Carboplatin induces apoptotic cell death through downregulation of constitutively active nuclear factor-kappaB in human HPV-18 E6-positive HEp-2 cells.
ABSTRACT Because the role of nuclear factor kappaB (NF-kappaB) is in cellular growth control and neoplasia, we explored the status of NF-kappaB and investigated its role in survival of human HPV-18 E6-positive HEp-2 cells. We observed accumulation of p65 in the nucleus. Moreover, without any external stimulus constitutive NF-kappaB DNA binding and transactivation activity were detected in HEp-2 cells. Treatment with NF-kappaB inhibitor curcumin (diferuloylmethane) and transient transfection of the mutant form of IkappaBalpha, IkappaBalpha super repressor (IkappaBalpha-SR), suppressed constitutive NF-kappaB activity as well as proliferation, suggesting that constitutive NF-kappaB activity is required for the survival of HEp-2 cells. Carboplatin treatment downregulated constitutive NF-kappaB activity and prevented nuclear retention of p65. Further, carboplatin also suppressed the constitutive IkappaBalpha phosphorylation leading to stabilization of IkappaBalpha protein in the cells. Carboplatin inhibited NF-kappaB binding to its response element present in Bcl-2 promoter resulting in downregulation of antiapoptotic Bcl-2 protein. Thus, our results for the first time indicate that constitutive NF-kappaB has a significant role in the survival of HPV-18 E6-positive HEp-2 cells. Moreover, inactivation of NF-kappaB is one of the mechanisms underlying the induction of carboplatin-mediated apoptosis in HPV-18 E6-positive cancer cells.
- SourceAvailable from: John A Blaho[show abstract] [hide abstract]
ABSTRACT: Wild-type herpes simplex virus type 1 (HSV-1) infection triggers apoptosis in human cells. The subsequent synthesis of infected cell proteins between 3 and 6 h postinfection (hpi) acts to block this process from killing the cells. The factors produced during this window also prevent cell death induced by environmental staurosporine or sorbitol (M. Aubert, J. O'Toole, and J. A. Blaho, J. Virol. 73:10359-10370, 1999). We now report that (i) during the prevention window, HSV-1(F) also inhibited apoptosis induced by tumor necrosis factor alpha (TNF-alpha) plus cycloheximide (CHX) treatment. While deciphering the mechanism of this inhibition, we observed that (ii) the transcription factor NF-kappaB translocated from the cytoplasm into the nuclei of infected cells, and (iii) this migration initiated at 3 hpi. (iv) The complete inhibition of protein synthesis at 3 hpi by the addition of CHX precluded NF-kappaB translocation, while CHX additions at 6 hpi or later did not elicit this effect. This result confirms that infected cell protein synthesis is required for the nuclear import of NF-kappaB. (v) The detection of NF-kappaB in nuclei correlated with the ability of HSV-1(F), HSV-1(KOS1.1), or HSV-1(R7032), a replication-competent recombinant virus containing a deletion in the gene encoding the gE glycoprotein, to prevent apoptosis. (vi) NF-kappaB did not bind its kappaB DNA recognition site and remained cytoplasmic in cells actively undergoing apoptosis following infection with HSV-1(vBSdelta27), a virus with the key regulatory protein ICP27 deleted. (vii) Prestimulation of NF-kappaB by the addition of a phorbol ester prevented HSV-1(vBSdelta27)-induced apoptosis. (viii) Retention of NF-kappaB in the cytoplasm by the addition of a pharmacological antagonist of its release from IkappaBalpha led to an increase in death factor processing during HSV-1(F) infection. (ix) A novel HEp-2 clonal cell line, termed IkappaBalphaDN, was generated which expresses a dominant-negative form of IkappaBalpha. Treatment of IkappaBalphaDN cells with TNF-alpha in the absence of CHX resulted in apoptotic death due to the inability of NF-kappaB to become activated in these cells. Finally, (x) infection of IkappaBalphaDN cells with HSV-1(F) or HSV-1(KOS1.1) resulted in apoptosis, demonstrating that (xi) the nuclear translocation of NF-kappaB between 3 and 6 hpi (the prevention window) is necessary to prevent apoptosis in wild-type HSV-1-infected human HEp-2 cells.Journal of Virology 08/2003; 77(13):7261-80. · 5.08 Impact Factor
- Urologic Oncology: Seminars and Original Investigations. 01/2004; 22(1):84.
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ABSTRACT: Primary hepatocellular carcinoma (HCC) is one of the 10 most common human carcinomas in the world. The mechanism by which HCC cells resist apoptosis induced by various treatment modalities is poorly understood. The authors sought to determine whether Rel/NF-kappaB transcription factors play a key role in controlling apoptosis in human HCCcells. We assessed constitutive and inducible activation of NF-kappaB in hepatitis B virus (HBV)-positive (Hep3B) and in hepatitis virus-negative (Chang, HepG2) HCC cells, as well as the role of known inhibitors of NF-kappaB activity. The current study data demonstrate that 1) RelA/NF-kappaB activity is activated constitutively in Hep3B cells, as determined by electrophoretic mobility shift assays; 2) RelA/NF-kappaB reporter gene activity is inhibited specifically by dominant-negative mutants of IkappaB(alpha), IKK1, IKK2, MEKK1, and MEKK3 and it is activated by overexpression of wild-type MEKK3, suggesting that upstream kinase cascades induce phosphorylation of IkappaB(alpha) and activate RelA/NF-kappaB in Hep3B cells; 3) overexpression of the HBV x gene fails to activate NF-kappaB in HepG2 and Chang cell lines; 4) The NF-kappaB-inducible gene, bcl-xl, is overexpressed in Hep3B cells and is inhibited by the proteosome inhibitor PS341, which prevents IkappaBalpha degradation and RelA/NF-kappaB activation; and 5) inhibition of constitutive RelA/NF-kappaB activity by PS341 sensitizes Hep3B cells to doxorubicin-induced apoptosis. These results are consistent with the role of RelA/NF-kappaB activity in the regulation of apoptosis through activation of its downstream target genes and suggest that signaling pathways that control RelA/NF-kappaB activity may be important targets for novel therapeutic approaches in the treatment of human HCC.Cancer 11/2002; 95(8):1696-705. · 5.20 Impact Factor