Article

Adenovirus-mediated TA-p73?? gene transfer increases chemosensitivity of human malignant melanomas

Department of Vectorology and Experimental Gene Therapy, University of Rostock, Schillingallee 70, Rostock, 18055, Germany.
APOPTOSIS (Impact Factor: 3.69). 03/2006; 11(2):235-43. DOI: 10.1007/s10495-006-3407-0
Source: PubMed

ABSTRACT

Malignant melanoma is the most aggressive form of skin cancer and has proven to be highly resistant to conventional chemotherapy. Intriguingly, the p53 tumor suppressor, a main mediator of chemoresistance in other tumor types, is rarely mutated in melanoma. However, we have previously shown that anti-apoptotic isoforms of p73 (deltaTA-p73), another member of the p53 family, are overexpressed in metastatic melanomas. DeltaTA-p73 can oppose the pro-apoptotic functions of p53 and full length p73, and thus it could contribute to melanoma chemoresistance. In this study, we use an efficient adenoviral-based gene transfer approach to introduce a transcriptionally active form of p73 (TA-p73beta) in melanoma cells, with the objective of overcoming drug resistance. Interestingly, TA-p73beta significantly sensitized 5 out of 7 aggressive melanoma cell lines to the standard therapeutic agents adriamycin and cisplatin. More importantly, TA-p73beta displayed a synergistic effect in vivo allowing adriamycin or cisplatin to block melanoma cell growth in mouse xenograft models (p < 0.05). In summary, our data show that Ad-mediated TA-p73beta gene expression can markedly sensitize a subset of melanoma cell lines to adriamycin and cisplatin in vitro and in vivo, suggesting a new chemosensitization strategy for malignant melanomas.

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    • "These cytotoxic effects may depend on the p53 status of the cell lines, as cytotoxicity of H-1PV has been shown to be more pronounced in p53-negative than in p53-positive cells [11]. SK29-Mel cells express the wildtype p53 gene [41] and a greater effect may be observed in p53-negative cell lines. On the other hand, Angelova et al showed that gemcitabine resistance could efficiently be overcome by H-1PV [38]. "
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    ABSTRACT: Parvovirus H-1 (H-1PV) infects and lyses human tumor cells including melanoma, hepatoma, gastric, colorectal, cervix and pancreatic cancers. We assessed whether the beneficial effects of chemotherapeutic agents or targeted agents could be combined with the oncolytic and immunostimmulatory properties of H-1PV. Using human ex vivo models we evaluated the biological and immunological effects of H-1PV-induced tumor cell lysis alone or in combination with chemotherapeutic or targeted agents in human melanoma cells +/- characterized human cytotoxic T-cells (CTL) and HLA-A2-restricted dendritic cells (DC). H-1PV-infected MZ7-Mel cells showed a clear reduction in cell viability of >50%, which appeared to occur primarily through apoptosis. This correlated with viral NS1 expression levels and was enhanced by combination with chemotherapeutic agents or sunitinib. Tumor cell preparations were phagocytosed by DC whose maturation was measured according to the treatment administered. Immature DC incubated with H-1PV-induced MZ7-Mel lysates significantly increased DC maturation compared with non-infected or necrotic MZ7-Mel cells. Tumor necrosis factor-α and interleukin-6 release was clearly increased by DC incubated with H-1PV-induced SK29-Mel tumor cell lysates (TCL) and was also high with DC-CTL co-cultures incubated with H-1PV-induced TCL. Similarly, DC co-cultures with TCL incubated with H-1PV combined with cytotoxic agents or sunitinib enhanced DC maturation to a greater extent than cytotoxic agents or sunitinib alone. Again, these combinations increased pro-inflammatory responses in DC-CTL co-cultures compared with chemotherapy or sunitinib alone. In our human models, chemotherapeutic or targeted agents did not only interfere with the pronounced immunomodulatory properties of H-1PV, but also reinforced drug-induced tumor cell killing. H-1PV combined with cisplatin, vincristine or sunitinib induced effective immunostimulation via a pronounced DC maturation, better cytokine release and cytotoxic T-cell activation compared with agents alone. Thus, the clinical assessment of H-1PV oncolytic tumor therapy not only alone but also in combination strategies is warranted.
    Full-text · Article · Oct 2011 · BMC Cancer
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    • "On the other hand, overexpression of E2F1, which stimulates transcription of the p73 gene, has been shown to induce apoptosis in melanoma cells [33] and sensitize melanoma cells to apoptosis [34]. Moreover, Tuve and colleagues have demonstrated that a transcriptionally active form of p73 (TA-p73β) increases chemosensitivity of human malignant melanomas to the standard therapeutic agents adriamycin and cisplatin [35]. Importantly, we have shown that TEF induces p73 accumulation, p73 nuclear translocation, and Noxa expression, apparently in a p53-independent manner. "
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    ABSTRACT: Previously we found that terfenadine, an H1 histamine receptor antagonist, acts as a potent apoptosis inducer in melanoma cells through modulation of Ca(2+) homeostasis. In this report, focusing our attention on the apoptotic mechanisms activated by terfenadine, we show that this drug can potentially activate distinct intrinsic signaling pathways depending on culture conditions. Serum-deprived conditions enhance the cytotoxic effect of terfenadine and caspase-4 and -2 are activated upstream of caspase-9. Moreover, although we found an increase in ROS levels, the apoptosis was ROS independent. Conversely, terfenadine treatment in complete medium induced ROS-dependent apoptosis. Caspase-4, -2, and -9 were simultaneously activated and p73 and Noxa induction were involved. ROS inhibition prevented p73 and Noxa expression but not p53 and p21 expression, suggesting a role for Noxa in p53-independent apoptosis in melanoma cells. Finally, we found that terfenadine induced autophagy, that can promote apoptosis. These findings demonstrate the great potential of terfenadine to kill melanoma cells through different cellular signaling pathways and could contribute to define new therapeutic strategies in melanoma.
    Full-text · Article · Aug 2011 · Apoptosis
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    ABSTRACT: The prototyical tumor suppressor p53 is able to arrest cells after DNA damage or as a response to oncogene expression. The transactivation-competent (TA) isoforms of the more recently discovered p53 family member p73 also prevent tumors, but the underlying mechanisms are less well understood. The work presented here addressed this issue by using a cell culture model of tumorigenesis in which normal human diploid fibroblasts are stepwise transduced with oncogenes. Cells in pretransformed stages were shown to harbour high levels of TAp73 mRNA and protein. This positive regulation was probably a result of pRB inactivation and derepression of E2F1, a key activator of TAp73. Consequences for such cells included an increased sensitivity to the cytostatic drug adriamycin, slower proliferation and reduced survival at high cell density, as demonstrated by rescue experiments using siRNA-mediated knockdown of TAp73. In order to identify potential effector pathways, the gene expression profile of siRNA treated, matched fibroblast cell lines with high and low TAp73 levels were compared in DNA microarrays. These findings support the notion of TAp73 up-regulation as an anti-proliferative defense mechanism, blocking the progress towards full transformation. This barrier could be overcome by the introduction of a constitutively active form of Ras which caused a switch from TAp73 to oncogenic DeltaNp73 expression, presumably through the phosphatidylinositol 3-kinase (PI3K) pathway. In summary, the results presented emphasize the tumor-suppressive function of TAp73 and indicate that its downregulation is a decisive event during the transformation of human cells by oncogenic Ras mutants. Der gut untersuchte Tumorsuppressor p53 vermag das Wachstum von Zellen nach DNA-Schädigung oder Onkogenaktivierung zu arretieren. Die transaktivierungsfähigen (TA) Isoformen von p73, eines kürzlich entdeckten Mitgliedes der p53-Familie, können ebenfalls die Tumorentstehung verhindern. Die Mechanismen sind hier aber noch sehr unvollkommen verstanden. Zu deren Untersuchung wurde in der vorliegenden Arbeit ein Zellkulturmodell der Tumorentstehung verwendet, bei dem normale humane diploide Fibroblasten schrittweise mit bestimmten Onkogenen transduziert wurden. Zellen in unvollständig transformierten Stadien hatten hohe Spiegel an TAp73-mRNA und -Protein. Diese positive Regulation war vermutlich eine Folge von pRB-Inaktivierung und der Derepression von E2F1, einem der wichtigsten Aktivatoren von TAp73. Beobachtete Konsequenzen für solche Zellen waren höhere Empfindlichkeit für Zytostatika wie Adriamycin, langsameres Wachstum und geringere Überlebensfähigkeit bei hoher Zelldichte, was durch Rescue-Experimente mit siRNA-vermitteltem TAp73-Knock down gezeigt werden konnte. Um mögliche Effektor-Signalwege zu identifizieren, wurden die Genexpressionsprofile von siRNA-behandelten Fibroblastenlinien, die sich nur im TAp73-Spiegel unterschieden, in DNA microarrays verglichen. Die Befunde daraus lassen den Schluss zu, dass die Hochregulation von TAp73 einen antiproliferativen Schutzmechanimus darstellt, der die vollständige Transformation verhindert. Diese Barriere konnte überwunden werden durch die zusätzliche Präsenz von aktiviertem Ras, das einen Wechsel der Expression von TAp73 zu der von onkogenem DeltaNp73 bewirkte. Dies ist vermutlich abhängig vom Phosphatidylinositol-Signalweg. Zusammenfassend wurde die Rolle von TAp73 als Tumorsuppressor weiter gefestigt, da die Niederregulierung des Proteins eine zentrale Rolle in der Transformation menschlicher Zellen durch onkogene Ras-Mutanten spielt.
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