Fabrizio Condorelli

BCR-ABL suppresses C/EBPα expression through inhibitory action of hnRNP E2

The arrest of differentiation is a feature of both chronic myelogenous leukemia cells in myeloid blast crisis and myeloid precursors that ectopically express the p210BCR-ABL oncoprotein; however, its underlying mechanisms remain poorly understood. Here we show that expression of BCR-ABL in myeloid precursor cells leads to transcriptional suppression of the granulocyte colony–stimulating factor receptor G-CSF-R (encoded by CSF3R), possibly through down-modulation of C/EBPα—the principal regulator of granulocytic differentiation. Expression of C/EBPα protein is barely detectable in primary marrow cells taken from individuals affected with chronic myeloid leukemia in blast crisis. In contrast, CEBPA RNA is clearly present. Ectopic expression of C/EBPα induces granulocytic differentiation of myeloid precursor cells expressing BCR-ABL. Expression of C/EBPα is suppressed at the translational level by interaction of the poly(rC)-binding protein hnRNP E2 with CEBPA mRNA, and ectopic expression of hnRNP E2 in myeloid precursor cells down-regulates both C/EBPα and G-CSF-R and leads to rapid cell death on treatment with G-CSF (encoded by CSF3). Our results indicate that BCR-ABL regulates the expression of C/EBPα by inducing hnRNP E2—which inhibits the translation of CEBPA mRNA.

Mitotic signaling by β-amyloid causes neuronal death

Aggregates of β‐amyloid peptide (βAP), the main constituent of amyloid plaques in Alzheimers brain, kill neurons by a not yet defined mechanism, leading to apoptotic death. Here, we report that both full‐length βAP(j_40) or (1–42) and its active fragment βAP(25_35) act as proliferative signals for differentiated cortical neurons, driving them into the cell cycle. The cycle followed some of the steps observed in proliferating cells, including induction of cyclin Di, phosphorylation of retinoblastoma, and induction of cyclin E and A, but did not progress beyond S phase. Inactivation of cyclin‐dependent protein kinase‐4 or ‐2 prevented both the entry into S phase and the development of apoptosis in βAP(25_35)‐treated neurons. We conclude that neurons must cross the G1/S transition before succumbing to βAP signaling, and therefore multiple steps within this pathway may be targets for neuroprotective agents.—Copani, A., Condorelli, F., Caruso, A., Vancheri, C., Sala, A., Giuffrida Stella, A. M., Canonico, P. L., Nicoletti, F., Sortino, M. A. Mitotic signaling by β ‐amyloid causes neuronal death. FASEB J. 13, 2225–2234 (1999)

The autophagy-associated factors DRAM1 and p62 regulate cell migration and invasion in glioblastoma stem cells

The aggressiveness of glioblastoma multiforme (GBM) is defined by local invasion and resistance to therapy. Within established GBM, a subpopulation of tumor-initiating cells with stem-like properties (GBM stem cells, GSCs) is believed to underlie resistance to therapy. The metabolic pathway autophagy has been implicated in the regulation of survival in GBM. However, the status of autophagy in GBM and its role in the cancer stem cell fraction is currently unclear. We found that a number of autophagy regulators are highly expressed in GBM tumors carrying a mesenchymal signature, which defines aggressiveness and invasion, and are associated with components of the MAPK pathway. This autophagy signature included the autophagy-associated genes DRAM1 and SQSTM1, which encode a key regulator of selective autophagy, p62. High levels of DRAM1 were associated with shorter overall survival in GBM patients. In GSCs, DRAM1 and SQSTM1 expression correlated with activation of MAPK and expression of the mesenchymal marker c-MET. DRAM1 knockdown decreased p62 localization to autophagosomes and its autophagy-mediated degradation, thus suggesting a role for DRAM1 in p62-mediated autophagy. In contrast, autophagy induced by starvation or inhibition of mTOR/PI-3K was not affected by either DRAM1 or p62 downregulation. Functionally, DRAM1 and p62 regulate cell motility and invasion in GSCs. This was associated with alterations of energy metabolism, in particular reduced ATP and lactate levels. Taken together, these findings shed new light on the role of autophagy in GBM and reveal a novel function of the autophagy regulators DRAM1 and p62 in control of migration/invasion in cancer stem cells.

Caspase cleavage enhances the apoptosis-inducing effects of BAD.

ABSTRACT The function of BAD, a proapoptotic member of the Bcl-2 family, is regulated primarily by rapid changes in phosphorylation that modulate its protein-protein interactions and subcellular localization. We show here that, during interleukin-3 (IL-3) deprivation-induced apoptosis of 32Dcl3 murine myeloid precursor cells, BAD is cleaved by a caspase(s) at its N terminus to generate a 15-kDa truncated protein. The 15-kDa truncated BAD is a more potent inducer of apoptosis than the wild-type protein, whereas a mutant BAD resistant to caspase 3 cleavage is a weak apoptosis inducer. Truncated BAD is detectable only in the mitochondrial fraction, interacts with BCL-XL at least as effectively as the wild-type protein, and is more potent than wild-type BAD in inducing cytochrome c release. Human BAD, which is 43 amino acids shorter than its mouse counterpart, is also cleaved by a caspase(s) upon exposure of Jurkat T cells to anti-FAS antibody, tumor necrosis factor alpha (TNF-α), or TRAIL. Moreover, a truncated form of human BAD lacking the N-terminal 28 amino acids is more potent than wild-type BAD in inducing apoptosis. The generation of truncated BAD was blocked by Bcl-2 in IL-3-deprived 32Dcl3 cells but not in Jurkat T cells exposed to anti-FAS antibody, TNF-α, or TRAIL. Together, these findings point to a novel and important role for BAD in maintaining the apoptotic phenotype in response to various apoptosis inducers.

Inhibitors of histone deacetylase (HDAC) restore the p53 pathway in neuroblastoma cells

Inhibitors of histone deacetylase (HDAC) are emerging as a promising class of anti‐cancer drugs, but a generic deregulation of transcription in neoplastic cells cannot fully explain their therapeutic effects. In this study we evaluated alternative molecular mechanisms by which HDAC inhibitors could affect neuroblastoma viability.

NAD depletion by FK866 induces autophagy.

NAD is a multifunctional molecule involved in both metabolic processes and signalling pathways. Such signalling pathways consume NAD which is replenished via one of several biosynthesis pathways. We show that influx of NAD across the plasma membrane may be able to contribute to the homeostasis of intracellular NAD levels. Indeed, extracellular application of NAD was able to replete NAD levels that had been lowered pharmacologically using the novel drug FK866 and was also able to rescue cells from FK866-induced cell death. A marked lag between the drop in NAD levels and cell death prompted us to investigate the mechanism of cell death. We were unable to find evidence of apoptosis as assessed by immunoblotting for the Caspase 3 activation fragment and immunostaining for cytochrome C and AIF translocation. We, therefore, investigated whether autophagy was initiated by FK866. Indeed, we were able to observe the formation of LC3-positive vesicles that had fused with lysosomes in FK866-treated but not control cells. Furthermore, this autophagic phenotype could be reverted by the addition of NAD to the extracellular medium. Addendum to: Billington RA, Travelli C, Ercolano E, Galli U, Blasi Roman C, Grolla AA, Canonico PL, Condorelli F, Genazzani AA. Characterization of NAD uptake in mammalian cells. J Biol Chem 2008; In Press.

Tumor Necrosis Factor-α Induces Apoptosis in Immortalized Hypothalamic Neurons: Involvement of Ceramide-Generating Pathways

To investigate possible effects that may contribute, together with a direct action on neurohormone secretion, to the impairment of gonadal axis function during inflammation, we evaluated the effect of TNF alpha on the growth and viability of GT1-7 hypothalamic neurons and the intracellular transduction pathways involved in these effects. TNF alpha caused a reduction of cell number and an induction of apoptotic death. These effects were mimicked by cell-permeable analogs of ceramide and by neutral or acidic sphingomyelinase. Exposure to acidic sphingomyelinase induced a persistent (up to 48 h) reduction of cell growth and apoptosis, whereas the effect of neutral sphingomyelinase was time limited. The involvement of acidic sphingomyelinase in TNF alpha action was demonstrated by the partial prevention of ceramide generation, apoptosis, and reduced cell growth by the inhibitor of the acidic sphingomyelinase-generating pathway, D609, whereas the involvement of ceramide was proved by complete prevention of TNF alpha-induced effects by treatment with okadaic acid at concentrations inhibiting ceramide-dependent protein phosphatase. The present data indicate that TNF alpha, through activation of ceramide-generating pathways, is able to affect GT1-7 cell viability, suggesting an additional effect that may contribute to the global action of this cytokine on neuroendocrine activities.

Solution-Phase Parallel Synthesis and Biological Evaluation of Combretatriazoles

Combretastatin A-4 is an antitumoral and antitubulin agent that is active only in its cis configuration. In the present manuscript, we have synthesized cis-locked combretastatins containing a triazole ring (combretatriazoles). To achieve this, we have developed a column chromatography-free parallel solution-phase synthesis that exploits the reaction between azides and alpha-keto phosphorus ylids, which is known to regioselectively generate the 1,5-disubstituted triazoles. The prepared compounds were screened as antitubulinic agents, allowing us to identify three new compounds with high potency, two of which show a new mechanism of action that induces cells to appear multinucleated and display a high number of mitotic spindles.

Relative Contribution of Different Receptor Subtypes in the Response of Neuroblastoma Cells to Tumor Necrosis Factor-α

Abstract: The effect of tumor necrosis factor‐α (TNF‐α) on neuronal viability has been investigated in the SK‐N‐BE neuroblastoma cell line. These cells undergo differentiation upon chronic treatment with retinoic acid. Exposure of SK‐N‐BE cells to TNF‐α produced a proliferative response in undifferentiated cells, whereas a reduced cell number was observed in retinoic acid (RA)‐differentiated cultures. This biphasic response may be related to the different expression of TNF‐α receptors (TNFRs); a significant increase in the density of TNFR1 was in fact observed following RA‐induced differentiation. Under these conditions, a pronounced increase in the formation of ceramide‐1‐phosphate (which was prevented by the selective inhibitor of phosphatidylcholine‐specific phospholipase C, D609) and an activation of caspase‐3 upon TNF‐α challenge were evident. Selective blockade of each TNFR subtype allowed a more detailed analysis of the effect observed. Preincubation with an anti‐TNFR1 antibody prevented the cytotoxic effect of TNF‐α in RA‐differentiated SK‐N‐BE cells, whereas the anti‐TNFR2 antibody blocked the proliferative activity of the cytokine in undifferentiated cultures.

Distinct effects of ceramide-generating pathways in prostate adenocarcinoma cells.

Ceramide, generated by the hydrolysis of sphingomyelin, mediates the actions of several cytokines such as tumour necrosis factor‐α (TNF‐α) interferon‐γ and interleukin‐1β (IL‐1β), including their inhibitory effect on tumour proliferation. We have evaluated the role of ceramide in the proliferation of prostate cancer by using the human prostate adenocarcinoma LNCaP cell line. Treatment of LNCaP cells with neutral or acidic sphingomyelinase or addition of C8‐ or C2‐ceramide, two cell permeable analogues of endogenous ceramide, induced a profound inhibition of cell proliferation. This effect appeared after 24 h, was still present after 72 h of exposure to the drugs and exhibited concentration‐dependency (10–200 and 5–200 mU ml−1 for neutral and acidic sphingomyelinase, respectively, and 1–25 μM for C8‐ceramide). The inhibitory effect on cell growth caused by neutral sphingomyelinase and ceramides was rapidly reversible as LNCaP cells rapidly regained their previous proliferation rate following withdrawal of the treatment. IL‐1β produced profound inhibition of LNCaP cell proliferation and caused enhanced ceramide formation. No clear features of apoptotic cell death were detectable by either oligonucleosome formation, cytofluorimetric analysis or nuclear staining following exposure of LNCaP cells to neutral sphingomyelinase, ceramide or IL‐1β. However, clear changes in LNCaP cell cycle distribution were detectable following these treatments. In contrast, treatment with acidic sphingomyelinase or TNF‐α induced apoptotic death detectable by flow cytometric analysis and bisbenzimide staining. In conclusion, our data demonstrate that preferential activation of distinct enzymatic pathways by cytokines may lead to different outcomes in the viability of LNCaP cells.