Arianna Gonelli

miR-221&222 regulate TRAIL resistance and enhance tumorigenicity through PTEN and TIMP3 downregulation.

Lung and liver cancers are among the most deadly types of cancer. Despite improvements in treatment over the past few decades, patient survival remains poor, underlining the need for development of targeted therapies. MicroRNAs represent a class of small RNAs frequently deregulated in human malignancies. We now report that miR-221&222 are overexpressed in aggressive non-small cell lung cancer and hepatocarcinoma cells, as compared with less invasive and/or normal lung and liver cells. We show that miR-221&222, by targeting PTEN and TIMP3 tumor suppressors, induce TRAIL resistance and enhance cellular migration through the activation of the AKT pathway and metallopeptidases. Finally, we demonstrate that the MET oncogene is involved in miR-221&222 activation through the c-Jun transcription factor.

Antiangiogenic Activity of the MDM2 Antagonist Nutlin-3

Nutlin-3, a nongenotoxic activator of the p53 pathway, dose-dependently (range 0.1 to 10 &mgr;mol/L) inhibited the formation of capillaries in an in vivo matrigel assay, as well as the formation of capillary-like structures in an in vitro coculture system composed of endothelial cells surrounded by fibroblasts. In contrast to the chemotherapeutic agent doxorubicin, nutlin-3 showed no induction of apoptosis in vitro either in the cocultures or in isolated vascular endothelial cells, even when used at the highest concentration (10 &mgr;mol/L). However, treatment with pharmacological inhibitors of the nuclear factor &kgr;B and phosphatidylinositol 3-kinase/Akt pathways sensitized endothelial cells to nutlin-3–induced apoptosis. Although nutlin-3 and doxorubicin induced a comparable p53 accumulation in endothelial cells, nutlin-3 was significantly more efficient than doxorubicin in upregulating the p53 target genes CDKN1A/p21, MDM2, and GDF-15, as well as in inhibiting cell cycle progression. However, the predominant in vitro effect of nutlin-3 was its strong antimigratory activity observed at concentrations significantly lower (0.1 &mgr;mol/L) than those required to inhibit endothelial cell cycle progression. Taken together, our data suggest that the antiangiogenic activity of nutlin-3 observed in vivo was mainly attributable to inhibition of endothelial cell migration, to some extent attributable to cell cycle arrest, and to a lesser extent attributable to induction of apoptosis.

Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand (TRAIL) Sequentially Upregulates Nitric Oxide and Prostanoid Production in Primary Human Endothelial Cells

&NA; —Endothelial cells express tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) receptors, but the function of TRAIL in endothelial cells is not completely understood. We explored the role of TRAIL in regulation of key intracellular signal pathways in endothelial cells. The addition of TRAIL to primary human endothelial cells increased phosphorylation of endothelial nitric oxide synthase (eNOS), NOS activity, and NO synthesis. Moreover, TRAIL induced cell migration and cytoskeleton reorganization in an NO‐dependent manner. TRAIL did not activate the NF‐&kgr;B or COX‐2 pathways in endothelial cells. Instead, TRAIL increased prostanoid production (PGE2=PGI2>TXA2), which was preferentially inhibited by the COX‐1 inhibitor SC‐560. Because NO and prostanoids play a crucial role in the state of blood vessel vasodilatation and angiogenesis, our data suggest that TRAIL might play an important role in endothelial cell function. (Circ Res. 2003;92:732–740.)

The MDM2 Inhibitor Nutlins as an Innovative Therapeutic Tool for the Treatment of Haematological Malignancies

At variance to solid tumors, which show percentage of p53 deletions and/or mutations close to 50%, more than 80% of haematological malignancies express wild-type p53 at diagnosis. Therefore, activation of the p53 pathway by antagonizing its negative regulator murine double minute 2 (MDM2) might offer a new therapeutic strategy for the great majority of haematological malignancies. Recently, potent and selective small-molecule MDM2 inhibitors, the Nutlins, have been identified. Studies with these compounds have strengthened the concept that selective, non-genotoxic p53 activation might represent an alternative to the current cytotoxic chemotherapy. Interestingly, Nutlins not only are able to induce apoptotic cell death when added to primary leukemic cell cultures, but also show a synergistic effect when used in combination with the chemotherapeutic drugs commonly used for the treatment of haematological malignancies. Of interest, Nutlins also display non-cell autonomous biological activities, such as inhibition of vascular endothelial growth factor, stromal derived factor-1/CXCL12 and osteprotegerin expression and/or release by primary fibroblasts and endothelial cells. Moreover, Nutlins have a direct anti-angiogenic and anti-osteoclastic activity. Thus, Nutlins might have therapeutic effects by two distinct mechanisms: a direct cytotoxic effect on leukemic cells and an indirect non-cell autonomous effect on tumor stromal and vascular cells, and this latter effect might be therapeutically relevant also for treatment of haematological malignancies carrying p53 mutations.

Stromal derived factor-1α (SDF-1α) induces CD4+ T cell apoptosis via the functional up-regulation of the Fas (CD95)/Fas ligand (CD95L) pathway

Stromal‐derived factor‐1α (SDF‐1α), the high‐affinity ligand of CXC‐chemokine receptor 4 (CXCR4), induced a progressive increase of apoptosis when added to the Jurkat CD4+/CXCR4+ T cell line. The SDF‐1α‐mediated Jurkat cell apoptosis was observed in serum‐free or serum‐containing cultures, peaked at SDF‐1α concentrations of 10–100 ng/ml, required 3 days to take place, and was completely blocked by the z‐VAD‐fmk tripeptide caspase inhibitor. Although SDF‐1α did not modify the expression of TNF‐α or that of TNF‐RI and TNF‐RII, it increased the expression of surface Fas/APO‐1 (CD95) and intracellular Fas ligand (CD95L) significantly. Moreover, the ability of SDF‐1α to induce apoptosis was inhibited by an anti‐CD95 Fab′ neutralizing antibody. These findings suggest a role for SDF‐1α in the homeostatic control of CD4+ T‐cell survival/apoptosis mediated by the CD95‐CD95L pathway.

TNF-Related Apoptosis-Inducing Ligand (TRAIL): A Potential Candidate for Combined Treatment of Hematological Malignancies

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF gene superfamily, which induces apoptosis through engagement of death receptors. TRAIL is unusual as compared to the other cytokines of this family, as it interacts with a complex system of receptors consisting of two pro-apoptotic death receptors (TRAIL-R1 and TRAIL-R2) and three decoy receptors (TRAIL-R3, TRAIL-R4 and osteoprotegerin). Moreover, with respect to other members of the TNF superfamily, such as CD95L and TNF-alpha, TRAIL has generated great interest as a potential tumor-specific cancer therapeutic because as a stable soluble trimer it selectively induces apoptosis in many transformed cells but not in normal cells. Of note, TRAIL cytotoxicity is at least partially independent of the major systems involved in resistance to chemotherapy, such as p53 wild-type function and multidrug resistance (MDR) genes. Since one fundamental problem of most cancers is the development of multiple mechanisms of resistance, which progressively reduce or suppress the therapeutic efficacy of conventional chemotherapy, new therapeutic approaches that either restore the pro-apoptotic activity of chemotherapeutic drugs or by-pass the mechanisms of resistance are highly desirable. This review will focus on the potential of TRAIL for its application in the therapy of hematological malignancies, used either alone or in combination with chemotherapy. The scenario emerging from the literature is that the treatment and management of hematological malignancies will require the rational combination of TRAIL plus conventional or new drugs in a regimen that would optimize the anti-neoplastic activity in malignant cells resistant to chemotherapy through restoration of the pro-apoptotic activity of TRAIL.

Potential Pathogenetic Implications of Cyclooxygenase-2 Overexpression in B Chronic Lymphoid Leukemia Cells

Evidence suggests that cyclooxygenase-2 (COX-2) increases tumorigenic potential by promoting resistance to apoptosis. Because B chronic lymphoid leukemia (B-CLL) cells exhibit a defective apoptotic response, we analyzed CD19(+) B lymphocytes purified from the peripheral blood of B-CLL patients. Microarray analysis showed a variable (up to 38-fold) increase in the steady-state mRNA levels of COX-2 in B-CLL lymphocytes compared with normal CD19(+) B lymphocytes. The up-regulation of COX-2 in B-CLL cells was confirmed by reverse transcriptase-polymerase chain reaction and Western blot analyses. Moreover, immunohistochemical analysis of B-CLL bone marrow infiltrates confirmed clear expression of COX-2 in leukemic cells. Ex vivo treatment with the COX-2 inhibitor NS-398 significantly decreased the survival of leukemic cells by increasing the rate of spontaneous apoptosis in 13 of 16 B-CLL samples examined, but it did not affect the survival of normal lymphocytes. Pretreatment with NS-398 significantly potentiated the cytotoxicity induced by chlorambucil in 8 of 16 B-CLL samples examined. Moreover, although recombinant tumor necrosis factor-related apoptosis inducing ligand (TRAIL)/Apo2L showed little cytotoxic effect in most B-CLL samples examined, pretreatment with NS-398 sensitized 8 of 16 B-CLL samples to TRAIL-induced apoptosis. Taken together, our data indicate that COX-2 overexpression likely represents an additional mechanism of resistance to apoptosis in B-CLL and that pharmacological suppression of COX-2 might enhance chemotherapy-mediated apoptosis.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNF-α promote the NF-κB-dependent maturation of normal and leukemic myeloid cells

Tumor necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL) and TNF‐α induced monocytic maturation of primary normal CD34‐derived myeloid precursors and of the M2/M3‐type acute myeloid leukemia HL‐60 cell line, associated to increased nuclear factor (NF)‐κB activity and nuclear translocation of p75, p65, and p50 NF‐κB family members. Consistently, both cytokines also induced the degradation of the NF‐κB inhibitors, IκBα and IκBɛ, and up‐regulated the surface expression of TRAIL‐R3, a known NF‐κB target. However, NF‐κB activation and IκB degradation occurred with different time‐courses, since TNF‐α was more potent, rapid, and transient than TRAIL. Of the two TRAIL receptors constitutively expressed by HL‐60 (TRAIL‐R1 and TRAIL‐R2), only the former was involved in IκB degradation, as demonstrated by using agonistic anti‐TRAIL receptor antibodies. Moreover, NF‐κB nuclear translocation induced by TRAIL but not by TNF‐α was abrogated by z‐IETD‐fmk, a caspase‐8‐specific inhibitor. The key role of NF‐κB in mediating the biological effects of TNF‐α and TRAIL was demonstrated by the ability of unrelated pharmacological inhibitors of the NF‐κB pathway (parthenolide and MG‐132) to abrogate TNF‐α‐ and TRAIL‐induced monocytic maturation. These findings demonstrate that NF‐κB is essential for monocytic maturation and is activated via distinct pathways, involving or not involving caspases, by the related cytokines TRAIL and TNF‐α.

Stroma-derived factor 1α induces a selective inhibition of human erythroid development via the functional upregulation of Fas/CD95 ligand

CXC chemokine receptor 4 (CXCR4), the high‐affinity receptor for stroma‐derived factor 1α (SDF‐1α), shows distinct patterns of expression in human CD34+ haematopoietic progenitor cells induced to differentiate in vitro along the granulocytic and erythroid lineages. In serum‐free liquid cultures supplemented with stem cell factor (SCF), interleukin 3 (IL‐3) and granulocyte colony‐stimulating factor, the expression of surface CXCR4 progressively increased in cells differentiating along the granulocytic lineage. The addition in culture of 200 ng/ml of SDF‐1α, a concentration which maximally activated intracellular Ca2+ flux, only modestly affected the expression levels of CD15 and CD11b granulocytic antigens, as well as the total number of viable cells. On the other hand, in liquid cultures supplemented with SCF, IL‐3 and erythropoietin, SDF‐1α induced the downregulation of glycophorin A erythroid antigen, accompanied by a progressive decline in the number of viable erythroblasts. Moreover, in semisolid assays, SDF‐1α significantly reduced the number of plurifocal erythroid colonies (erythroid blast‐forming units; BFU‐E), whereas it did not affect granulocyte–macrophage colony‐forming units (CFU‐GM). We also demonstrated that the inhibitory effect of SDF‐1α on glycophorin A+ erythroid cell development was mediated by the functional upregulation of CD95L in erythroid cultures. These data indicate that SDF‐1α plays a role as a negative regulator of erythropoiesis.

TEMPORAL MAPPING OF TRANSCRIPTS IN HUMAN HERPESVIRUS-7

Transcription of human herpesvirus-7 (HHV-7) in cultures of productively infected T-cells was studied. Transcription of HHV-7 was regulated by the typical herpesvirus cascade in which alpha, beta and gamma genes are sequentially transcribed. Transcripts of U10, U14, U18, U31, U39, U41, U42, U53, U73 and U89/90 were detected 3 h after infection and were not inhibited by the absence of protein synthesis and therefore were alpha functions. U19 and U18/20 were beta genes; their transcription was inhibited by cycloheximide but not by phosphonoacetate, an inhibitor of DNA synthesis. U60/66 and U98/100 were gamma genes since their spliced transcripts were not detected in cells treated with phosphonoacetate. HHV-7 transcription was regulated by complex mechanisms, which involve the temporal coordinated activation of specific viral promoters and post-transcriptional processing. Splice mechanisms were also temporally regulated. Transcription of U89/90 pre-mRNA and splice took place simultaneously in the immediate-early phase. On the other hand, U16/17 pre-mRNA was synthesized with typical alpha kinetics, but the spliced product was regulated as a beta function. Likewise, the primary transcripts of U60/66 and U98/100 were alpha and beta, respectively, but both spliced products were synthesized in the late phase of virus replication. Finally, HHV-7 supported a bona fide latent infection in the adult population, since viral transcripts were not detected in peripheral blood mononuclear cells of healthy donors infected with HHV-7.