S phase dependence and involvement of NF-kappaB activating kinase to NF-kappaB activation by camptothecin.
Camptothecin (CPT) and derivatives are topoisomerase I poisons currently used as anticancer drugs. Their cytotoxicity is maximal for cells in S phase. Using asynchronous and S phase-synchronized HeLa cells, we showed that both the nuclear factor-kappaB (NF-kappaB) activation and its transcriptional activity, induced by CPT treatment, are enhanced in S phase cells. After CPT treatment, NF-kappaB activation reached a maximum within 2-3 hr and was still detectable after 24 hr. The nature of the complex evolved with time, forming mostly p50/p65 after 2 hr to almost exclusively p52 after 24 hr. In HeLa cells, the different steps of the induction were readily observable in S phase synchronized cells, whereas they were barely noticeable in a randomly growing cell population. The signal progressed through the activation of the IKK complex, the phosphorylation of IkappaBalpha, and the degradation of phosphorylated-IkappaBalpha and -IkappaBbeta. The stable expression of wild-type HA-tagged-IkappaBalpha or mutated HA-tagged-IkappaBalpha (S32,36A) allowed us to confirm the essential role of Ser32 and Ser36. NF-kappaB-activating kinase (NIK) could play a role upstream of the IKK complex, as the transient expression of a kinase inactive mutant NIK(K429,430A) abolished the activation of NF-kappaB by CPT. A kinase inactive mutant of mitogen-activated protein/ERK kinase kinase 1 (MEKK1), another kinase susceptible of acting upstream of the signalsome, did not. Cytotoxicity studies with clonal populations expressing different amounts of wild-type or mutated IkappaBalpha revealed that the overexpression of wild-type IkappaBa in large amount increases the sensitivity of HeLa cells to CPT more efficiently than a lower level of expression of non-phosphorylable IkappaBalpha.
Habraken
Y
Y
Laboratory of Virology & Immunology, University of Liège, Institute of Pathology, Belgium.
Piret
B
B
Piette
J
J
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D002166:Camptothecin / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004354:Drug Screening Assays, Antitumor; D004789:Enzyme Activation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency / Q000378:metabolism / Q000502:physiology*; D016196:S Phase / Q000187:drug effects*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013997:Time Factors; D014162:Transfection; D000097782:NF-kappaB-Inducing Kinase
D000972:Antineoplastic Agents, Phytogenic; D002166:Camptothecin; D004247:DNA; D004268:DNA-Binding Proteins; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; D017346:Protein Serine-Threonine Kinases; D012694:Serine; D000097782:NF-kappaB-Inducing Kinase; C000606204:NFKBIA protein, human; C496559:CHUK protein, human; C496560:IKBKB protein, human; C496565:IKBKE protein, human; C061553:ATP dependent 26S protease