This report demonstrates that Gadd45, a p53-responsive stress protein, can facilitate topoisomerase relaxing and cleavage activity in the presence of core histones. A correlation between reduced expression of Gadd45 and increased resistance to topoisomerase I and topoisomerase II inhibitors in a variety of human cell lines was also found. Gadd45 could potentially mediate this effect by destabilizing histone-DNA interactions since it was found to interact directly with the four core histones. To evaluate this possibility, we investigated the effect of Gadd45 on preassembled mononucleosomes. Our data indicate that Gadd45 directly associates with mononucleosomes that have been altered by histone acetylation or UV radiation. This interaction resulted in increased DNase I accessibility on hyperacetylated mononucleosomes and substantial reduction of T4 endonuclease V accessibility to cyclobutane pyrimidine dimers on UV-irradiated mononucleosomes but not on naked DNA. Both histone acetylation and UV radiation are thought to destabilize the nucleosomal structure. Hence, these results imply that Gadd45 can recognize an altered chromatin state and modulate DNA accessibility to cellular proteins.

The levels of S100 Ca 2؉ -binding proteins correlate with the progression of certain tumors, but their role, if any, in carcinogenesis is still poorly understood. S100B protein associates with both the p53 oligomerization domain (residues 325-355) and the extreme C terminus of the tumor suppressor p53 (residues 367-392). Consequently, S100B inhibits p53 tetramer formation and p53 phosphorylation mediated by protein kinase C, on p53 C-terminal end. In this report, we show that the S100B protein decreases p53 DNA binding and transcriptional activity. The effect of S100B is reflected in vivo by a reduced accumulation of p53, p21, and MDM2 protein levels in co-transfection assays and in response to bleomycin. The S100B can still interact with p53 in the absence of p53 extreme C-terminal end and reduce the expression of p53 downstream effector genes. These data indicate that S100B does not require p53 extreme C-terminal end to inhibit p53 activity. Collectively, these findings imply that elevated levels of S100B in tumors such as astrocytomas and gliomas could inhibit p53 functions and contribute to cancer progression.The S100 proteins are dimeric Ca 2ϩ -binding proteins (ϳ10 kDa/subunit) initially characterized by their solubility in 100% ammonium sulfate (S100) (1). Deregulation of Ca 2ϩ homeostasis has been associated with different pathologies including neurodegenerative disorders, hypertension, and cancer (2). The S100 proteins are overexpressed in many tumor cells and have been used as a marker for the classification of tumors (3). A possible role for the S100 proteins in carcinogenesis has often been suspected, but their specific involvement is still ill defined. Evidence has indicated that S100B interacts with the tumor suppressor p53 (4). p53 plays a pivotal role in the maintenance and regulation of normal cellular functions, and its inactivation can affect cell cycle checkpoints, apoptosis, gene amplification, centrosome duplication, and ploidy (5). p53 interacts with a number of proteins to mediate its pleiotropic effects. The interactions of p53 with S100 calcium-binding proteins are of particular interest because like p53, the S100 proteins affect cell cycle progression, are overexpressed in numerous tumor cells, and are associated with tumor progression (2). The S100B protein interacts with the p53 C-terminal end and inhibits both,p53 tetramerization and phosphorylation by PKC 1 (4). Because these two events are known to be important for p53 activation (6), we wanted to determine the effect of S100B on p53 transcriptional activity in vivo. Our data indicate that overexpression of S100B can reduce p53 transcriptional activity by more than 50%. This effect is correlated with a decrease in p53 DNA binding activity and a reduction in the accumulation of MDM2 and p21 protein levels. Interaction of the S100B protein with p53 may thus impede p53 cellular functions. Such an interaction could especially be detrimental in astrocytomas and gliomas, where S100B levels are significantly elevated (7). EXPERIMENTAL PR...

Neem leaf extract (NLE) has medicinal properties, which have been attributed to its limonoid content. We identified the NLE tetranorterpenoid, nimbolide, as being the key limonoid responsible for the cytotoxicity of NLE in various preclinical models of human B-lymphocyte cancer. Of the models tested, Waldenströms macroglobulinemia (WM) cells were most sensitive to nimbolide, undergoing significant mitochondrial mediated apoptosis. Notably, nimbolide toxicity was also observed in drug-resistant (bortezomib or ibrutinib) WM cells. To identify putative targets of nimbolide, relevant in WM, we used chemoinformatics-based approaches comprised of virtual in silico screening, molecular modeling and target–ligand reverse docking. In silico analysis revealed the antiapoptotic protein BCL2 was the preferential binding partner of nimbolide. The significance of this finding was further tested in vitro in RS4;11 (BCL2-dependent) tumor cells, in which nimbolide induced significantly more apoptosis compared with BCL2 mutated (Jurkat BCL2Ser70-Ala) cells. Lastly, intraperitoneal administration of nimbolide in WM tumor xenografted mice, significantly reduced tumor growth and IgM secretion in vivo, while modulating the expression of several proteins as seen on immunohistochemistry. Overall, our data demonstrate that nimbolide is highly active in WM cells, as well as other B-cell cancers, and engages BCL2 to exert its cytotoxic activity.