The interaction of interleukin-1␤ (IL-1␤) with its type 1 cell surface receptor initiates a cascade of intracellular reactions leading to the activation of transcription factors and the expression of target genes. One of the major transcription factors mediating IL-1␤ biological activities is NF-B (for reviews, see references 2, 3, and 22). This factor is sequestered in the cytoplasm by an inhibitor from the IB family. IL-1␤ cellular stimulation leads to a rapid phosphorylation and degradation of IB␣, the most common NF-B inhibitor. This reaction allows NF-B to translocate to the nucleus, to bind DNA, and to activate the transcription of specific genes (2, 55).Following its interaction with IL-1, the type 1 IL-1 receptor recruits the IL-1 receptor-associated kinase (IRAK) protein, which subsequently interacts with the TRAF6 adapter protein (15,16,30,61,62,65). TRAF6 is required for IL-1␤-induced NF-B activation, as demonstrated in 293 cells (16).However, the signaling pathways leading to NF-B activation from the IL-1␤ receptors are still controversial. It has been demonstrated that TRAF6 interacts with a MAP kinase kinase kinase (MAPKKK) known as NIK and that NIK is required for IL-1␤-or tumor necrosis factor alpha (TNF-␣)-dependent 56). Large (500 to 900 kDa) multimeric protein kinase complexes have been purified from HeLa cells and transmit the signal from the TNF receptor type 1 (TNFR-1) and type 1 IL-1 receptors to the NF-B/IB cytoplasmic complex (17,20,33,41). From these complexes three IB kinases, IKK-␣, IKK-␤, and IKK-␥, have been purified, and their genes were cloned (20,42,49,66). Other investigators have cloned IKK kinases by virtue of their association with the NIK protein kinase (47, 64). Moreover, inactivation of these kinases by dominant negative mutants suppresses IL-1␤ and TNF-␣ induction of NF-B. These studies indicate that the activated NIK kinase phosphorylates and activates the IKK protein kinases. IKK protein kinases can in turn phosphorylate the IB␣ protein on serines located at positions 32 and 36, a reaction which targets IB␣ for ubiquitination and rapid degradation by the proteasome (12,58,59). These reactions are extremely rapid, and the cellular IB␣ protein is completely degraded within minutes following TNF-␣ or IL-1␤ cell stimulation (4, 13).Despite this simplified linear receptor-TRAF-NIK-IKK axis for IB␣ phosphorylation and degradation, other intermediates might be involved in NF-B activation by TNF-␣ or IL-1␤. First, several components of the large signaling complex remain to be identified, as the three IKK protein kinases do not account for the molecular weight of the whole complex. Second, a large number of studies, some of them being a matter of controversy, have identified other intermediates which seem to be required for TNF-␣-or IL-1␤-mediated NF-B activation. These intermediates are Raf-1, MAP kinases, the PKC and / isoforms, Rho and Rac proteins, and ceramide or reactive oxygen intermediates (ROIs) (19,24,25,32,33,38,46,(50)(51)(52)(53)57). Such a large number of controver...

Cryptolepine hydrochloride is an indoloquinoline alkaloid isolated from the roots of Cryptolepis sanguinolenta. It is characterized by a multiplicity of host-mediated biological activities, including antibacterial, antiviral, and antimalarial properties. To date, the molecular basis for its diverse biological effects remains largely uncertain. Several lines of evidence strongly suggest that DNA might correspond to its principal cellular target. Consequently, we studied the strength and mode of binding to DNA of cryptolepine by means of absorption, fluorescence, circular, and linear dichroism, as well as by a relaxation assay using DNA topoisomerases. The results of various optical and gel electrophoresis techniques converge to reveal that the alkaloid binds tightly to DNA and behaves as a typical intercalating agent. In DNAase I footprinting experiments it was found that the drug interacts preferentially with GC-rich sequences and discriminates against homo-oligomeric runs of A and T. This study has also led to the discovery that cryptolepine is a potent topoisomerase II inhibitor and a promising antitumor agent. It stabilizes topoisomerase II-DNA covalent complexes and stimulates the cutting of DNA at a subset of preexisting topoisomerase II cleavage sites. Taking advantage of the fluorescence of the indoloquinoline chromophore, fluorescence microscopy was used to map cellular uptake of the drug. Cryptolepine easily crosses the cell membranes and accumulates selectively into the nuclei rather than in the cytoplasm of B16 melanoma cells. Quantitative analyses of DNA in cells after Feulgen reaction and image cytometry reveal that the drug blocks the cell cycle in G2/M phases. It is also shown that the alkaloid is more potent at inhibiting DNA synthesis rather than RNA and protein synthesis. Altogether, the results provide direct evidence that DNA is the primary target of cryptolepine and suggest that this alkaloid is a valid candidate for the development of tumor active compounds.