cell kinetics and induction of inflammatory cytokines.

In vivo functional characterization of the SARS-Coronavirus 3a protein in Drosophila☆

Abstract The Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) 3a locus encodes a 274 a.a. novel protein, and its expression has been confirmed in SARS patients. To study functional roles of 3a, we established a transgenic fly model for the SARS-CoV 3a gene. Misexpression of 3a in Drosophila caused a dominant rough eye phenotype. Using a specific monoclonal antibody, we demonstrated that the 3a protein displayed a punctate cytoplasmic localization in Drosophila as in SARS-CoV-infected cells. We provide genetic evidence to support that 3a is functionally related to clathrin-mediated endocytosis. We further found that 3a misexpression induces apoptosis, which could be modulated by cellular cytochrome c levels and caspase activity. From a forward genetic screen, 78 dominant 3a modifying loci were recovered and the identity of these modifiers revealed that the severity of the 3a-induced rough eye phenotype depends on multiple cellular processes including gene transcriptional regulation.

Comparative proteomic analysis identifies protein disulfide isomerase and peroxiredoxin 1 as new players involved in embryonic interdigital cell death

In this study, we used comparative proteomics to identify proteins that were involved in the regulation of interdigital cell death. The protein profiles of embryonic day (E) 12.5 and 13.5 mouse hindlimb interdigital tissues were compared to identify proteins that were differentially expressed. The interdigital cells are irreversibly committed to programmed cell death (PCD) at E13.5, whereas they are developmentally plastic at E12.5. We established that protein disulfide isomerase (PDI) expression was up‐regulated at E13.5, while peroxiredoxin 1 (Prdx1) expression was down‐regulated at this time point. Semiquantitative reverse transcriptase‐polymerase chain reaction and Western blot analyses confirmed the data obtained from the two‐dimensional electrophoresis gels. Furthermore, we were able to up‐regulate PDI expression by manipulating the E12.5 interdigital tissues to die during culture, although this up‐regulation was not possible when cell survival was promoted. In addition, we could inhibit interdigital cell death and expression of proapoptotic genes (Bmp‐4 and Bambi) by treating interdigital tissues with PDI antibodies and bacitracin (a PDI enzyme inhibitor). These findings suggested that PDI was involved in the activation and maintenance of interdigital cell death. Conversely, we determined that Prdx1 expression was maintained when interdigital cultures were manipulated to survive but down‐regulated when the cultures were permitted to die. The result suggested that Prdx1 was involved in maintaining interdigital cell survival. However, we were unable to induce interdigital cell death by means of RNA interference‐mediated silencing of Prdx1 expression, indicating that Prdx1 down‐regulation is not sufficient for PCD to occur. Proteomic analysis of the Prdx1 knock‐down cells revealed that the level of NF‐kappaB inhibitor epsilon (IκBε) was dramatically reduced. Furthermore, we found an increase in NFκB activation and reactive oxygen species (ROS) levels in the cytoplasm as a result of Prdx1 knockdown. We also found that silencing Prdx1 made the interdigital cells more susceptible to ROS‐induced cell death. Taken together, our study identifies two new players in interdigital cell death and highlights that PCD is regulated by a delicate balance of proapoptotic and survival‐promoting activities. Developmental Dynamics 233:266–281, 2005.

KIAA0495/PDAM Is frequently downregulated in oligodendroglial tumors and its knockdown by siRNA induces cisplatin resistance in glioma cells

Co‐deletion of chromosomes 1p and 19q is a common event in oligodendroglial tumors (OTs), suggesting the presence of OT‐related genes. The aim of this study was to identify the target genes residing in the minimally deleted regions on chromosome 1p36.31–p36.32 that might be involved in OTs. A novel gene KIAA0495/p53‐dependent apoptosis modulator (PDAM) was found frequently deregulated, with 37 of 58 (63.8%) OTs examined showing reduced expression compared with normal brain. Chromosome 1p loss and epigenetic modifications were the major mechanisms contributing to PDAM downregulation. The role of PDAM in chemosensitivity was also evaluated. PDAM knockdown had no effect on sensitivity to vincristine, lomustine, temozolomide and paclitaxel, but could induce cisplatin resistance in glioma cells harboring wild‐type p53. B‐cell CCL/lymphoma 2 (BCL2)‐like 1 (BCL2L1) exhibited significant upregulation, while BCL2 showed partial derepression in PDAM‐silenced cells after cisplatin treatment, suggesting that alteration of anti‐apoptotic genes contributed in part to cisplatin resistance. Knockdown of BCL2L1 abrogated the induced cisplatin‐resistant phenotype. Moreover, our data suggested that PDAM might function as a non‐protein‐coding RNA. Collectively, these findings suggest that PDAM deregulation may play a role in OT development and that PDAM may possess the capacity to modulate apoptosis via regulation of p53‐dependent anti‐apoptotic genes.

A human and a mouse anti-idiotypic antibody specific for human T14(+) anti-DNA antibodies reconstructed by phage display.

Little is known about human anti-idiotypic antibodies. Phage display methodology was used to reconstruct these antibodies from lupus patients, which recognize a subset (T14(+)) of anti-DNA antibodies. Antigen-specific B cells were isolated from the blood using a peptide based on a complementarity determining region (V(H)CDR3) of the prototypic T14(+) antibody. cDNA fragments of the V(H) and V(L) genes prepared from the cells were expressed as phage displayed single chain Fv (scFv) fragments using the pCANTAB-5E phagemid vector. From a reactive clone obtained, the Ig genes used were identified to be V(H)3, D5-D3, J(H)4b, V(kappa)I and J(kappa)2. The heavy chain was highly mutated, especially in CDR3, which bears mutations mostly of the replacement type; this region is also unusual in being extremely long due to a D-D fusion. In contrast, a mouse hybridoma antibody, made to the same T14(+) peptide and transformed as a scFv fragment, uses a short V(H)CDR3 comprising five amino acids, three of which are tyrosines. Tyrosines may be important for antigen binding because two of these also exist in the human V(H)CDR3. The light chains of both antibodies may also contribute to the specificity of the protein, because their V(L) segments, including the CDRs, are highly homologous to each other.

BRE over-expression promotes growth of hepatocellular carcinoma

BRE, also known as TNFRSF1A modulator and BRCC45, is an evolutionarily highly conserved protein. It is a death receptor-associated protein in cytoplasm and a component of BRCA1/2-containing DNA repair complex in nucleus. BRE was found to have anti-apoptotic activity. Over-expression of BRE by transfection promoted survival of cell lines against apoptotic induction; whereas depletion of the protein by siRNA resulted in the opposite. In vivo anti-apoptotic activity of BRE was demonstrated by significant attenuation of Fas-induced acute fulminant hepatitis in transgenic mice expressing the human protein specifically in the liver. BRE was also implicated in tumor promotion by the accelerated tumor growth of Lewis Lung carcinoma transfected with human BRE; and by high expression of BRE specifically in the tumoral regions of human hepatocellular carcinoma (HCC). The present study was to test directly if transgenic expression of BRE in livers could promote HCC development in neonatal diethylnitrosamine model. By 8months after tumor induction, the maximal sizes of tumor nodules of transgenic mice were significantly larger than those of the non-transgenic controls, although the numbers of tumor nodules between the two groups did not significantly differ. Importantly, as in human HCC, the mouse endogenous BRE level was up-regulated in mouse HCC nodules. These results show that BRE over-expression can indeed promote growth, though not initiation, of liver tumors. Furthermore, the common occurrence of BRE over-expression in human and mouse HCC suggests that up-regulation of BRE is functionally important in liver tumor development.

Silencing BRE expression in human umbilical cord perivascular (HUCPV) progenitor cells accelerates osteogenic and chondrogenic differentiation.

BRE is a multifunctional adapter protein involved in DNA repair, cell survival and stress response. To date, most studies of this protein have been focused in the tumor model. The role of BRE in stem cell biology has never been investigated. Therefore, we have used HUCPV progenitor cells to elucidate the function of BRE. HUCPV cells are multipotent fetal progenitor cells which possess the ability to differentiate into a multitude of mesenchymal cell lineages when chemically induced and can be more easily amplified in culture. In this study, we have established that BRE expression was normally expressed in HUCPV cells but become down-regulated when the cells were induced to differentiate. In addition, silencing BRE expression, using BRE-siRNAs, in HUCPV cells could accelerate induced chondrogenic and osteogenic differentiation. Hence, we postulated that BRE played an important role in maintaining the stemness of HUCPV cells. We used microarray analysis to examine the transcriptome of BRE-silenced cells. BRE-silencing negatively regulated OCT4, FGF5 and FOXO1A. BRE-silencing also altered the expression of epigenetic genes and components of the TGF-β/BMP and FGF signaling pathways which are crucially involved in maintaining stem cell self-renewal. Comparative proteomic profiling also revealed that BRE-silencing resulted in decreased expressions of actin-binding proteins. In sum, we propose that BRE acts like an adaptor protein that promotes stemness and at the same time inhibits the differentiation of HUCPV cells.