The antineoplastic drug sorafenib ) is a multikinase inhibitor that targets the serine-threonine kinase B-Raf as well as several tyrosine kinases. Given the numerous molecular targets of sorafenib, there are several potential anticancer mechanisms of action, including induction of apoptosis, cytostasis, and antiangiogenesis. We observed that sorafenib has broad activity in viability assays in several human tumor cell lines but selectively induces apoptosis in only some lines. Sorafenib was found to decrease Mcl-1 levels in most cell lines tested, but this decrease did not correlate with apoptotic sensitivity. Sorafenib slows cell cycle progression and prevents irradiated cells from reaching and accumulating at G 2 -M. In synchronized cells, sorafenib causes a reversible G 1 delay, which is associated with decreased levels of cyclin D1, Rb, and phosphorylation of Rb. Although sorafenib does not affect intrinsic radiosensitivity using in vitro colony formation assays, it significantly reduces colony size. In HCT116 xenograft tumor growth delay experiments in mice, sorafenib alters radiation response in a schedule-dependent manner. Radiation treatment followed sequentially by sorafenib was found to be associated with the greatest tumor growth delay. This study establishes a foundation for clinical testing of sequential fractionated radiation followed by sorafenib in gastrointestinal and other malignancies. [Cancer Res 2007;67(19):9443-54]

The estrogen receptor (ER), a member of the nuclear hormone receptor superfamily important in human physiology and disease, recruits coactivators which modify local chromatin structure. Here we describe effects of ER on large-scale chromatin structure as visualized in live cells. We targeted ER to gene-amplified chromosome arms containing large numbers of lac operator sites either directly, through a lac repressor-ER fusion protein (lac rep-ER), or indirectly, by fusing lac repressor with the ER interaction domain of the coactivator steroid receptor coactivator 1. Significant decondensation of large-scale chromatin structure, comparable to that produced by the ϳ150-fold-stronger viral protein 16 (VP16) transcriptional activator, was produced by ER in the absence of estradiol using both approaches. Addition of estradiol induced a partial reversal of this unfolding by green fluorescent protein-lac rep-ER but not by wild-type ER recruited by a lac repressor-SRC570-780 fusion protein. The chromatin decondensation activity did not require transcriptional activation by ER nor did it require ligand-induced coactivator interactions, and unfolding did not correlate with histone hyperacetylation. Ligand-induced coactivator interactions with helix 12 of ER were necessary for the partial refolding of chromatin in response to estradiol using the lac rep-ER tethering system. This work demonstrates that when tethered or recruited to DNA, ER possesses a novel large-scale chromatin unfolding activity.Nuclear hormone receptors are ligand-inducible transcriptional activators that play a large number of physiological roles (37). These proteins activate transcription through a multistage process involving binding to DNA response elements (26), recruitment of coactivators (18, 25), remodeling of chromatin (11, 32), and assembly of general transcription factors and RNA polymerase II (42). Gene expression, the last step of the process, is typically used as a readout of transcription factor function. Few experimental approaches allow dissection of the chromatin-related activities of a transcriptional activator. In addition, conclusions about how steroid hormone receptors and their associated coregulators function have often been based on studies of transiently expressed reporter genes which lack native chromatin structure.It has become clear in recent years that the ability of steroid hormone receptors to activate transcription of endogenous genes likely depends upon their ability to affect chromatin structure. Indeed, many steroid hormone receptors interact with coregulator proteins that are implicated in the remodeling of local chromatin structure and the acetylation of histones (reviewed in references 32, 33, and 41). In fact, enhancement of transcription by adding ligand to estrogen receptor (ER) was observed using chromatinized template DNA but not when using naked DNA lacking histones (27). In addition, genes integrated into chromosomes have been shown to be regulated differently from genes located in transiently transfected plasm...

We report on the identification of novel, nonsteroidal ligands that show pronounced subtype-selective differences in ligand binding and transcriptional potency or efficacy for the two estrogen receptor (ER) subtypes, ER alpha and ER beta. An aryl-substituted pyrazole is an ER alpha potency-selective agonist, showing higher binding affinity for ER alpha and 120-fold higher potency in stimulation of ER alpha vs. ER beta in transactivation assays in cells. A tetrahydrochrysene (THC) has a 4-fold preferential binding affinity for ER beta; it is an agonist on ER alpha, but a complete antagonist on ER beta. Intriguingly, the antagonist activity of THC is associated with the R,R-enantiomer (R,R-THC). The S,S-enantiomer (S,S-THC) is an agonist on both ER alpha and ER beta but has a 20-fold lower affinity for ER beta than R,R-THC. This difference in binding affinity accounts for the full ER beta antagonist activity of the THC racemate (a 1:1 mixture of R,R-THC and S,S-THC). These compounds should be useful in probing the conformational changes in these two ERs that are evoked by agonists and antagonists, and in evaluating the distinct roles that ER beta and ER alpha may play in the diverse target tissues in which estrogens act.

We have identified a novel DEAD box RNA helicase (97 kDa, DP97) from a breast cancer cDNA library that interacts in a hormone-dependent manner with nuclear receptors and represses their transcriptional activity. DP97 has RNA-dependent ATPase activity, and mapping studies localize the interacting regions of DP97 and nuclear receptors to the C-terminal region of DP97 and the hormone binding/activation function-2 region of estrogen receptors (ER), as well as several other nuclear receptors. Repression by DP97 maps to a small region (amino acids 589 -631) that has homology to a repression domain in the corepressor protein NCoR2/SMRTe. This region of DP97 is necessary and sufficient for its intrinsic repression activity. The N-terminal helicase region of DP97 is, however, dispensable for its transcriptional repressor activity. The knockdown of endogenous cellular DP97 by antisense DP97 or RNA interference (siRNA for DP97) results in significant enhancement of the expression of estradiol-ER-stimulated genes and attenuation of the repression of genes inhibited by the estradiol-ER. This implies that endogenous DP97 normally dampens stimulation and intensifies repression of estradiol-ER-regulated genes. Our findings add to the growing evidence that RNA helicases can associate with nuclear receptors and function as coregulators to modulate receptor transcriptional activity.Nuclear receptors comprise a superfamily of transcription factors that activate or repress gene transcription in a manner that is dependent on the nature of the hormonal ligand and coregulator proteins (coactivators or corepressors) that are recruited to the ligand-receptor complex (1). Among the steroid hormone receptors, estrogen receptors (ERs) 1 mediate the diverse stimulatory and repressive biological actions of estrogens and antiestrogens. These ligands, which are naturally occurring as well as synthetic, display a spectrum of activities ranging from full agonist to full antagonist that are reflective of changes in receptor conformation engendered by the ligand and the distinct coactivator/corepressor proteins recruited. These observations have led to the designation of some of these ligands as selective ER modulators (SERMs) (2, 3). Many coactivator proteins have been identified, and these assemble into several dynamic multiprotein complexes (1, 4 -7). These coactivator complexes include the SRC/p160 family of proteins, CREB-binding protein (CBP) and/or p300, and other factors that are recruited in a temporally ordered fashion (4) and up-regulate nuclear receptor activity, at least in part, through enhanced histone acetyltransferase activity (5-7). ATPdependent chromatin remodeling complexes, such as BRG1/ hBrm, and the TRAP-DRIP-ARC complex, which act sequentially or combinatorially, also enhance gene transcription by facilitating RNA polymerase II recruitment to promoters (4).In contrast to the coactivators, far fewer corepressors are known. Most fully characterized are NCoR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoi...

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