Dendritic cell differentiation blocked by primary effusion lymphoma-released factors is partially restored by inhibition of P38 MAPK.

To better understand the molecular mechanisms underlying the dendritic cell (DC) defects in cancer, we analyzed which signaling pathway is implicated in the abnormal monocyte differentiation into DC determined by the presence of Primary effusion lymphoma (PEL) released factors. Our results indicate that the DC, obtained in this condition, together with phenotypic abnormalities and reduced allostimulatory function, showed hyperphosphorylation of signal transducer and activator of transcription 3 (STAT3) and p38 mitogen-activated protein kinase (MAPK) molecules, in comparison to the DC differentiated in the absence of PEL-released factors. The inhibition of p38 MAPK but not of STAT3 phosphorylation, with specific inhibitors, was able to revert the effect of the PEL-released factors on the DC phenotype. This study suggests that p38 MAPK signaling pathway is an important contributor to the abnormal differentiation of DC in PEL.

Identification and characterization of the product encoded by ORF69 of Kaposi's sarcoma-associated herpesvirus.

We report the identification and characterization of p33, the product of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 69 (ORF69), a positional homolog of the conserved herpesvirus protein UL31. p33 is expressed upon induction of viral lytic cycle with early kinetics. Immunofluorescence analysis revealed that in infected cell lines, the protein is localized in the nucleus, both in dotted spots and along the nuclear membrane. Nuclear fractionation experiments showed that p33 partitions with the nuclear matrix, and both immunoblotting of purified virions and immunoelectron microscopy indicated that the novel protein is not a component of the mature virus. Following ectopic expression in KSHV-negative cells, the protein was never associated with the nuclear membrane, suggesting that p33 needs to interact with additional viral proteins to reach the nuclear rim. In fact, after cotransfection with the ORF67 gene, the KSHV positional homolog of UL34, the p33 intranuclear signal changed and the two proteins colocalized on the nuclear membrane. A similar result was obtained when ORF69 was cotransfected with BFRF1, the Epstein-Barr virus (EBV) positional homolog of UL34 and ORF67. Finally, upon cotransfection, ORF69 significantly increased nuclear membrane reduplications induced by BFRF1. The above results indicate that KSHV p33 shares many similarities with its EBV homolog BFLF2 and suggest that functional cross-complementation is possible between members of the gammaherpesvirus subfamily.

Calcium modulation activates Epstein-Barr virus genome in latently infected cells.

In many viral infections the host cell carries the viral genome without producing viral particles, a phenomenon known as viral latency. The cellular mechanisms by which viral latency is maintained or viral replication is induced are not known. The modulation of intracellular calcium concentrations by calcium ionophores induced Epstein-Barr viral antigens in lymphoblastoid cell lines that carry the virus. When calcium ionophores were used in conjunction with direct activators of protein kinase C (12-O-tetradecanoyl phorbol-13-acetate and a synthetic diacylglycerol), a greater induction of viral antigens was observed than with either agent alone. Activation of protein kinase C may be required for the expression of the viral genome.

EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation.

EBV has been reported to impair monocyte in vitro differentiation into dendritic cells (DCs) and reduce cell survival. In this study, we added another layer of knowledge to this topic and showed that these effects correlated with macroautophagy/autophagy, ROS and mitochondrial biogenesis reduction. Of note, autophagy and ROS, although strongly interconnected, have been separately reported to be induced by CSF2/GM-CSF (colony stimulating factor 2) and required for CSF2-IL4-driven monocyte in vitro differentiation into DCs. We show that EBV infects monocytes and initiates a feedback loop in which, by inhibiting autophagy, reduces ROS and through ROS reduction negatively influences autophagy. Mechanistically, autophagy reduction correlated with the downregulation of RAB7 and ATG5 expression and STAT3 activation, leading to the accumulation of SQSTM1/p62. The latter activated the SQSTM1-KEAP1- NFE2L2 axis and upregulated the anti-oxidant response, reducing ROS and further inhibiting autophagy. ROS decrease correlated also with the reduction of mitochondria, the main source of intracellular ROS, achieved by the downregulation of NRF1 and TFAM, mitochondrial biogenesis transcription factors. Interestingly, mitochondria supply membranes and ATP required for autophagy execution, thus their reduction may further reduce autophagy in EBV-infected monocytes. In conclusion, this study shows for the first time that the interconnected reduction of autophagy, intracellular ROS and mitochondria mediated by EBV switches monocyte differentiation into apoptosis, giving new insights into the mechanisms through which this virus reduces immune surveillance. Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; BAF: bafilomycin A1; BECN1: beclin 1; CAT: catalase; CSF2: colony stimulating factor 2; CT: control; CYCS (cytochrome C: somatic); DCs: dendritic cells; EBV: Epstein-Barr virus; GSR: glutathione-disulfide reductase; KEAP1: kelch like ECH associated protein 1; IL4: interleukin 4; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MET: metformin; NAC: N-acetylcysteine; NFE2L2/NRF2 nuclear factor: erythroid 2 like 2; NRF1 (nuclear respiratory factor 1); clPARP1: cleaved poly(ADP-ribose) polymerase; Rapa: Rapamycin; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TFAM: (transcription factor A: mitochondrial); TUBA1A: tubulin alpha 1a.

EBV early lytic protein BFRF1 alters emerin distribution and post-translational modification.

The nuclear envelope (NE), a structural element of fundamental importance for the cell, is the first barrier that meets a virus in the early stages of viral maturation. Therefore, in order to allow the passage of nucleocapsids, viruses are known to modulate the architecture of the nuclear membrane to permit a proficient viral infection. Epstein-Barr Virus (EBV), a pathogen from Herpesvirus family, possesses two well conserved proteins, BFRF1 and BFLF2, which together form the heterodimeric nuclear egress complex (NEC) that is involved in the early steps of nuclear egress. Here we show that EBV replication causes the delocalization of emerin, a cellular LEM-domain protein normally distributed on the nuclear rim. We also demonstrate that the early lytic protein BFRF1 is responsible for emerin delocalization. Expression of BFRF1 alone or in combination with BFLF2 induces emerin hyperphosphorylation. Altogether, these results suggest a novel mechanism by which EBV exploits the cellular machinery for nucleocapsid egress.

Chloroquine supplementation increases the cytotoxic effect of curcumin against Her2/neu overexpressing breast cancer cells in vitro and in vivo in nude mice while counteracts it in immune competent mice.

Autophagy is usually a pro-survival mechanism in cancer cells, especially in the course of chemotherapy, thus autophagy inhibition may enhance the chemotherapy-mediated anti-cancer effect. However, since autophagy is strongly involved in the immunogenicity of cell death by promoting ATP release, its inhibition may reduce the immune response against tumors, negatively influencing the overall outcome of chemotherapy. In this study, we evaluated the in vitro and in vivo anti-cancer effect of curcumin (CUR) against Her2/neu overexpressing breast cancer cells (TUBO) in the presence or in the absence of the autophagy inhibitor chloroquine (CQ). We found that TUBO cell death induced by CUR was increased in vitro by CQ and slightly in vivo in nude mice. Conversely, CQ counteracted the Cur cytotoxic effect in immune competent mice, as demonstrated by the lack of in vivo tumor regression and the reduction of overall mice survival as compared with CUR-treated mice. Immunohistochemistry analysis revealed the presence of a remarkable FoxP3 T cell infiltrate within the tumors in CUR/CQ treated mice and a reduction of T cytotoxic cells, as compared with single CUR treatment. These findings suggest that autophagy is important to elicit anti-tumor immune response and that autophagy inhibition by CQ reduces such response also by recruiting T regulatory (Treg) cells in the tumor microenvironment that may be pro-tumorigenic and might counteract CUR-mediated anti-cancer effects.

Epstein-Barr virus-related lymphocyte stimulation inhibitor: a possible prognostic tool for undifferentiated nasopharyngeal carcinoma.

It is demonstrated in this study that a serum factor, a lymphocyte stimulation inhibitor (LSI), which inhibits Epstein-Barr virus (EBV)-induced lymphocyte stimulation, is a potentially useful tool in the diagnosis and monitoring of nasopharyngeal carcinoma (NPC). In a study of 25 patients with undifferentiated NPC, 20 healthy controls, and 20 patients with other head and neck tumors, LSI was found only in the NPC patients with active disease. In a more complete study of 8 patients longitudinally followed up for at least 20 months, a comparison of LSI with antibodies to a variety of EBV antigens including viral capsid antigen, early antigen, and nuclear antigen indicated that LSI levels provided a reliable and sensitive indicator of disease activity that should be added to clinical markers currently in use as monitors of disease activity in NPC.

KSHV reduces autophagy in THP-1 cells and in differentiating monocytes by decreasing CAST/calpastatin and ATG5 expression.

We have previously shown that Kaposi sarcoma-associated herpesvirus (KSHV) impairs monocyte differentiation into dendritic cells (DCs). Macroautophagy/autophagy has been reported to be essential in such a differentiating process. Here we extended these studies and found that the impairment of DC formation by KSHV occurs through autophagy inhibition. KSHV indeed reduces CAST (calpastatin) and consequently decreases ATG5 expression in both THP-1 monocytoid cells and primary monocytes. We unveiled a new mechanism put in place by KSHV to escape from immune control. The discovery of viral immune suppressive strategies that contribute to the onset and progression of viral-associated malignancies is of fundamental importance for finding new therapeutic approaches against them.

HCV derived from sera of HCV-infected patients induces pro-fibrotic effects in human primary fibroblasts by activating GLI2.

Hepatitis C virus (HCV) infection is a leading cause of liver fibrosis, especially in developing countries. The process is characterized by the excess accumulation of ECM that may lead, over time, to hepatic cirrhosis, liver failure and also to hepatocarcinoma. The direct role of HCV in promoting fibroblasts trans-differentiation into myofibroblasts, the major fibrogenic cells, has not been fully clarified. In this study, we found that HCV derived from HCV-infected patients infected and directly induced the trans-differentiation of human primary fibroblasts into myofibroblasts, promoting fibrogenesis. This effect correlated with the activation of GLI2, one of the targets of Hedgehog signaling pathway previously reported to be involved in myofibroblast generation. Moreover, GLI2 activation by HCV correlated with a reduction of autophagy in fibroblasts, that may further promoted fibrosis. GLI2 inhibition by Gant 61 counteracted the pro-fibrotic effects and autophagy inhibition mediated by HCV, suggesting that targeting HH/GLI2 pathway might represent a promising strategy to reduce the HCV-induced fibrosis.

Expression of Egr-1 correlates with the transformed phenotype and the type of viral latency in EBV genome positive lymphoid cell lines.

In this paper we have investigated the role of Egr-1 in B cell growth regulation by examining the gene expression in a panel of B cell lines, including both EBV genome negative and EBV carrying cell lines. Egr-1 expression correlates with the cellular phenotype and the specific pattern of viral latency established within the individual cell lines. Thus, constitutive activation of Egr-1 gene is invariably associated with unrestricted expression of viral latent genes in all group III EBV genome carrying cell lines. In contrast, Egr-1 expression is abrogated in group I Burkitt tumor cells, irrespective of the EBV genome carrying status. Activated viral gene expression associated with phenotypic conversion of group I cell lines in to group II or III restores the Egr-1 gene expression. Several forms of EGR-1 protein are found within the different groups of cell lines, and the binding activity to DNA consensus sequences was investigated. Finally, time course analysis of Egr-1 expression during the early steps of EBV infection in vitro demonstrated that Egr-1 is upregulated within minutes from the initial interaction with the B lymphocyte.

HMGB1-Induced Cross Talk between PTEN and miRs 221/222 in Thyroid Cancer.

High mobility group box 1 (HMGB1) is an ubiquitous protein that plays different roles in the nucleus, cytoplasm, and extracellular space. It is an important DAMP molecule that allows communication between damaged or tumor cells and the immune system. Tumor cells exploit HMGB1's ability to activate intracellular pathways that lead to cell growth and migration. Papillary thyroid cancer is a well-differentiated tumor and is often used to study relationships between cells and the inflammatory microenvironment as the latter is characterized by high levels of inflammatory cells and cytokines. Anaplastic thyroid cancer is one of the most lethal human cancers in which many microRNAs and tumor suppressor genes are deregulated. Upregulation of microRNAs 221 and 222 has been shown to induce the malignant phenotype in many human cancers via inhibition of PTEN expression. In this study we suggest that extracellular HMGB1 interaction with RAGE enhances expression of oncogenic cluster miR221/222 that in turn inhibits tumor suppressor gene PTEN in two cell lines derived from human thyroid anaplastic and papillary cancers. The newly identified pathway HMGB1/RAGE/miR221/222 may represent an effective way of tumor escape from immune surveillance that could be used to develop new therapeutic strategies against anaplastic tumors.

Evidence for three binding sites for C3 (hemolytically inactive), C3b and C3d on a CR2-positive Burkitt lymphoma-derived cell line (Raji).

The present investigation shows that C3 (hemolytic inactive) as well as C3b and C3d bind Raji, a CR2-positive Burkitt lymphoma-derived cell line. Pretreatment of the cells with OKB-7 inhibited the binding of C3, whereas pretreatment with HB-5 inhibited the binding of C3b. Furthermore, the cells coated either with OKB-7 or HB-5 bound high amounts of C3d. TPA-treated cells showed binding for C3b and weak binding for C3 and C3d. Taken together, the data suggest that Raji cells may express three binding sites for C3, C3b and C3d which can be differently modulated by anti-CR2 MoAbs and TPA.

Cross-linking of at least three binding sites mediates signal transduction in a CR2-positive Burkitt lymphoma derived cell line (Raji).

In this study we demonstrate that Raji cells, a CR2-positive Burkitt lymphoma-derived cell line, during cell growth, need the cross-linking of multiple OKB7 binding sites or C3d determinants to mediate signal transduction. The loss of one of these affects the cellular response. Moreover, OKB7, the anti-CR2 MoAb, recognizes C3d determinants on the cell surface and inhibits signal transduction induced by anti-C3d polyclonal antibody. Since Raji cells are always CR2 positive during cell growth, we suppose that at least another protein, along with CR2, may be involved in setting up a cell surface complex able to receive and transduce the signal triggered by OKB7. In our experimental system the protein that offers a third binding site to OKB7, may be represented by a 33 kDa protein bearing C3d determinants.

Proliferating and quiescent cells exhibit different subcellular distribution of protein kinase C activity.

The activity of calcium, phospholipid-dependent protein kinase (PKc), which is thought to play an important role in cell proliferation, has been measured in the particulate and soluble fractions of cultured cells, under different proliferative conditions. Our results indicate that proliferating cells display higher PKc activity than quiescent cells. Furthermore, in both normal and transformed cells, PKc is preferentially associated with the particulate fraction when the cells are proliferating, while in mitotically quiescent cells the majority of the enzyme activity is found in the soluble fraction. These data suggest tha PKc activity and subcellular distribution undergo spontaneous changes according to the proliferative state of the cells.

Partition of epidermal growth factor receptors on freeze-fractured plasma membranes of A431 cells is affected by the ligand.

The fracture immunolabel technique, which permits assessment of the partition of transmembrane proteins with the inner or outer leaflets of the freeze-fractured membrane, was used to analyze the behavior on fracture of epidermal growth factor (EGF) receptors over the plasma membranes of A431 cells. The receptors partition mainly with the outer leaflet of the freeze-fractured plasma membranes, whereas they become associated with the inner leaflet when they are occupied by the ligand. This modified partition is even more evident after receptor clustering induced by incubation with EGF at 37 degrees C. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) decreases the number of receptors over both inner and outer leaflets. An effect similar to that induced by the ligand is obtained when receptor aggregation is achieved using anti-receptor monoclonal antibodies (MAb). The modified partition therefore indicates receptor activation and appears to be a consequence of receptor cross-linking rather than to reflect a conformational change of the receptor molecule. Parallel immunolabeling with anti-phosphotyrosine antibodies of freeze-fractured EGF-treated A431 cells reveals that the receptors, when activated, are associated only with the inner leaflet of the plasma membrane.

Epstein-Barr virus DNA is present both in CD10/CD77 positive and negative subsets of human tonsillar lymphocytes.

We report the fractionation of freshly isolated subsets of tonsillar lymphocytes according to cell density and double sorting for the differential expression of CD10 and CD77, and their analysis for the presence of Epstein Barr virus genome by nested PCR. All the subsets of tonsillar lymphocytes gave unequivocal evidence for the presence of EBV DNA, when obtained from EBV seropositive individuals. Confirmation of all cases examined demonstrates that B lymphocytes from the germinal centers of tonsils are also involved in carrying the EBV infection in vivo.

Infection of human T lymphoid cells by human herpesvirus 6 is blocked by two unrelated protein tyrosine kinase inhibitors, biochanin A and herbimycin.

Human herpesvirus 6 is a T lymphotropic herpesvirus that causes exanthem subitum in infants and is considered a potential cofactor in AIDS etiopathogenesis and progression owing to its in vivo and in vitro interactions with human immunodeficiency virus. We report that no differences in phosphorylation on tyrosine residues of cellular proteins were detectable at early times following HHV-6 infection in comparison to uninfected cells. On the contrary, several cellular proteins appeared phosphorylated on tyrosine at 24-48 hr postinfection. In addition, when tyrosine phosphorylation induced by HHV-6 infection was inhibited by the tyrosine kinase inhibitor biochanin A, the infection of HSB-2 cells was also coordinately reduced, as judged by inhibition of cytopathic effect and by inhibition of early and late viral antigen expression. Similar results were obtained with a second unrelated tyrosine kinase inhibitor, herbimycin. The inhibitors seem to act at a late stage of the viral infectious cycle, since neither viral binding nor internalization were affected. Thus, our results indicate that HHV-6 infection leads to the phosphorylation of protein tyrosine kinases, which may play a role in the course of viral infection, probably by participating in the cytopathic effect induced by the virus.

Human herpesvirus 6 variant A, but not variant B, infects EBV-positive B lymphoid cells, activating the latent EBV genome through a BZLF-1-dependent mechanism.

Human herpesvirus 6, a predominantly T lymphotropic virus, has been recently shown to infect some EBV-positive B cell lines, and to induce in them the activation of the EBV lytic cycle. Here we have confirmed and extended such observations, showing that (1) this phenomenon is restricted to the variant A of HHV-6: in fact two isolates belonging to the HHV-6 variant B (BA92 and Z29) were neither able to infect any B cell line, independently of the EBV status, nor to induce the EBV genome expression. The only exception is represented by the P3HR1 cells, in which, however, the infection by the variant B does not determine induction of EBV antigens; (2) the presence of the EBV genome contributes to the susceptibility of the B cell lines to HHV-6 infection, increasing the binding sites and the percentage of infectable cells, as detected by immunoelectron microscopy; and (3) HHV-6 infected T cells, transfected with plasmids bearing the promoter regions of the EBV early genes BZLF1 and BMRF1, show a strong transactivation of these promoters.

Human herpesvirus 6 envelope glycoproteins B and H-L complex are undetectable on the plasma membrane of infected lymphocytes.

Membrane immunofluorescence analysis of cells infected with either variant (A or B) of human herpesvirus 6 revealed a typical punctate staining, after labeling with several HHV-6-positive human sera or with two monoclonal antibodies directed to gB and gH. Immunoprecipitation studies showed a sharp difference in glycoprotein content in whole-cell extracts versus on the cell surface, suggesting the occurrence of gB in the extracellular virions juxtaposed to plasma membranes. By immunoelectron microscopy, the extracellular virions still attached to the cell surface appeared consistently and specifically labeled, whereas the plasma membrane was always unlabeled, independent of viral variant, antibody, or target cell used. These findings may reflect an atypical maturation pathway of HHV-6, and could have important implications in the control of cellular immune response to HHV-6-infected lymphocytes.

Infection by human herpesvirus 6 (HHV-6) of human lymphoid T cells occurs through an endocytic pathway.

We have analyzed by immunoelectron microscopy the early events of binding and internalization of human herpesvirus 6 (HHV-6, strain GS) on a susceptible T-lymphoblastoid cell line, HSB-2. The virions bound to the cell surface at 4 degrees C were tightly associated with the plasma membrane. Gold immunolabeling of the viral envelope proteins was strong and specific. Warming at 37 degrees C for different times showed viral internalization through smooth surfaced pits and vesicles. Fusion events of the virions with the cell plasma membrane were never observed. Gold immunolabeling performed in parallel experiments before or after viral internalization showed: (1) absence of viral envelope proteins on the cell plasma membranes at all times of internalization, again excluding fusion events; (2) entry of the virions with their envelopes. Treatment of the cells with chloroquine, a drug known to affect the endocytic pathway, led to an almost complete inhibition of viral infectivity, suggesting that the endocytosed virions are responsible for a successful infection. Comparable results were obtained using a second strain of HHV-6 (BA92), with biologic and molecular characteristics similar to the prototype strain Z29. The chloroquine inhibition was effective on two different T cell lines (HSB-2 and J-Jhan), as well as on phytohemagglutinin-stimulated peripheral blood mononuclear cells.