bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 1080: Molecular and cellular pharmacology of BI 811283, a potent inhibitor of Aurora B kinase
Authors: Tontsch-Grunt Ulrike , Gürtler Ulrich , Zahn Stephan K. , Boehmelt Guido , Jarvis Marc , Adolf Guenther R. , Solca Flavio 
Published: February 2011

Abstract
Background. The serine/threonine kinase Aurora B is involved in the regulation of several mitotic processes, including chromosome condensation, congression and segregation as well as cytokinesis. These essential functions of Aurora B and its overexpression in many cancer types render this protein kinase an attractive target for anticancer drug development.
Methods. BI 811283 was profiled in enzymatic kinase assays as well as in proliferation assays on various human cancer cell lines. Cell cycle status was assessed by DNA content analysis (Cellomics ArrayScan, FACScalibur). Histone H3 phosphorylation was determined by immunofluorescence (Cellomics ArrayScan). Apoptosis was detected by Western blotting for cleaved PARP and microscopic enumeration of DAPI-stained cells showing nuclear fragmentation. Senescent cells were identified by staining for SA-ß-Gal activity.
Results. BI 811283 inhibited human Aurora B kinase activity with an IC50 value of 9 nM, Aurora A and C kinases with 70 nM and 17 nM, respectively. In a panel of 46 additional kinases representative of the human kinome, BI 811283 at 1000 nM inhibited 7/46 kinases by more than 50%. EC50 values for inhibition of proliferation of &gt;20 human cancer cell lines were in the range of 2 to 14 nM. In the non-small cell lung cancer cell line NCI-H460, treatment with BI 811283 resulted in a rapid (&lt;1 h) inhibition of histone H3 phosphorylation. Within 48 h of treatment, the fraction of polyploid cells increased from &lt;5% to &gt;80%, paralleled by a marked increase in cell volume. An increase of cleaved poly (ADP-ribose) polymerase and a concomitant increase in the fraction of cells with nuclear fragmentation from &lt;1% to 7% was observed after 72 h and 96 h of treatment. Within 96 h, the fraction of senescent cells markedly increased from &lt;3% to 25% of the population.
Conclusions. BI 811283 is a potent and selective Aurora kinase inhibitor that inhibits proliferation of cancer cells independent of tissue origin or oncogenome status. Treated cells exhibit a polyploid phenotype characteristic for Aurora B inhibition and show hallmarks of senescence as well as a slow onset of apoptosis in a small fraction of cells. In vivo activity of BI 811283 has been demonstrated in multiple cancer xenograft models in nude mice (see accompanying poster). Phase I clinical trials are ongoing.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1080.
Abstract 1086: The role of HEF1 protein in stabilization and activation of AurA kinase in metastatic breast cancer
Authors: Pugacheva Elena N. , McLaughlin Sarah , Hindman Bridget , Golemis Erica A. 
Published: February 2011

Abstract
AurA kinase expression is linked to human disease since the extent of AurA up-regulation was significantly associated with advanced tumor stage and the occurrence of metastasis. AurA inhibitors have recently entered phase II clinical trials for cancer treatment, but application of these inhibitors for treatment of advanced human cancers has demonstrated limited efficacy. The reasons why potent in vitro inhibitors of AurA are inefficient under these conditions are unknown. In our current study, we define the mechanisms by which AurA is upregulated in breast tumor cells and what impact HEF1 expression has on AurA-associated occurrence of metastasis. Our hypothesis is that over-expression of HEF1 protein in breast cancer cells will protect AurA from proteolytic degradation, resulting in activation of AurA kinase. Here we demonstrate that direct binding of HEF1 stabilizes AurA by blocking the cdh1 binding site and limiting AurA ubiquitination. HEF1 binding decreases the efficacy of AurA inhibitors in cell culture. Deletion of HEF1 by gene knockout and depletion by siRNA in cell lines cause dramatic decreases in AurA protein level and kinase activity - indicating HEF1 is the major AurA regulator in vivo. We further show that deletion of HEF1 reduces tumor cell proliferation and invasion in mouse mammary tumor models. These data provide insights into the potential development of small molecule inhibitors that could be used therapeutically to target the HEF1/AurA pathway and thereby inhibit tumor formation.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1086.
Abstract 1092: Alterations of the retinoblastoma gene in stage III breast cancers
Authors: Berge Elisabet O. , Knappskog Stian , Geisler Stephanie , Staalesen Vidar , Puntervoll Pål , Pacal Marek , Børresen-Dale Anne-Lise , Lillehaug Johan R. , Lønning Per E. 
Published: February 2011

Abstract
We report the novel finding of point mutations affecting the RB1 gene in breast cancer tissue. Analyzing 73 locally advanced (stage III) breast cancers, we identified two somatic and one germline single nucleotide changes, each leading to amino acid substitution; Leu607Ile, Arg698Trp, and Arg621Cys, respectively. In addition, MLPA analysis revealed two large multiexon deletions (exons 13 to 27 and exons 21 to 23) with the exons 21-23 deletion occurring in the tumor also harboring Leu607Ile. All three RB1 point mutations encoded stable nuclear proteins with impaired ability to induce apoptosis compared to wild-type pRb in vitro. Interestingly, Leu607Ile and Arg621Cys are both located within the spacer region of the protein. Mutations in this part of the RB1 gene have previously been identified in retinoblastoma-prone families. Multiple sequence alignment across species indicates this area to be evolutionary conserved. Notably, three out of four tumors harboring RB1 mutations displayed primary resistance to treatment with either mitomycin or doxorubicin while only 14 out of 64 tumors without mutations were resistant (p = 0.046). Although rare, our findings suggest RB1 mutations to be of biological importance in breast cancer.
Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1092.
Abstract 1094: Transcriptional activation by pRB/p107 and coordination with SWI/SNF
Authors: Flowers Stephen , Beck George R. , Moran Elizabeth 
Published: February 2011

Abstract
The retinoblastoma susceptibility gene, Rb1, is closely linked with osteosarcoma as well as retinoblastoma. The basis of such ties between individual susceptibility genes and tumors of specific tissue origin is a central question in cancer biology. Functional analysis of pRB and the closely related family members, p107 and p130, has tended to focus on repression of proliferation, but genetic evidence from our lab and others indicates an active requirement for pRB in osteoblast differentiation that might relate more directly with osteosarcoma susceptibility. Our previous studies linked pRB with activation of the early osteoblast marker, alkaline phosphatase (the product of the Akp2 gene). Here we have investigated the functional relationships between pRB and the SWI/SNF complex in Akp2 activation. Chromatin immunoprecipitation (ChIP) assays reveal the Akp2 promoter is targeted directly by all three pRB family members. Only p130 occupies the promoter prior to induction, while pRB and p107 target Akp2 directly during activation, and concurrent with association of the global co-activator p300. Akp2 is also a direct target of SWI/SNF chromatin remodeling complexes. These ATPase-powered complexes help orchestrate the programmatic shifts in gene expression necessary for progression from a self-renewing precursor state to a state of terminal differentiation. In osteoblasts, SWI/SNF complexes that repress osteogenic gene expression in order to maintain the precursor state are characterized by inclusion of the BRM ATPase. The alternative ATPase, BRG1, characterizes complexes that activate osteogenic genes during terminal differentiation. Thus, the onset of osteoblast differentiation is marked by a switch from BRM to BRG1 complexes on the Akp2 promoter. Our analysis shows that the BRM to BRG1 switch is accomplished in a precise two-step mechanism, with dissociation of BRM-containing SWI/SNF dependent on p300, and association of BRG1-containing SWI/SNF dependent on pRB/p107. Binding of RNA polymerase-II is dependent in turn on BRG1. Thus, in contrast to their widely studied role in cell cycle repression, pRB/p107 play a directly activating role in osteogenic gene expression, specifically in recruitment of the activating SWI/SNF complex.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1094.
Abstract 1098: Cys141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines
Authors: Yusuf Mohammad A. , Srivenugopal Kalkunte S. 
Published: February 2011

Abstract
Purpose: We previously described that human p53 is functionally inactivated by S-glutathionylation at Cys 141 during oxidative and genotoxic stresses (Biochemistry 46, 7765-7780, 2007). To study the dynamics of this posttranslational modification in human cancers and normal tissues, polyclonal antibodies specifically recognizing the thiolated p53 were generated and characterized.
Experimental Design: An 18 residue p53 peptide harboring a mixed disulfide with GSH at Cys141 was synthesized, and antibodies were raised in 5 rabbits. After initial assessment of the sera for the presence of specific (p53-glut) antibodies, it was affinity purified. The antibodies were tested in ELISA, immunoblotting and immunohistochemistry applications using human cancer cell lines and tumor tissue microarrays.
Results: The p53-glut antibodies recognized the antigenic peptide but not the unmodified counterpart, with high sensitivity in ELISA. In immunoblot analyses, the purified antibodies did not react with unmodified p53 or recombinant p53 (rp53), but readily detected the glutathionylated or cysteinylated or small molecule ethanediol-modified rp53 only under non-reducing conditions. Western blotting and immunocytochemistry in HCT116 cells showed low basal levels of thiolated p53 in untreated cells which increased markedly after acute oxidative stress induced by H2O2, diamide and DNA damage by cisplatin or doxorubicin. The glut-p53 decreased sharply after passage of cells to oxidant-free media. The mutant p53 present in HT29 cells was also recognized. In human prostatatic tissues, the apparently normal prostate adjacent to the tumor and most prostate cancers showed abundant presence of glut-p53 in luminal epithelium, a site well known to generate reactive oxygen species, hydrogen peroxide, and lack antioxidant enzymes such as GST-pi. The melanoma and colon cancer were also positive while gastric cancers were negative. Further, the glut-p53 was degraded faster than unmodified p53 in the presence of rabbit reticulocyte lysates.
Conclusions: Our findings provide first evidence for the presence of glut-p53 in human cells and cancer specimens. Glut-p53 found in prostate tissues may reflect the chronic oxidative stress and an alternative route to functional inactivation of p53. This is a first description of thiolation-specific antibodies for any protein. The glut-p53 antibodies should provide a valuable tool to study p53 inactivation in sustained redox imbalance states found in cancer and degenerative diseases, as well as the therapy-induced negative regulation of p53.
Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1098.
Abstract 1105: Dysregulated ΔNp63α displaces p73 from the promoter of the tumor suppressor gene maspin in keratinocytes
Authors: King Kathryn E. , Muraleedharan Deepti , Ponnamperuma Roshini M. , Gerdes Michael , Weinberg Wendy C. 
Published: February 2011

Abstract
The p53 homologue p63 is critical for normal epidermal development. While overexpression of p63, in particular ΔNp63α, has been reported in many squamous cell cancers including those of the skin, the role of p63 in cancer pathogenesis remains unclear. We have previously shown that elevated levels of ΔNp63α aberrantly maintain keratinocyte proliferation under differentiating conditions. We performed microarray analyses of primary murine epidermal keratinocytes overexpressing adenoviral (Ad)-driven ΔNp63α vs. β-gal to identify target genes impacted by dysregulated ΔNp63α. Our studies revealed a coordinated downregulation of RNA transcripts for multiple protease inhibitors, including two members of the serine protease inhibitor (serpin) family, maspin (serpinB5) and plasminogen activator inhibitor-2, PAI-2 (serpinB2), in addition to the metalloproteinase inhibitor, tissue inhibitor of metalloproteinase-3 (TIMP-3). Downregulation of all three protease inhibitors was confirmed by RT-PCR and examined further by western analysis. TIMP-3 protein was detected in keratinocyte conditioned medium but not in whole cell lysates, supporting a primarily extracellular function for TIMP-3. PAI-2 and maspin were detected in both whole cell lysates and conditioned medium, consistent with both intracellular and extracellular roles for these proteins. Secreted levels of TIMP-3 and PAI-2 declined in Ad-ΔNp63α-cultures compared to Ad-β-gal-cultures, whereas maspin levels remained stable. Intracellular expression of both PAI-2 and maspin was negatively regulated by ΔNp63α. To investigate the mechanism by which ΔNp63α down-regulates maspin, we performed electrophoretic mobility shift assays (EMSAs) using a known p53 consensus DNA binding sequence in the maspin promoter. An endogenous complex was seen by EMSA in normal keratinocytes that was disrupted in the presence of increasing levels of ΔNp63α. Supershift analysis revealed that this endogenous complex was interrupted by the addition of a p73 antibody, but no supershift or block was seen with antibodies to p63 or p53. EMSA analysis of nuclear extracts prepared from p73(-/-) keratinocytes confirmed that p73 is required for this complex formation. The current data suggest additional roles for ΔNp63α in cancer pathogenesis by disrupting p73 transcriptional activity to down-modulate protease inhibitors.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1105.
Abstract 1108: The Drosophila Caliban suppresses tumor formation through both DNA damage repair and apoptosis
Authors: Bi Xiaolin , Wang Zhe , Mortin Mark A. , Wang Yajie , Stultz Brian , Hursh Deborah A. 
Published: February 2011

Abstract
The tumor suppressor gene sdccag1 is down-regulated in human colon cancer cell lines, its Drosophila homolog caliban was screened using a functional HDA domain of homeobox gene prospero as bait. In our previous work, we had suggested Caliban is a bipartite nuclear export mediator, working through the Exportin pathway, and itself can shuttle between cytoplasm and nucleus. To study its tumor suppression functions, we generated knock-out Drosophila Melanogaster of caliban by gene targeting strategy. The caliban knock-out flies are viable and fertile, similar as tumor suppressor gene Dmp53 knock-out flies. While caliban null flies have retarded growth by three days, this delayed growth was rescued by Dmp53 mutation. The caliban flies are sensitive to irradiation, and its DNA damage sensitivity depends on p53. High dosage treatment with irradiation causes tumor formation in caliban null flies, while atm and caliban double mutant flies have autonomous tumor formation, which is similar as atm p53 double mutant. The tumor suppression function of caliban was also studied in an in vivo mouse model where lung tumor cells A549 were implanted subcutaneously into athymic nude mice, we observed the greatly reduced tumor size in caliban expressing A549 cells. These preliminary results suggest caliban may compensate with p53 to suppress the tumor predisposition through both DNA damage repair and apoptosis.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1108.
Abstract 1111: Enhancer of Zeste homologue 2 (EZH2) regulates BRCA1 transiently nuclear export during the cell cycle, and induces tetraploidy of breast cells through AKT1
Authors: Gonzalez Maria E. , Li Xin , Toy Kathy , Prie Mattew Du , Ventura Alejandra , Kruger Heather , Kleer Celina G. 
Published: February 2011

Abstract
EZH2 is overexpressed in breast carcinomas with high propensity to metastasize. EZH2 is an oncogene essential to promote proliferation and capable to trigger neoplastic conversion in the breast. We have reported that EZH2 promotes proliferation of breast cancer through downregulation of BRCA1 protein, but the mechanism is unknown. BRCA1 shuttles between the cytoplasm and nucleus to maintain genomic stability. AKT1 up regulation disrupts BRCA1 function by altering its nuclear localization and inducing genomic instability. We hypothesize that EZH2 regulates nuclear-cytoplasmic shuttling of BRCA1 in breast cells through AKT1.
Methods: We developed two cell culture models: an inducible EZH2 up regulation system in MCF10A benign mammary cells and a lentivirus-mediated shRNA interference targeting EZH2 model in CAL51 breast cancer cells (tetraploid, hormone receptor negative, and with intact p53 and BRCA1 genes). Our recently developed EZH2 transgenic mouse model was analyzed to determine the in vivo relevance of our in vitro data obtained by Western blots, immunofluorescence, proliferation, and FACS.
Results: EZH2 shRNA knock-down in CAL51 cells increased BRCA1 nuclear localization in early S phase, decreased cell growth by delaying cell cycle progression, and tetraploidy. EZH2 overexpression in MCF10A cells decreased BRCA1 nuclear localization, increased proliferation and caused mitotic spindle defects and tetraploidy. EZH2 overexpression increased total and phoshporylated AKT1 at serine 473. Specific pharmacologic inhibition of AKT reverted the effect of EZH2 on BRCA1 nuclear localization, proliferation, mitotic defects and tetraploidy. We also found that EZH2 directly interacts with AKT1 protein. Mammary glands of EZH2 transgenic mice show up-regulation of AKT1 and a decreased in nuclear BRCA1 protein compared to wild type mice.
Conclusion: EZH2 regulates the nuclear-cytoplasmic shuttling of BRCA1 protein and influences BRCA1 functions on proliferation, mitotic spindle control, and maintenance of genomic stability in benign breast cells and in breast cancer cells. The regulation of BRCA1 localization and function by EZH2 requires AKT.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1111.
Abstract 1117: Characterization of BAT1 (UAP56) interaction with BARD1
Authors: Carvalho Renato S. , Nepomuceno Thales C. , Sweet Michael , Monteiro Alvaro N. , Carvalho Marcelo A. 
Published: February 2011

Abstract
BARD1 (BRCA1-associated ring domain 1) was originally identified in a yeast-two-hybrid screen as a binding partner of BRCA1. The functional heterodimer BRCA1-BARD1 is required for several of the cellular and tumour-suppressor functions of BRCA1. Both proteins interact through the N-terminal RING domain to form a heterodimeric E3 ubiquitin ligase that constitutes the major catalytic activity of the BRCA1-BARD1 complex. BARD1 is also associated to p53-mediated apoptosis, in a BRCA1-independent manner. The carboxy terminus of BRCA1 is highly acidic and contains two tandem BRCA1 C-terminal (BRCT) domains, which are characteristic of members of a large superfamily of proteins involved in DNA repair and cell cycle checkpoint control. These domains are protein-protein interacting regions that mediate the association with a number of other proteins with a role in DNA replication, DNA damage repair (DDR) pathway, transcription, cell cycle control, and ubiquitination. BARD1 also contains two tandem BRCT repeats at its C-terminus, a region shown to bind serine-phosphorylated peptides in vitro. In order to identify putative interaction-proteins with BARD1 C-terminal region we performed a yeast two hybrid (Y2H) screening using BARD1 BRCT tandem domain (aa 554-777) to screen a human testis cDNA library. We identified, among other hits, a cDNA coding for residues 308-418 of BAT1 (UAP56, C-terminal region [OMIM 142560]). UAP56 is a member of DEAD box family of ATP-dependent RNA helicases. Several studies indicate that UAP56 protein is not only essential in pre-mRNA splicing but is also important in mRNA nuclear export and cytoplasmic mRNA localization. We confirmed BAT1-BARD1 interaction by conducting co-IP analysis of ectopic expressed BARD1 and BAT1 in HEK293T cells. We are using a Y2H system approach to determine the minimal region of interaction between BARD1 and BAT1; in parallel, we are also checking for the presence of a multicomponent complex, investigating the presence of BRCA1. In order to assess the biological significance of the interaction between BARD1 and BAT1 we will determine whether this interaction is important for the activation of, or recovery from, the G2/M cell cycle checkpoint after IR and also investigate its putative involvement in p53-dependent apoptosis pathway.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1117.