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 I kappaB alpha, and the degradation of phosphorylated-I kappaB alpha and -I kappaB beta. The stable expression of wild-type HA-tagged-I kappaB alpha or mutated HA-tagged-I kappaB alpha (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 I kappaB alpha revealed that the overexpression of wild-type I kappaB alpha in large amount increases the sensitivity of HeLa cells to CPT more efficiently than a lower level of expression of non-phosphorylable I kappaB alpha. (C) 2001 Elsevier Science Inc. All rights reserved.