Interference effect of epigallocatechin-3-gallate on targets of nuclear factor kappaB signal transduction pathways activated by EB virus encoded latent membrane protein 1

AIM: To elucidate the interference effect of epigallocatechin-3-gallate (EGCG) on targets of nuclear factor kappaB (NF-kappaB) signal transduction pathway activated by EB virus encoded latent membrane protein 1 (LMP1) in nasopharyngeal carcinoma (NPC) cell lines. METHODS: The survival rates of CNE1 and CNE-LMP1 cell lines after the EGCG treatment were determined by MTT assay. NF-kappaB activation in CNE1 and CNE-LMP1 cells after EGCG treatment was analyzed by promoter luciferase reporter system. And then nuclear translocation of NF-kappaB (p65) after the EGCG treatment was analyzed by immunofluorescence and western blotting. Meanwhile, the changes of IkappaBalpha phosphorylation were observed after the EGCG treatment. EGFR promoter activity was analyzed by promoter luciferase reporter system and EGFR phosphorylation was observed by western blotting after the EGCG treatment. RESULTS: EGCG inhibited the survival rates of CNE1 and CNE-LMP1 cells and NF-kappaB activation caused by LMP1 in CNE-LMP1 cells. EGCG also suppressed the nuclear translocation of NF-kappaB (p65) and IkappaBalpha phosphorylation. Meanwhile, EGCG inhibited EGFR promoter activity and EGFR phosphorylation. CONCLUSIONS: EGCG inhibited not only the dose-dependent survival rate of NPC cells, but also the dose-dependent activation of NF-kappaB. This inhibition of LMP1-caused NF-kappaB activation was mediated via the phosphorylative degradation of its inhibitory protein IkappaBalpha, and then EGCG inhibited EGFR activity which was a downstream gene from NF-kappaB. This study suggests that interference effect of EGCG on targets of signal transduction pathway plays an important role in the anticancer function.     

TNFalpha induces rapid activation and nuclear translocation of telomerase in human lymphocytes

Maintenance of telomeres regulates chromosomal stability and cellular mitosis through a checkpoint mechanism. Continuous cell proliferation requires telomerase to maintain chromosomal stability and to counteract the cellular mitotic clock. Importantly, nuclear expression of telomerase activity is required for elongation of telomere sequences. In this study, we show that tumor necrosis factor alpha (TNFalpha) induces telomerase activity in the cytoplasm of peripheral blood lymphocytes (PBL) at 60 min, followed by translocation of activated telomerase to the nucleus at 120 min. Conversely, the phosphoinositol 3-kinase (PI3K) inhibitor wortmannin blocks TNFalpha-induced activation of telomerase, whereas the specific NF-kappaB translocation inhibitor SN-50 blocks TNFalpha-induced nuclear translocation of activated telomerase. These studies suggest that activation and nuclear translocation of telomerase are regulated by PI3K/Akt/NF-kappaB signaling pathways in PBL.     

A human TERT C-terminal polypeptide sensitizes HeLa cells to H2O2-induced senescence without affecting telomerase enzymatic activity

We have constructed a 27-kDa hTERT C-terminal polypeptide (hTERTC27) devoid of domains required for telomerase activity and demonstrated that it is capable of nuclear translocation/telomere-end targeting. Here we showed that expression of a low level of hTERTC27 renders hTERT positive HeLa cells sensitive to H(2)O(2)-induced oxidative stress and subsequent cell senescence. The senescence-associated gene, the cyclin/cdk inhibitor p21(Waf1), was up-regulated. This occurs without changing the expression of endogenous hTERT, causing significant telomere shortening or inhibiting telomerase activity. Results from this study suggest for the first time that in addition to telomerase activity, the C-terminus of hTERT also plays a role in hTERT-mediated cellular resistance to oxidative stress.     

Upregulated expression of kappa light chain by Epstein-Barr virus encoded latent membrane protein 1 in nasopharyngeal carcinoma cells via NF-kappaB and AP-1 pathways

B lymphocytes are generally considered to be the only source of immunoglobulins. However, increasing evidence revealed that some human epithelial cancer cell lines, including nasopharyngeal carcinoma (NPC) cell lines, expressed immunoglobulins. Moreover, we previously found that expression of kappa light chain in NPC cells could be upregulated by EBV-encoded latent membrane protein 1 (LMP1). Here, Western blot and flow cytometric analysis of intracellular kappa staining indicated that upregulation of the expression of kappa was inhibited by using LMP1-targeted DNAzyme and that Bay11-7082 and SP600125, inhibitors of JNK and NF-kappaB, respectively, inhibited LMP1-augmented kappa light chain expression in NPC cells. LMP1-positive NPC cells expressing the dominant-negative mutant of IkappaBalpha (DNMIkappaBalpha) or of c-Jun (TAM67) exhibited significantly decreasing kappa production compared with their parental cells. These results suggest that LMP1 elevated kappa light chain through activation of the NF-kappaB and AP-1 signaling pathways. The present study provided some hints of possible mechanisms by which human cancer cells of epithelial origin produced immunoglobulins.     

Involvement of 14-3-3 proteins in nuclear localization of telomerase

Maintenance of telomeres is implicated in chromosome stabilization and cell immortalization. Telomerase, which catalyzes de novo synthesis of telomeres, is activated in germ cells and most cancers. Telomerase activity is regulated by gene expression for its catalytic subunit, TERT, whereas several lines of evidence have suggested a post-translational regulation of telomerase activity. Here we identify the 14-3-3 signaling proteins as human TERT (hTERT)-binding partners. A dominant-negative 14-3-3 redistributed hTERT, which was normally predominant in the nucleus, into the cytoplasm. Consistent with this observation, hTERT-3A, a mutant that could not bind 14-3-3, was localized into the cytoplasm. Leptomycin B, an inhibitor of CRM1/exportin 1-mediated nuclear export, or disruption of a nuclear export signal (NES)-like motif located just upstream of the 14-3-3 binding site in hTERT impaired the cytoplasmic localization of hTERT. Compared with wild-type hTERT, hTERT-3A increased its association with CRM1. 14-3-3 binding was not required for telomerase activity either in vitro or in cell extracts. These observations suggest that 14-3-3 enhances nuclear localization of TERT by inhibiting the CRM1 binding to the TERT NES-like motif.     

Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor through effects on Bcl-3 and STAT3

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) activates multiple signaling pathways. Two regions, C-terminal-activating region 1 (CTAR1) and CTAR2, have been identified within the cytoplasmic carboxy terminal domain that activates NF-kappaB. CTAR2 activates the canonical NF-kappaB pathway, which includes p50/p65 complexes. CTAR1 can activate both the canonical and noncanonical pathways to produce multiple distinct NF-kappaB dimers, including p52/p50, p52/p65, and p50/p50. CTAR1 also uniquely upregulates the epidermal growth factor receptor (EGFR) in epithelial cells. Increased p50-Bcl-3 complexes have been detected by chromatin precipitation on the NF-kappaB consensus motifs within the egfr promoter in CTAR1-expressing epithelial cells and nasopharyngeal carcinoma cells. In this study, the mechanism responsible for the increase in Bcl-3 has been further investigated. The data indicate that LMP1-CTAR1 induces Bcl-3 mRNA and increases the nuclear translocation of both Bcl-3 and p50. LMP1-CTAR1 constitutively activates STAT3, and this activation was not due to the induction of interleukin 6 (IL-6). In LMP1-CTAR1-expressing cells, increased levels of activated STAT3 were detected by chromatin immunoprecipitation on STAT-binding sites located within both the promoter and the second intron of Bcl-3. A STAT3 inhibitor significantly reduced the activation of STAT3, as well as the CTAR1-mediated upregulation of Bcl-3 and EGFR. These data suggest that LMP1 activates distinct forms of NF-kappaB through multiple pathways. In addition to activating the canonical and noncanonical pathways, LMP1-CTAR1 constitutively activates STAT3 and increases Bcl-3. The increased nuclear Bcl-3 and p50 homodimer complexes positively regulate EGFR expression. These results indicate that LMP1 likely regulates distinct cellular genes by activating specific NF-kappaB pathways.     

DNA-protein kinase catalytic subunit-interacting protein KIP binds telomerase by interacting with human telomerase reverse transcriptase

Telomere homeostasis, a process that is essential for continued cell proliferation and genomic stability, is regulated by endogenous telomerase and a collection of associated proteins. In this paper, a protein called KIP (previously reported as a protein that binds specifically to DNA-dependent protein kinase), has been identified as a telomerase-regulating activity based on the following pieces of evidence. First, complexes between KIP and the catalytic subunit of telomerase (hTERT) were identified using the yeast two-hybrid technique. Second, antibodies specific to KIP immunoprecipitate human telomerase in cell-free extracts. Third, immunolocalization experiments demonstrate that KIP is a nuclear protein that co-localizes with hTERT in cells. Fourth, KIP binds to hTERT both in vitro and in vivo in the absence of human telomerase RNA or telomeric DNA, thus defining the catalytic subunit of telomerase as the site of physical interaction. Fifth, co-immunoprecipitation experiments suggest that KIP-hTERT complexes form readily in cells and that overexpression of KIP in telomerase-positive cells increases endogenous telomerase activity. Finally, continued overexpression of KIP (60 population doublings) resulted in cells with elongated telomeres; thus, KIP directly or indirectly stimulates telomerase activity through hTERT and contributes to telomere lengthening. The collective data in this paper suggest that KIP plays a positive role in telomere length maintenance and/or regulation and may represent a novel target for anti-cancer drug development.     

Involvement of hTERT in apoptosis induced by interference with Bcl-2 expression and function

Here, we investigated the role of telomerase on Bcl-2-dependent apoptosis. To this end, the 4625 Bcl-2/Bcl-xL bispecific antisense oligonucleotide and the HA14-1 Bcl-2 inhibitor were used. We found that apoptosis induced by 4625 oligonucleotide was associated with decreased Bcl-2 protein expression and telomerase activity, while HA14-1 triggered apoptosis without affecting both Bcl-2 and telomerase levels. Interestingly, HA14-1 treatment resulted in a profound change from predominantly nuclear to a predominantly cytoplasmic localization of hTERT. Downregulation of endogenous hTERT protein by RNA interference markedly increased apoptosis induced by both 4625 and HA14-1, while overexpression of wild-type hTERT blocked Bcl-2-dependent apoptosis in a p53-independent manner. Catalytically and biologically inactive hTERT mutants showed a similar behavior as the wild-type form, indicating that hTERT inhibited the 4625 and HA14-1-induced apoptosis regardless of telomerase activity and its ability to lengthening telomeres. Finally, hTERT overexpression abrogated 4625 and HA14-1-induced mitochondrial dysfunction and nuclear translocation of hTERT. In conclusion, our results demonstrate that hTERT is involved in mitochondrial apoptosis induced by targeted inhibition of Bcl-2.     

Epstein-Barr virus LMP2A alters in vivo and in vitro models of B-cell anergy, but not deletion, in response to autoantigen

A significant percentage of the population latently harbors Epstein-Barr virus (EBV) in B cells. One EBV-encoded protein, latent membrane protein 2A (LMP2A), is expressed in tissue culture models of EBV latent infection, in human infections, and in many of the EBV-associated proliferative disorders. LMP2A constitutively activates proteins involved in the B-cell receptor (BCR) signal transduction cascade and inhibits the antigen-induced activation of these proteins. In the present study, we investigated whether LMP2A alters B-cell receptor signaling in primary B cells in vivo and in vitro. LMP2A does not inhibit antigen-induced tolerance in response to strong stimuli in an in vivo tolerance model in which B cells are reactive to self-antigen. In contrast, LMP2A bypasses anergy induction in response to low levels of soluble hen egg lysozyme (HEL) both in vivo and in vitro as determined by the ability of LMP2A-expressing HEL-specific B cells to proliferate and induce NF-kappaB nuclear translocation after exposure to low levels of antigen. Furthermore, LMP2A induces NF-kappaB nuclear translocation independent of BCR cross-linking. Since NF-kappaB is required to bypass tolerance induction, this LMP2A-dependent NF-kappaB activation may complete the tolerogenic signal induced by low levels of soluble HEL. Overall, the findings suggest that LMP2A may not inhibit BCR-induced signals under all conditions as previously suggested by studies with EBV immortalized B cells.     

Inhibition of nuclear factor-kappaB activation is essential for membrane-associated TNF-alpha-induced apoptosis in HL-60 cells

The killing of tumour cells that are resistant to soluble TNF-alpha (sTNF-alpha) by the membrane-bound form of TNF-alpha (mTNF-alpha) suggests that different intracellular signalling pathways are involved. We found that mTNF-alpha induced apoptosis in HL-60 cells and failed to cause degradation of inhibitor of kappa B alpha (IkappaB-alpha) and translocation and activation of nuclear factor kappa B (NF-kappaB), whereas sTNF-alpha failed to induce apoptosis, but lowered cytoplasmic inhibitor of kappa B alpha, induced translocation of NF-kappaB to the nucleus and experimentally increased activity of the regulated luciferase. Furthermore, mTNF-alpha upregulated the expression of TNF receptor associated factor (TRAF) 1 and failed to induce TRAF1 and TRAF2 membrane translocation, but led to cytoplasmic colocalization. In contrast, sTNF-alpha stimulated the expression of TRAF1 and TRAF2, recruiting both molecules onto the cell membrane poststimulation. These results suggest that the increased susceptibility of HL-60 cells to mTNF-alpha may be due to the failure of TRAF2 membrane translocation caused by the upregulation of TRAF1 expression and formation of a TRAF1/TRAF2 complex in the cytoplasm, thereby inhibiting NF-kappaB activation and inducing apoptosis.     

Proteasome subunit LMP2 is required for matrix metalloproteinase-2 and -9 expression and activities in human invasive extravillous trophoblast cell line

The ubiquitin-proteasome pathway (UPP) is involved in the degradation of the extracellular matrix (ECM) and trophoblastic invasion during early pregnancy. Our previous studies demonstrated that inhibition of UPP suppresses expression of matrix metalloproteinase (MMP)-2 and -9. LMP2 is an important proteasome subunit that is critical for proteasome activity. This study investigated the regulatory mechanism of LMP2 on the expression and activities of MMP-2 and MMP-9. Our results showed that transfection of LMP2 siRNA plasmid into the human invasive extravillous trophoblast cell line (HTR8/Svneo) could significantly suppress expression of LMP2 mRNA and protein. The mRNA expression of MMP-2 and MMP-9 and their activities were markedly decreased in the LMP2-inhibited cells. Inhibition of LMP2 could also reduce IkappaBalpha mRNA level, although the expression of phosphorylated IkappaBalpha was increased. In the LMP2-inhibited cells, expression of mRNA encoding NF-kappaB subunits p50 and p65 remained normal, but the p50 protein level was significantly decreased in the cytosolic and nuclear extracts, while p65 protein was markedly reduced only in the nuclear extract. We also demonstrated that blockage of the NF-kappaB pathway by the NF-kappaB translocation inhibitor SN50 markedly reduced the expression of MMP-2 and MMP-9 in HTR8/Svneo cells, a result that is fully consistent with the results from the LMP2-inhibited HTR8/Svneo cells. These data suggest that LMP2 contributes to IkappaBalpha degradation and p50 generation, and that inhibition of LMP2 suppresses expression and activities of MMP-2 and MMP-9 by blocking the transfer of active NF-kappaB heterodimers into the nucleus.