Transfection of a dominant-negative mutant NF-kB inhibitor (IkBm) represses p53-dependent apoptosis in acute lymphoblastic leukemia cells: interaction of IkBm and p53.
To investigate the possible role of inhibiting NF-kB activation in sensitizing tumor cells to chemotherapy-induced apoptosis, we transfected the dominant-negative mutant inhibitor of NF-kB (IkBm) into the EU-1 cell line, an acute lymphoblastic leukemia (ALL) line with constitutive NF-kB activation. Overexpression of IkBm significantly reduced constitutive NF-kB activity in EU-1 cells, resulting in decreased cell growth. In response to apoptosis induced by chemotherapeutic drugs, IkBm-transfected cells (EU-1/IkBm) exhibited increased sensitivity to vincristine (VCR), whereas sensitivity to doxorubicin (Dox) was not changed as compared to neo-transfected control (EU-1/neo) cells. To further evaluate the link between IkBm and sensitivity to Dox and VCR, we demonstrated that both endogenous IkBalpha and ectopic IkBm bind to p53. In response to Dox, the cytosolic p53.IkBalpha complex rapidly dissociated due to downregulation of IkBalpha. However, the p53.IkBm complex did not dissociate under these conditions. Although treatment of EU-1/IkBm cells with Dox increased the expression of p53, the nondissociating p53.IkBm complex resulted in decreased p53 function, as demonstrated by absence of cell-cycle arrest and induction of p53 target genes. Contrastingly, VCR-induced cell death neither downregulated IkBalpha nor induced p53, as shown by the lack of NF-kB activation and p53-mediated gene expression in VCR-treated cells. Our data suggest that IkBm simultaneously downregulates NF-kB activation and sequesters p53 in the cytoplasm, thus enhancing NF-kB-regulated apoptosis but blocking p53-dependent apoptosis.
Zhou
Muxiang
M
Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA. mzhou@emory.edu
Gu
Lubing
L
Zhu
Ningxi
N
Woods
William G
WG
Findley
Harry W
HW
D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics* / Q000378:metabolism; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000378:metabolism*; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D020672:I-kappa B Proteins; D016328:NF-kappa B; D016159:Tumor Suppressor Protein p53