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Glioblastoma multiforme (GBM) is the most common and aggressive brain cancer, and despite treatment advances, patient prognosis remains poor. During routine animal studies, we serendipitously observed that fenbendazole, a benzimidazole antihelminthic used to treat pinworm infection, inhibited brain tumor engraftment. Subsequent in vitro and in vivo experiments with benzimidazoles identified mebendazole as the more promising drug for GBM therapy. In GBM cell lines, mebendazole displayed cytotoxicity, with half-maximal inhibitory concentrations ranging from 0.1 to 0.3 µM. Mebendazole disrupted microtubule formation in GBM cells, and in vitro activity was correlated with reduced tubulin polymerization. Subsequently, we showed that mebendazole significantly extended mean survival up to 63% in syngeneic and xenograft orthotopic mouse glioma models. Mebendazole has been approved by the US Food and Drug Administration for parasitic infections, has a long track-record of safe human use, and was effective in our animal models with doses documented as safe in humans. Our findings indicate that mebendazole is a possible novel anti-brain tumor therapeutic that could be further tested in clinical trials.

Cellular CCAAT/enhancer binding protein alpha (C/EBPalpha) promotes cellular differentiation and has antimitotic activities involving cell cycle arrest at G(1)/S through stabilization of p21(CIP-1)/WAF1 and through transcriptional activation of the p21 promoter. The Epstein-Barr virus lytic-cycle transactivator protein ZTA is known to arrest the host cell cycle at G(1)/S via a p53-independent p21 pathway, but the detailed molecular mechanisms involved have not been defined. To further evaluate the role of ZTA in cell cycle arrest, we constructed a recombinant adenovirus vector expressing ZTA (Ad-ZTA), whose level of expression at a low multiplicity of infection in normal human diploid fibroblast (HF) cells was lower than or equal to the physiological level seen in Akata cells lytically induced by EBV (EBV-Akata cells). Fluorescence-activated cell sorting analysis of HF cells infected with Ad-ZTA confirmed that G(1)/S cell cycle arrest occurred in the majority of ZTA-positive cells, but not with an adenovirus vector expressing green fluorescent protein. Double-label immunofluorescence assays (IFA) performed with Ad-ZTA-infected HF cells revealed that only ZTA-positive cells induced the expression of both endogenous C/EBPalpha and p21 and blocked the progression into S phase, as detected by a lack of incorporation of bromodeoxyuridine. The stimulation of endogenous ZTA protein expression either through treatment with tetradecanoyl phorbol acetate in D98/HR1 cells or through B-cell receptor cross-linking with anti-immunoglobulin G antibody in EBV-Akata cells also coincided with the induction of both C/EBPalpha and p21 and their mRNAs, as assayed by Northern blot, Western blot, and IFA experiments. Mechanistically, the ZTA protein proved to directly interact with C/EBPalpha by coimmunoprecipitation in EBV-Akata cells and with DNA-bound C/EBPalpha in electrophoretic mobility shift assay experiments, and the in vitro interaction domain encompassed the basic leucine zipper domain of ZTA. ZTA also specifically protected C/EBPalpha from degradation in a protein stability assay with a non-EBV-induced Akata cell proteasome extract. Furthermore, both C/EBPalpha and ZTA were found to specifically associate with the C/EBPalpha promoter in chromatin immunoprecipitation assays, but the interaction with ZTA appeared to be mediated by C/EBPalpha because it was abolished by clearing with anti-C/EBPalpha antibody. ZTA did not bind to or activate the C/EBPalpha promoter directly but cooperatively enhanced the positive autoregulation of the C/EBPalpha promoter by cotransfected C/EBPalpha in transient luciferase reporter gene assays with Vero and HeLa cells as well as with DG75 B lymphocytes. Similarly, ZTA alone had little effect on the p21 promoter in transient reporter gene assays, but in the presence of cotransfected C/EBPalpha, ZTA enhanced the level of C/EBPalpha activation. This effect proved to require a previously unrecognized region in the proximal p21 promoter that contains three high-affinity C/EBPalpha binding sites. Finally, in C/EBPalpha-deficient mouse embryonic fibroblasts (MEF), Ad-ZTA was unable to induce either p21 or G(1) arrest, whereas it was able to induce both in wild-type MEF. Overall, we conclude that C/EBPalpha is essential for at least one pathway of ZTA-induced G(1) arrest during EBV lytic-cycle DNA replication and that this process involves a physical piggyback interaction between ZTA and C/EBPalpha leading to greatly enhanced C/EBPalpha and p21 levels through both transcriptional and posttranslational mechanisms.

Human cytomegalovirus (HCMV) major immediate-early protein IE1 is an abundant 72-kDa nuclear phosphoprotein that is thought to play an important role in efficient triggering of the lytic cycle, especially at low multiplicity of infection. The best-known properties of IE1 at present are its transient targeting to punctate promyelocytic leukemia protein (PML)-associated nuclear bodies (PML oncogenic domains [PODs] or nuclear domain 10 [ND10]), with associated displacement of the cellular PML tumor suppressor protein into a diffuse nucleoplasmic form and its association with metaphase chromosomes. Recent studies have shown that the targeting of PML (and associated proteins such as hDaxx) to PODs is dependent on modification of PML by ubiquitin-like protein SUMO-1. In this study, we provide direct evidence that IE1 is also covalently modified by SUMO-1 in both infected and cotransfected cells, as well as in in vitro assays, with up to 30% of the protein representing the covalently conjugated 90-kDa form in stable U373/IE1 cell lines. Lysine 450 was mapped as the major SUMO-1 conjugation site, but a point mutation of this lysine residue in IE1 did not interfere with its targeting to and disruption of the PODs. Surprisingly, unlike PML or IE2, IE1 did not interact with either Ubc9 or SUMO-1 in yeast two-hybrid assays, suggesting that some additional unknown intranuclear cofactors must play a role in IE1 sumoylation. Interestingly, stable expression of either exogenous PML or exogenous Flag-SUMO-1 in U373 cell lines greatly enhanced both the levels and rate of in vivo IE1 sumoylation during HCMV infection. Unlike the disruption of PODs by the herpes simplex virus type 1 IE110(ICP0) protein, the disruption of PODs by HCMV IE1 proved not to involve proteasome-dependent degradation of PML. We also demonstrate here that the 560-amino-acid PML1 isoform functions as a transcriptional repressor when fused to the GAL4 DNA-binding domain and that wild-type IE1 inhibits the repressor function of PML1 in transient cotransfection assays. Furthermore, both IE1(1-346) and IE1(L174P) mutants, which are defective in displacing PML from PODs, failed to inhibit the repression activity of PML1, whereas the sumoylation-negative IE1(K450R) mutant derepressed as efficiently as wild-type IE1. Taken together, our results suggest that proteasome-independent disruption of PODs, but not IE1 sumoylation, is required for efficient IE1 inhibition of PML-mediated transcriptional repression.

Transcriptional activation of c-fos in response to both serum stimulation and DNA damage requires the serum response element. The inability of in vitro aged or senescent fibroblasts to proliferate in response to serum has been shown to be associated with repressed c-fos expression and reduced AP-1 binding activity. In contrast, we have observed similar levels of c-fos mRNA and protein expression in young (early passage) and old (late passage) cells following their treatment with ultraviolet (UV) irradiation or methyl methanesulfonate (MMS). Thus, the early events in the signal transduction pathway leading to transcriptional activation of c-fos following DNA damage are distinct from those mediating the gene's expression in response to mitogenic stimulation. Despite normal levels of c-fos expression, we observed a reduced level of AP-1 binding activity in old cells relative to young cells treated with UV irradiation or MMS. Reduced AP-1 binding activity is associated with reduced expression of the AP-1-dependent gene, collagenase, in old cells treated with DNA damaging agents. Since other DNA damage-inducible genes also contain an AP-1 regulatory element presumed to play a role in their expression, reduced AP-1 binding activity is likely to have a major impact on the old cell's ability to respond appropriately to DNA damage.

Expression from the promoter of the herpes simplex virus type 2 (HSV-2) large subunit of ribonucleotide reductase (ICP10) is stimulated by co-transfection with DNA that encodes the virion protein Vmw65 previously shown to activate in trans the transcription of all IE genes (Wymer et al., 1989). Specific cis response elements involved in ICP10 transcriptional regulation were studied by chloramphenicol acetyltransferase analysis with hybrid ICP10 promoter/CAT structural gene constructions containing wild type or site-directed mutations of the promoter sequences. The data indicate that Vmw65 activation requires an intact TAAT-GARAT motif while complex formation requires an intact Oct-1 element, and the AP-1 consensus elements in the ICP10 promoter are functional in vitro. Thus, expression from the wild type and GA-rich mutant constructions was enhanced 10-20-fold by co-transfection with DNA encoding Vmw65. The GARAT and POU homeobox (PHB) binding motifs were required for Vmw65 mediated activation but the mutant in the POU specific box (PSB) binding motif was activated at higher concentrations of Vmw65 DNA (1.0-3.0 micrograms). The PHB and PSB binding motifs were necessary for complex formation as determined by gel retardation analysis with in vitro synthesized OTF-1 and Vmw65 proteins. The GARAT and GA-rich elements were not required. CAT expression from pICP10-cat was enhanced by co-transfection with jun and fos encoding DNA, and the ICP10 promoter complexed with in vitro synthesized jun protein.

Expression of the immediate-early (IE) genes of herpes simplex virus (HSV) is specifically stimulated by a 65-kilodalton virion transcription factor (VF65 or VP16) that is introduced as a component of infecting virions. In both the IE175(ICP4) and IE110(ICP0) promoters, this activation requires an upstream cis-acting target response element that contains a single TAATGARAT consensus element. Furthermore, many HSV IE TAATGARAT elements overlap with ATGCTAAT octamer motifs that are similar to the OTF-1-binding sites found in both immunoglobulin and histone H2b genes and to the nuclear factor III (NFIII)-binding site within the adenovirus type 2 origin of DNA replication. Purified HeLa cell NFIII protein proved to form specific DNA-protein complexes with several upstream regions from both the IE110 and IE175 promoters, and this interaction was subject to efficient competition with an adenovirus type 2 DNA fragment containing an intact NFIII-binding site. Surprisingly, the NFIII protein bound to synthetic oligonucleotides containing only the TAATGARAT consensus elements as well as to those containing the ATGCTAAT octamer sequence, although the former exhibited lower affinity and gave complexes with slightly different electrophoretic mobility. The ATGCTAAT oligonucleotide also competed more efficiently than the TAATGARAT sequence itself for binding to a TAATGARAT probe, indicating that the same protein species binds to both sites. The oligonucleotides also formed novel supershifted complexes with lysed virion proteins, but only in the presence of a crude nuclear extract and not with affinity-purified NFIII alone. We conclude that the cellular NFIII protein can recognize both the ATGCTAAT and TAATGARAT elements independently but that only the interaction with TAATGARAT represents an intermediate step in the transcriptional stimulation of IE genes by the HSV virion factor.