Induction of differentiation and apoptosis by interferon-γ in human neuroblastoma cells in vitro as a dual and alternative early biological response

Montaldo, Paolo G.; Chiesa, Valeria; Bado, M.; Raffaghello, Lizzia; Rozzo, Carla; Ponzoni, Mirco

doi:10.1038/sj.cdd.4400215

Cited by 15 publications

(26 citation statements)

References 31 publications

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“…GILIN and LAN-5 cells, 2 NB cell lines that differ in their in vitro response to chemotherapeutic drugs and cytokines, 14 -16 were cultivated in the presence of cathepsin inhibitors under conditions (concentration and time of incubation) that effectively impair the proteolytic activity of CB and CD. 18,20 Two main conclusions can be drawn from our study: (i) the proteolytic function of endosomal-lysosomal organelles is essen- 15,16 were highly susceptible to lysosomal cathepsin inhibitors. It should be noted that in these cells CA074Me could induce cell death by necrosis or by apoptosis, depending on concentration used and time of incubation.…”

Section: Discussionmentioning

confidence: 68%

Lysosomal proteases as potential targets for the induction of apoptotic cell death in human neuroblastomas

Castino¹,

Pace²,

Démoz³

et al. 2001

Intl Journal of Cancer

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Neuroblastoma is the most common type of cancer in infants. In children this tumor is particularly aggressive; despite various new therapeutic approaches, it is associated with poor prognosis. Given the importance of endosomallysosomal proteolysis in cellular metabolism, we hypothesized that inhibition of lysosomal protease would impact negatively on neuroblastoma cell survival. Treatment with E-64 or CA074Me (2 specific inhibitors of cathepsin B) or with pepstatin A (a specific inhibitor of cathepsin D) was cytotoxic for 2 neuroblastoma cell lines having different degrees of malignancy. Cell death was associated with condensation and fragmentation of chromatin and externalization of plasma membrane phosphatidylserine, 2 hallmarks of apoptosis. Concomitant inhibition of the caspase cascade protected neuroblastoma cells from cathepsin inhibitor-induced cytotoxicity. These data indicate that prolonged inhibition of the lysosomal proteolytic pathway is incompatible with cell survival, leading to apoptosis of neuroblastoma cells, and that the cathepsin-mediated and caspase-mediated proteolytic systems are connected and cooperate in the regulation of such an event. Since modern antitumor chemotherapy is aimed at restoring the normal rate of apoptosis in neoplastic tissues, the demonstration that endosomal-lysosomal cathepsins are involved in this process may constitute a basis for novel strategies that include cathepsin inhibitors in the therapeutic regimen. © 2002 Wiley-Liss, Inc. Key words: apoptosis; neuroblastoma; caspases; cathepsins; protease inhibitorsAltered regulation of cell survival and death, including apoptosis, is considered an important factor in tumor development and progression, as well as in the response to antineoplastic therapy. 1,2 The molecular pathways controlling apoptosis include a complex network of intracellular proteases that act in an orderly sequence on cellular substrates and lead to characteristic modifications of cell morphology with eventual DNA cleavage and apoptotic body formation. Several proteolytic systems have been shown to participate in the apoptotic process, depending on the cell type and stimuli adopted. With few exceptions, the caspase system seems to be the most universally involved one. 3,4 Recently, proteases resident within the endosomal-lysosomal compartment (cathepsins) have also been associated with apoptosis. 5-9 These studies demonstrated the need for a cathepsin-mediated proteolytic event in the apoptotic pathway triggered by cytokines or antiblastic drugs. In addition, the active participation of the autophagic proteolytic pathway, particularly of lysosomal cathepsins B and D (CB, CD), has been envisaged in rat pheochromocytoma PC12 cell death after nutrient and serum factor deprivation. In this model of caspasedependent apoptosis, CD acted as a death factor, whereas CB acted as a pro-survival factor. 10 We followed an opposite approach, assuming that the endosomal-lysosomal proteolytic pathway serves crucial functions for cell viability. Indeed, experiments usi...

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Section: Discussionmentioning

confidence: 68%

Lysosomal proteases as potential targets for the induction of apoptotic cell death in human neuroblastomas

Castino¹,

Pace²,

Démoz³

et al. 2001

Intl Journal of Cancer

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“…27 In the present study, we showed that all neuroblastoma cell lines tested were partially or completely negative for CD95 and CD95L expression and that both mRNA and protein levels of CD95 and CD95L were enhanced by IFNg. Although IFNg has been reported to modulate apoptosis in a variety of cell types including neuroblastoma cell lines, 25,26 the molecular mechanism whereby the cytokine induces cell death is still unclear. Our results show that IFNginduced cell death in neuroblastoma cells is regulated by CD95 and CD95L induction: (i) apoptosis induced by IFNg strongly correlates with cell surface expression of CD95 and with CD95L levels; (ii) a clone in which CD95L is not induced did not undergo apoptosis; (iii) blocking reagents inhibiting the interaction between CD95 and CD95L were able to at least partially reduce cell death.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, IFNg may act by exerting a powerful differentiative effect on several neuroblastoma cell lines. 24,26 Further studies are required to assess CD95/ CD95L expression in neuroblastoma tumour specimens and to evaluate the susceptibility to IFNg-mediated cytotoxicity of neuroblastoma in vivo; indeed, several questions still remain to be explored, such as the signalling pathway which balances IFNg-induced apoptosis and differentiation.…”

Section: Complexity Of the Apoptotic Signalling Of Ifngmentioning

confidence: 99%

“…24 It has also been shown to induce apoptosis in a variety of cell types including neuroblastoma cell lines. 25,26 Although the molecular mechanism whereby the cytokine induces cell death is still largely unclear, IFNg and TNFa have been shown to up-regulate CD95 expression thus increasing tumour sensitivity to anti-CD95 antibodymediated apoptosis. 16,25,27 Recently, IFNg has been found to positively modulate CD95L expression in human embryonal carcinoma cells.…”

Section: Introductionmentioning

confidence: 99%

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Induction of apoptosis by IFNγ in human neuroblastoma cell lines through the CD95/CD95L autocrine circuit

et al. 1999

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The CD95 (APO-1/Fas) system can mediate apoptosis in immune cells as well as in tumour cells, where it may contribute to tumour immune-escape. On the other hand, its induction by anticancer drugs may lead to tumour reduction. Interferong (IFNg) increases the sensitivity of tumour cell lines to anti-CD95 antibody-mediated apoptosis. We describe induction of apoptosis by IFNg through the expression of CD95 and its ligand (CD95L) in human neuroblastoma cell lines. Neuroblastoma cells showed low constitutive expression of CD95 and CD95L. Subsequent to IFNg-modulated increase in CD95 and CD95L mRNA as well as protein levels, apoptosis was observed. Our results demonstrated that cytokine-mediated apoptosis was mediated through the activation of the CD95/CD95L autocrine circuit since: (i) cell death occurred following CD95/CD95L expression and correlated with CD95 and CD95L expression levels, (ii) failed to occur in a clone which weakly upregulated CD95 and lacked CD95L induction after IFNg stimulation, (iii) was at least partially inhibited by using blocking F(ab') 2 anti-CD95 antibody fragments and the recombinant Fas-Fc protein, that prevented the interaction between CD95 and CD95L. The intracellular molecular mechanisms elicited by IFNg are clearly highly complex, with several signalling pathways being activated, including the CD95 system. These findings suggest that IFNg may have a significant potential in the therapy of neuroblastoma in vivo.

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“…In some experiments, NB cell lines were incubated with or without rIFN-g (1000 IU ml À1 ) (Imuchin s , Boehringher Ingelheim Italia, Florence, Italy) for 72 h before undergoing molecular or immunophenotypic studies. The rIFN-g concentration and the culture time were selected on the ground of previous studies (Montaldo et al, 1997).…”

Section: Cell Separation and Culturementioning

confidence: 99%

Expression of costimulatory molecules in human neuroblastoma. Evidence that CD40+ neuroblastoma cells undergo apoptosis following interaction with CD40L

et al. 2003

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Tumour cells display low to absent expression of costimulatory molecules. Here, we have investigated the expression of costimulatory molecules (CD40, CD80, CD86, PD-1L, B7H2, OX40L and 4-1BBL) in human neuroblastoma (NB) cells, since virtually no information is available on this issue. Both established NB cell lines and primary tumours were tested by RT -PCR and flow cytometry. Neuroblastoma cell lines expressed the transcripts of all costimulatory molecule genes, but not the corresponding proteins. Culture of NB cell lines with human recombinant (r)IFN-g induced surface expression of CD40 in half of them. Primary NB cells showed CD40, CD80, CD86, OX40L, 4-1BBL, but not PD-1L and B7H2, mRNA expression. Surface CD40 was consistently detected on primary NB cells by flow cytometry. Interferon-g gene-transfected NB cells expressed constitutively surface CD40 and were induced into apoptosis by incubation with rCD40L through a caspase-8-dependent mechanism. CD40 may represent a novel therapeutic target in NB.

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Induction of differentiation and apoptosis by interferon-γ in human neuroblastoma cells in vitro as a dual and alternative early biological response

Cited by 15 publications

References 31 publications

Lysosomal proteases as potential targets for the induction of apoptotic cell death in human neuroblastomas

Lysosomal proteases as potential targets for the induction of apoptotic cell death in human neuroblastomas

Induction of apoptosis by IFNγ in human neuroblastoma cell lines through the CD95/CD95L autocrine circuit

Expression of costimulatory molecules in human neuroblastoma. Evidence that CD40+ neuroblastoma cells undergo apoptosis following interaction with CD40L

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