Prisco Mirandola

NK Cells and Cancer

In this review, we overview the main features and functions of NK cells, focusing on their role in cell-mediated immune response to tumor cells. In parallel, we discuss the information available in the field of NK cell receptors and offer a wide general overview of functional aspects of cell targeting and killing, focusing on the recent acknowledgments on the efficacy of NK cells after cytokine and mAb administration in cancer therapy. Since efficacy of NK cell-based immunotherapy has been proven in KIR-mismatch regimens or in TRAIL-dependent apoptosis, the ability to manipulate the balance of activating and inhibitory receptors on NK cells and of their cognate ligands, as well as the sensitivity of tumor cells to apoptosis, opens new perspectives for NK cell-based immunotherapy.

Human herpesvirus 6: An emerging pathogen.

Infections with human herpesvirus 6 (HHV-6), a ß-herpesvirus of which two variant groups (A and B) are recognized, is very common, approaching 100% in seroprevalence. Primary infection with HHV-6B causes roseola infantum or exanthem subitum, a common childhood disease that resolves spontaneously. After primary infection, the virus replicates in the salivary glands and is shed in saliva, the recognized route of transmission for variant B strains; it remains latent in lymphocytes and monocytes and persists at low levels in cells and tissues. Not usually associated with disease in the immunocompetent, HHV-6 infection is a major cause of opportunistic viral infections in the immunosuppressed, typically AIDS patients and transplant recipients, in whom HHV-6 infection/reactivation may culminate in rejection of transplanted organs and death. Other opportunistic viruses, human cytomegalovirus and HHV-7, also infect or reactivate in persons at risk. Another disease whose pathogenesis may be correlated with HHV-6 is multiple sclerosis. Data in favor of and against the correlation are discussed.

Caffeine inhibits adenosine-induced accumulation of hypoxia-inducible factor-1alpha, vascular endothelial growth factor, and interleukin-8 expression in hypoxic human colon cancer cells.

Frequent coffee consumption has been associated with a reduced risk of colorectal cancer in a number of case-control studies. Coffee is a leading source of methylxanthines, such as caffeine. The induction of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) is an essential feature of tumor angiogenesis, and the hypoxia-inducible factor-1 (HIF-1) transcription factor is known to be a key regulator of this process. In this study, we investigated the effects of caffeine on HIF-1 protein accumulation and on VEGF and IL-8 expression in the human colon cancer cell line HT29 under hypoxic conditions. Our results show that caffeine significantly inhibits adenosine-induced HIF-1α protein accumulation in cancer cells. We show that HIF-1α and VEGF are increased through A3 adenosine receptor stimulation, whereas the effects on IL-8 are mediated via the A2B subtype. Pretreatment of cells with caffeine significantly reduces adenosine-induced VEGF promoter activity and VEGF and IL-8 expression. The mechanism of caffeine seems to involve the inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and Akt, leading to a marked decrease in adenosine-induced HIF-1α accumulation, VEGF transcriptional activation, and VEGF and IL-8 protein accumulation. From a functional perspective, we observe that caffeine also significantly inhibits the A3 receptor-stimulated cell migration of colon cancer cells. Conditioned media prepared from colon cells treated with an adenosine analog increased human umbilical vein endothelial cell migration. These data provide evidence that adenosine could modulate the migration of colon cancer cells by an HIF-1α/VEGF/IL-8-dependent mechanism and that caffeine has the potential to inhibit colon cancer cell growth.

Hydrogen sulfide prevents apoptosis of human PMN via inhibition of p38 and caspase 3.

Hydrogen sulfide, together with carbon monoxide and nitric oxide, is now considered a gasotransmitter able to induce specific cellular responses. As hydrogen sulfide is a component of several natural compounds known to be effective in many inflammatory pathologies, particularly of the respiratory tract, we studied its effects in vitro on the survival and bactericidal activity of purified human neutrophils. We found that (1) HS− ions promote the survival of granulocytes, but not that of lymphocytes or eosinophils, cultured in serum-free medium; (2) the pro-survival effect of HS− is due to inhibition of caspase-3 cleavage and p38 MAP kinase phosphorylation; (3) the bactericidal activity of neutrophils is not impaired by hydrogen sulfide. We conclude that HS− promotes the short-term survival of neutrophils potentially accelerating the resolution of inflammatory processes and preventing the occurrence of new ones.

Adenosine receptors in colon carcinoma tissues and colon tumoral cell lines: Focus on the A3 adenosine subtype

Adenosine may affect several pathophysiological processes, including cellular proliferation, through interaction with A1, A2A, A2B, and A3 receptors. In this study we characterized adenosine receptors in human colon cancer tissues and in colon cancer cell lines Caco2, DLD1, HT29. mRNA of all adenosine subtypes was detected in cancer tissues and cell lines. At a protein levels low amount of A1, A2A, and A2B receptors were detected, whilst the A3 was the most abundant subtype in both cancer tissues and cells, with a pharmacological profile typical of the A3 subtype. All the receptors were coupled to stimulation/inhibition of adenylyl‐cyclase in cancer cells, with the exception of A1 subtype. Adenosine increased cell proliferation with an EC50 of 3–12 µM in cancer cells. This effect was not essentially reduced by adenosine receptor antagonists. However dypiridamol, an adenosine transport inhibitor, increased the stimulatory effect induced by adenosine, suggesting an action at the cell surface. Addition of adenosine deaminase makes the A3 agonist 2‐chloro‐N6‐(3‐iodobenzyl)‐N‐methyl‐5′‐carbamoyladenosine (Cl‐IB‐MECA) able to stimulate cell proliferation with an EC50 of 0.5–0.9 nM in cancer cells, suggesting a tonic proliferative effect induced by endogenous adenosine. This effect was antagonized by 5‐N‐(4‐methoxyphenyl‐carbamoyl)amino‐8‐propyl‐2(2furyl)‐pyrazolo‐[4,3e]‐1,2,4‐triazolo [1,5‐c] pyrimidine (MRE 3008F20) 10 nM. Cl‐IB‐MECA‐stimulated cell proliferation involved extracellular‐signal‐regulated‐kinases (ERK1/2) pathway, as demonstrated by reduction of proliferation with 1,4‐diamino‐2,3‐dicyano‐1,4‐bis‐[2‐amino‐phenylthio]‐butadiene (U0126) and by ERK1/2 phosphorylation. In conclusion this study indicates for the first time that in colon cancer cell lines endogenous adenosine, through the interaction with A3 receptors, mediates a tonic proliferative effect. J. Cell. Physiol. 211: 826–836, 2007.

HIV-1 Tat-mediated Inhibition of the Tyrosine Hydroxylase Gene Expression in Dopaminergic Neuronal Cells

Treatment of dopaminergic rat PC12 cells with human immunodeficiency virus, type 1 (HIV-1) Tat protein ortat cDNA inhibited the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme for the dopamine biosynthetic pathway, as well as the production and release of dopamine into the culture medium. Moreover, the Tat addition to PC12 cells up-regulated the expression of the inducible cAMP early repressor (ICER), a specific member of the cAMP-responsive element modulator transcription factor family, in a cAMP-dependent manner. In turn, ICER overexpression abrogated the transcription activity of the TH promoter in PC12 cells, strongly suggesting ICER involvement in Tat-mediated inhibition of TH gene expression. In vivoinjection of synthetic HIV-1 Tat protein into the striatum of healthy rats induced a subclinical Parkinsons-like disease that became manifested only when the animals were treated with amphetamine. As early as one week postinjection, the histochemical examination of the rat substantia nigra showed a reduced staining of neurons expressing TH followed by a loss of TH+ neurons at later time points. As Tat protein can be locally released into the central nervous system by HIV-1-infected microglial cells, our findings may contribute to the explanation of the pathogenesis of the motorial abnormalities often reported in HIV-1 seropositive individuals.

Persistence of Human Herpesvirus 7 in Normal Tissues Detected by Expression of a Structural Antigen

Human herpesvirus 7 (HHV-7) infection in histologically normal human tissues was investigated by immunohistochemical detection of the 85-kDa tegument phosphoprotein (pp85) encoded by the U14 gene. So far, two cell types were recognized as sites of HHV-7 infection in vivo: CD4+ T lymphocytes, believed to be the site of latent infection, and epithelial cells of salivary glands, the site of productive infection and viral shedding. Unexpectedly, cells expressing the HHV-7 structural antigen were detectable in lungs, skin, and mammary glands. Morphologically and phenotypically, they were distinct from lymphocytes. Liver, kidney, and tonsils were positive, although the number of HHV-7-positive cells was low. Large intestine, spleen, and brain were negative. Different from the current notion of the state of HHV-7 in humans, the results show that a variety of tissues harbor cells at a late stage of infection and suggest that HHV-7 causes a persistent rather than a true latent infection.

Hypoxia Inhibits Paclitaxel-Induced Apoptosis through Adenosine-Mediated Phosphorylation of Bad in Glioblastoma Cells

Solid tumors contain hypoxic cells that are resistant to radiotherapy and chemotherapy. The resistance in glioblastoma has been linked to the expression of antiapoptotic Bcl-2 family members. In this study, we found that in human glioblastoma cells hypoxia induces the phosphorylation of the Bcl-2 family protein Bad, thus protecting hypoxic cells from paclitaxel-induced apoptosis. Akt activation is required for the hypoxia-induced protection. In contrast, the extracellular signal-regulated kinase 1/2 activities have only a partial effect, being able to modulate Bad phosphorylation but not paclitaxel-induced apoptosis in hypoxia. We also demonstrated that the degradation of adenosine with adenosine deaminase, the knockdown of A3 adenosine receptor expression by gene silencing, and the blockade of this receptor through A3 receptor antagonists blocked the hypoxia-induced phosphorylation of Bad and the prolonged cell survival after treatment with paclitaxel in hypoxia. Thus, the adenosinergic signaling may be an essential component in the hypoxia survival pathway. These results suggest that hypoxia-induced chemoresistance of human glioblastoma cells may occur in a novel mechanism involving activation of adenosine-A3 receptor-Akt pathway, which mediates Bad inactivation and favors cell survival.

HIV-1 Tat protein down-regulates CREB transcription factor expression in PC12 neuronal cells through a phosphatidylinositol 3-kinase/AKT/cyclic nucleoside phosphodiesterase pathway

The addition of low concentrations (0.1–1 nM) of extracellular HIV‐1 Tat protein to PC12 neuronal cells stimulated a rapid (peak at 5 min) elevation of the cAMP intracellular levels, which in turn induced the phosphorylation of CREB transcription factor (peak at 15 min) on serine‐133 (Ser‐133). On the contrary, at later time points (60–120 min) Tat induced a significant decline of intracellular cAMP with respect to the basal levels observed in control cells treated with bovine serum albumin. In blocking experiments performed with pharmacological inhibitors, Tat decreased the intracellular levels of cAMP and CREB Ser‐133 phosphorylation through a signal transduction pathway involving the sequential activation of phosphatidylinositol 3‐kinase, AKT, and cyclic nucleoside phosphodiesterases. Moreover, in transient transfection experiments, Tat inhibited transcription of CREB promoter in a manner strictly dependent on the presence of the cAMP‐responsive elements (CRE) in the CREB promoter. Consistently, the expression of endogenous CREB protein was significantly reduced in PC12 cells by prolonged (24–48 h) treatment with Tat. This decline in the expression of CREB, which plays an essential role in the survival and function of neuronal cells, anticipated a progressive increase of apoptosis in Tat‐treated cells. Although obtained in a neuronal cell line, our findings might help to explain some aspects of the pathogenesis of HIV‐1‐associated dementia.—Zauli, G., Milani, D., Mirandola, P., Mazzoni, M., Secchiero, P., Miscia, S., Capitani, S. HIV‐1 Tat protein downregulates CREB transcription factor expression in PC12 neuronal cells through a phosphatidylinositol 3‐kinase/AKT/cyclic nucleoside phosphodiesterase pathway. FASEB J. 15, 483‐491 (2001)

Extracellular HIV-1 Tat protein differentially activates the JNK and ERK/MAPK pathways in CD4 T cells.

OBJECTIVE To investigate the intracellular signals elicited by extracellular HIV-1 Tat protein in lymphoid CD4 T cells. METHODS CD4 Jurkat T cells were treated with a series of glutathione S-transferase (GST)-Tat fusion proteins: full-length two-exon GST-Tat (GST-Tat2E); one-exon Tat, in which the second exon of Tat was deleted (GST-Tat1E); two-exon Tat, in which the seven arginine residues have been changed to alanine residues (GST-TatArg(mut)), GST-TatdeltaN, which shows a deletion of the N-terminal 21 amino acids. The cells were either treated with soluble GST-Tat proteins or seeded on plates coated with GST-Tat proteins immobilized on plastic. At various time points, Jurkat cells were lysed and examined for c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) activity. RESULTS Soluble and immobilized GST-Tat2E, but not GST-Tat1E, GST-TatArg(mut) and GST-TatdeltaN, activated JNK in a dose-dependent manner, induced a rapid phosphorylation of c-Jun on Ser63 and promoted the de novo synthesis of c-Jun protein. Moreover, both GST-Tat2E and GST-Tat1E also stimulated ERK/MAPK. However, the activation of JNK was maximal at concentrations of 100 nM of GST-Tat2E and was blocked by the S6-kinase inhibitor rapamycin, whereas the activation of ERK/MAPK was already maximal at 1 nM of GST-Tat2E and was enhanced by rapamycin. CONCLUSIONS Tat-mediated activation of JNK requires the second exon of Tat, which is dispensable for the activation of ERK/MAPK. The ability to stimulate JNK and ERK/MAPK does not require Tat internalization.