The dioxin receptor is a gene regulatory protein which exhibits many structural and functional similarities to steroid hormone receptors. In this study we compare the subunit composition of two forms of the dioxin receptor, sedimenting at approximately 9S and approximately 6S respectively, which are present in nuclear extract from wild‐type Hepa 1c1c7 mouse hepatoma cells following treatment in vivo with dioxin. The nuclear approximately 9S receptor form contained the 90 kd heat shock protein, hsp90. As assessed by a gel mobility shift assay, this receptor form did not bind to the xenobiotic response element (XRE) of the target gene cytochrome P‐450 IA1. In contrast, the smaller approximately 6S receptor form did not contain any immunochemically detectable hsp90. Moreover, this receptor form specifically bound to the XRE recognition sequence. Thus, the specific DNA binding activity of the dioxin receptor was inhibited by association with hsp90, and the approximately 9S dioxin receptor species could be regarded as a nonactive receptor form. Neither the approximately 9S nor the approximately 6S receptor forms were detected in nuclear extract from a dioxin treated mutant clone of Hepa 1 that expresses a nuclear translocation deficient receptor phenotype. We conclude that activation of the dioxin receptor is, at least, a two step process involving binding of the ligand and dissociation of hsp90 from the ligand‐binding receptor protein. Inhibition of the DNA binding activity of transcription factors by protein‐‐protein interaction has also been described for several steroid hormone receptors and for the NF kappa B factor.(ABSTRACT TRUNCATED AT 250 WORDS)

The approval of histone deacetylase inhibitors for treatment of lymphoma subtypes has positioned histone modifications as potential targets for the development of new classes of anticancer drugs. Histones also undergo phosphorylation events, and Haspin is a protein kinase the only known target of which is phosphorylation of histone H3 at Thr3 residue (H3T3ph), which is necessary for mitosis progression. Mitotic kinases can be blocked by small drugs and several clinical trials are underway with these agents. As occurs with Aurora kinase inhibitors, Haspin might be an optimal candidate for the pharmacological development of these compounds. A high-throughput screening for Haspin inhibitors identified the CHR-6494 compound as being one promising such agent. We demonstrate that CHR-6494 reduces H3T3ph levels in a dose-dependent manner and causes a mitotic catastrophe characterized by metaphase misalignment, spindle abnormalities and centrosome amplification. From the cellular standpoint, the identified small-molecule Haspin inhibitor causes arrest in G2/M and subsequently apoptosis. Importantly, ex vivo assays also demonstrate its anti-angiogenetic features; in vivo, it shows antitumor potential in xenografted nude mice without any observed toxicity. Thus, CHR-6494 is a first-in-class Haspin inhibitor with a wide spectrum of anticancer effects that merits further preclinical research as a new member of the family of mitotic kinase inhibitors.

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Human papillomavirus type 16 (HPV16) is the most common cause of cervical carcinoma. Cervical cancer develops from low-grade lesions that support the productive stages of the virus life cycle. The 16E1 ∧ E4 protein is abundantly expressed in such lesions and can be detected in cells supporting vegetative viral genome amplification. Using an inducible mammalian expression system, we have shown that 16E1 ∧ E4 arrests HeLa cervical epithelial cells in G 2 . 16E1 ∧ E4 also caused a G 2 arrest in SiHa, Saos-2 and Saccharomyces pombe cells and, as with HeLa cells, was found in the cytoplasm. However, whereas 16E1 ∧ E4 is found on the keratin networks in HeLa and SiHa cells, in Saos-2 and S. pombe cells that lack keratins, 16E1 ∧ E4 had a punctate distribution. Mutagenesis studies revealed a proline-rich region between amino acids 17 and 45 of 16E1 ∧ E4 to be important for arrest. This region, which we have termed the "arrest domain," contains a putative nuclear localization signal, a cyclin-binding motif, and a single cyclin-dependent kinase (Cdk) phosphorylation site. A single point mutation in the putative Cdk phosphorylation site (T23A) abolished 16E1 ∧ E4-mediated G 2 arrest. Arrest did not involve proteins regulating the phosphorylation state of Cdc2 and does not appear to involve the activation of the DNA damage or incomplete replication checkpoint. G 2 arrest was also mediated by the E1 ∧ E4 protein of HPV11, a low-risk mucosal HPV type that also causes cervical lesions. The E1 ∧ E4 protein of HPV1, which is more distantly related to that of HPV16, did not cause G 2 arrest. We conclude that, like other papillomavirus proteins, 16E1 ∧ E4 affects cell cycle progression and that it targets a conserved component of the cell cycle machinery.Papillomaviruses are small DNA viruses that infect the epithelial tissue of a wide range of vertebrates, including humans (47). Infection begins in cells of the basal layer, with the productive stages of the virus life cycle being initiated as these infected cells migrate toward the epithelial surface. In this way, the virus life cycle is tightly linked to the differentiation of the epithelium, which makes human papillomaviruses (HPVs) difficult to study in the laboratory (37). More than 200 papillomavirus types have been identified (12). These share a common organization of their ϳ8-kb genomes but differ in the types of epithelium they infect and the pathologies that each virus causes (47). Infection occurs at cutaneous epithelial sites, i.e., the skin, for viruses such as HPV type 1 (HPV1) or at mucosal sites (e.g., the anogenital tract or the cervix) for viruses such as HPV11 and HPV16 (47). HPVs are additionally classified from low to high risk, with low-risk viruses such as HPV11 (and HPV1) being associated solely with benign lesions (often known as warts), whereas lesions caused by highrisk viruses such as HPV16 can progress to malignancy (47). HPV16 is the most prevalent high-risk virus and is the most common causative agent of cervical cancers (68).Although the 16E1 ∧ E4 ...

Human papillomavirus type 16 (HPV16) can cause cervical cancer. Expression of the viral E1∧ E4 protein is lost during malignant progression, but in premalignant lesions, E1∧ E4 is abundant in cells supporting viral DNA amplification. Expression of 16E1 ∧ E4 in cell culture causes G 2 cell cycle arrest. Here we show that unlike many other G 2 arrest mechanisms, 16E1∧ E4 does not inhibit the kinase activity of the Cdk1/cyclin B1 complex. Instead, 16E1∧ E4 uses a novel mechanism in which it sequesters Cdk1/cyclin B1 onto the cytokeratin network. This prevents the accumulation of active Cdk1/cyclin B1 complexes in the nucleus and hence prevents mitosis. A mutant 16E1∧ E4 (T22A, T23A) which does not bind cyclin B1 or alter its intracellular location fails to induce G 2 arrest. The significance of these results is highlighted by the observation that in lesions induced by HPV16, there is evidence for Cdk1/cyclin B1 activity on the keratins of 16E1 ∧

Upon binding of 2,3,7,8-tetrachlorodibenzop-dioxin (called dioxin or TCDD), the dioxin receptor exhibits increased affinity for the cell nucleus in vivo and for DNA in vitro. To derme the recognition sequence of the dioxin receptor and its relationship with that of the glucocorticoid receptor, oligonucleotides derived from dioxin-responsive elements of the rat cytochrome P-450c gene were tested for their ability to form specific protein-DNA complexes in a gel retardation assay. We found that a previously defined sequence motif that is similar to the glucocorticoid-responsive element and exhibits strong enhancer activity in response to dioxin receptor ligands bound a dioxin-inducible factor with high specificity but was not recognized by the DNA-binding domain of the glucocorticoid receptor. Binding to this element was only observed in nuclear extracts of wild-type mouse hepatoma cells in a time-and dose-dependent manner and not in nuclear extracts from a nonresponsive mutant cell line deficient in DNA binding of the dioxin receptor. The specific DNA-binding activity in wild-type nuclear extracts comigrated in a Superose size-exclusion column and cosedimented on sucrose gradients with the in vivo labeled dioxin receptor. These experiments strongly suggest that the dioxin receptor is a sequence-specific DNA-binding protein and is not only biochemically but also functionally similar to the steroid receptor family.The effect of dioxin on specific cytochrome P-450c gene transcription is mediated by an intracellular receptor protein to which dioxin and related compounds bind with high affinity and selectivity (1,2). In analogy to the mechanism of action of steroid hormones, binding of dioxin to its receptor induces an increased affinity ofthe receptor for nuclear target sites in vivo and for nonspecific DNA in vitro (ref. 3 and references therein). Several biochemical properties of the dioxin receptor are similar to those of the glucocorticoid receptor (refs. 4 and 5). However, the dioxin and glucocorticoid receptors do not appear to share any common ligandbinding specificity (6), and the endogenous ligand for the dioxin receptor, if any, has not yet been identified. Although steroid hormone receptors are known to activate gene expression by binding to specific hormone-dependent enhancers, little is known about the function of the dioxin receptor. Attempts to determine whether the dioxin receptor binds directly to specific D1A sequences have been hampered by difficulties in purifying the receptor (7). While this work was in progress, dioxin-inducible protein-DNA interactions with an unidentified sequence motif in the 5' flank of the murine cytochrome P1-450 gene were reported (8).In the rat cytochrome P-450c gene, two classes of sequence elements have been defined by deletion analysis to mediate dioxin induction of gene expression. The "drug regulatory elements" (DREs; ref. 9) exhibit a rather weak enhancer activity, whereas the second class of elements, referred to as "xenobiotic-responsive elements" (XREs),...

Regulation of transcriptional responses in growth-arrested human cells under conditions that promote potentially lethal damage repair after ionizing radiation (IR) is poorly understood. Sp1/retinoblastoma control protein (RCP) DNA binding increased within 30 min and peaked at 2-4 h after IR (450-600 cGy) in confluent radioresistant human malignant melanoma (U1-Mel) cells. Increased phosphorylation of Sp1 directly corresponded to Sp1/RCP binding and immediate-early gene induction, whereas pRb remained hypophosphorylated. Transfection of U1-Mel cells with the human papillomavirus E7 gene abrogated Sp1/RCP induction and G(0)/G(1) cell cycle checkpoint arrest responses, increased apoptosis and radiosensitivity, and augmented genetic instability (i.e., increased polyploidy cells) after IR. Increased NF-kappaB DNA binding in U1-Mel cells after IR treatment lasted much longer (i.e., >20 h). U1-Mel cells overexpressing dominant-negative IkappaBalpha S32/36A mutant protein were significantly more resistant to IR exposure and retained both G(2)/M and G(0)/G(1) cell cycle checkpoint responses without significant genetic instability (i.e., polyploid cell populations were not observed). Nuclear p53 protein levels and DNA binding activity increased only after high doses of IR (>1200 cGy). Disruption of p53 responses in U1-Mel cells by E6 transfection also abrogated G(0)/G(1) cell cycle checkpoint arrest responses and increased polyploidy after IR, but did not alter radiosensitivity. These data suggest that abrogation of individual components of this coordinate IR-activated transcription factor response may lead to divergent alterations in cell cycle checkpoints, genomic instability, apoptosis, and survival. Such coordinate transcription factor activation in human cancer cells is reminiscent of prokaryotic SOS responses, and further elucidation of these events should shed light on the initial molecular events in the chromosome instability phenotype.-Yang, C.-R., Wilson-Van Patten, C., Planchon, S. M., Wuerzberger-Davis, S. M., Davis, T. W., Cuthill, C., Miyamoto, S., Boothman, D. A. Coordinate modulation of Sp1, NF-kappa B, and p53 in confluent human malignant melanoma cells after ionizing radiation.

To reconstitute the molecular mechanisms underlying the cellular response to soluble receptor ligands, we have exploited a cell-free system that exhibits signal-(dioxin-)induced activation of the latent cytosolic dioxin receptor to an active DNA-binding species. The DNA-binding properties of the in vitro-activated form were qualitatively indistinguishable from those of in vivo-activated nuclear receptor extracted from dioxin-treated cells. In vitro activation of the receptor by dioxin was dose dependent and was mimicked by other dioxin receptor ligands in a manner that followed the rank order of their relative affinities for the receptor in vitro and their relative potencies to induce target gene transcription in vivo. Thus, in addition to triggering the initial release of inhibition of DNA binding and presumably allowing nuclear translocation, the ligand appears to play a crucial role in the direct control of the level of functional activity of a given ligand-receptor complex.The dioxin receptor regulates transcription of the cytochrome P-450IA1 gene and several other genes encoding drug-metabolizing enzymes in response to the environmental contaminant dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) or related compounds. This process is mediated by the binding of dioxin to an intracellular receptor protein presumably in the cytosolic compartment of target cells (for reviews, see references 20 and 29). Upon ligand binding, the dioxinreceptor complex becomes activated by a poorly understood structural alteration to a form which is retained on DNA cellulose in vitro (references 5 and 34 and references therein). In wild-type hepatoma cells, ligand-induced receptor activation results in the apparent translocation of the dioxin receptor from the cytosolic to the nuclear compartment (17). Transcription of the cytochrome P-45OIA1 target gene requires the activated, nuclear dioxin-receptor complex, since it is not observed in mutant Hepa 1 cells (nt-) which form normal ligand-receptor complexes but fail to accumulate these complexes in the nucleus (reviewed in reference 20). More recently, the in vivo-activated wild-type Hepa 1 but not the mutant nt-dioxin receptor has been implicated in recognition of specific DNA sequences, termed xenobiotic response elements (XREs), which modulate the activity of linked promoters, thereby serving as dioxin-inducible enhancers (10-12, 14, 22). Thus, the mechanism of action of dioxin is similar to that of steroid hormones in that an intracellular soluble receptor protein transduces the extracellular signal to the transcriptional unit it regulates. More importantly, the dioxin receptor together with most, if not all, steroid receptors belongs to a class of gene regulatory proteins that require activation for function.In the case of steroid hormone receptors, the mechanism of activation to a functional species is not yet understood, and a number of different regulatory mechanisms have been proposed to control this process. These models include, among others, intramolecular alterations of r...