المؤلفون: Luke H. Stockwin, Trevor Glaros, Michael E. Mullendore, Bethanie L. Morrison, Brian J. Smith, Dianne L. Newton

المصدر: Cancer Chemotherapy and Pharmacology. 70:207-212

مصطلحات موضوعية: Cancer Research, Time Factors, DNA damage, Survivin, Blotting, Western, Repressor, Antineoplastic Agents, Tripartite Motif-Containing Protein 28, Toxicology, Inhibitor of Apoptosis Proteins, Histones, chemistry.chemical_compound, Cell Line, Tumor, Neoplasms, Humans, Pharmacology (medical), RNA, Neoplasm, Pharmacology, Dose-Response Relationship, Drug, Molecular Structure, biology, Chemistry, Imidazoles, DNA, Neoplasm, HCT116 Cells, Molecular biology, Repressor Proteins, Blot, Histone, Oncology, Cell culture, Protein Biosynthesis, biology.protein, Cancer research, K562 Cells, HT29 Cells, DNA, DNA Damage, Naphthoquinones, K562 cells

الوصف: To establish whether NSC80467, a novel fused naphthquinone imidazolium, has a similar spectrum of activity to the well-characterized "survivin suppressant" YM155 and to extend mechanistic studies for this structural class of agent.NSC80467 and YM155 were analyzed in parallel using assays measuring viability, survivin suppression, inhibition of DNA/RNA/protein synthesis and the cellular response to DNA damage.GI(50) values generated for both compounds in the NCI-60 screen yielded a correlation coefficient of 0.748, suggesting significant concordance. Both agents were also shown to inhibit protein expression of survivin [BIRC5]. COMPARE analysis identified DNA damaging agents chromomycin A3 and bisantrene HCl and one DNA-directed inhibitor of transcription, actinomycin D, as correlating with the activity of NSC80467 and YM155. Furthermore, both agents were shown to preferentially inhibit DNA, over RNA and protein synthesis. Thus, the ability of NSC80467 and YM155 to induce a DNA damage response was examined further. Treatment of PC3 cells with either agent resulted in dose-dependent induction of γH2AX and pKAP1, two markers of DNA damage. The concentrations of agent required to stimulate γH2AX were considerably lower than those required to inhibit survivin, implicating DNA damage as an initiating event. The DNA damage response was then confirmed in a panel of cell lines treated with NSC80467 or YM155, suggesting that γH2AX and pKAP1 have potential as response biomarkers.These data provide the first evidence that NSC80467 and YM155 are DNA damaging agents where suppression of survivin is a secondary event, likely a consequence of transcriptional repression.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::d04c644abd4a1296196c33493e4ee597Test
https://doi.org/10.1007/s00280-012-1868-0Test

المؤلفون: Bethanie L. Morrison, Young H. Lee, Donald P. Bottaro

المصدر: The Journal of biological chemistry. 289(30)

مصطلحات موضوعية: MAPK/ERK pathway, MAP Kinase Signaling System, medicine.medical_treatment, Breast Neoplasms, Biology, urologic and male genital diseases, Biochemistry, Gene Expression Regulation, Enzymologic, Epidermal growth factor, Cell Line, Tumor, medicine, Humans, Neoplasm Invasiveness, Protein Phosphatase 2, Protein kinase A, Molecular Biology, Protein kinase B, Carcinoma, Renal Cell, Protein kinase C, Protein Kinase C, Diacylglycerol kinase, Hepatocyte Growth Factor, Growth factor, Dual Specificity Phosphatase 2, Cell Biology, female genital diseases and pregnancy complications, Cell Hypoxia, Kidney Neoplasms, Gene Expression Regulation, Neoplastic, Von Hippel-Lindau Tumor Suppressor Protein, Cancer research, Hepatocyte growth factor, Female, Reactive Oxygen Species, Proto-Oncogene Proteins c-akt, medicine.drug, Signal Transduction

الوصف: Hepatocyte growth factor (HGF) signaling promotes tumor invasiveness in renal cell carcinoma (RCC) and other cancers. In clear cell RCC, VHL loss generates pseudohypoxia that exacerbates HGF-driven invasion through β-catenin deregulation. Hypoxia also enhances HGF-driven invasiveness by papillary RCC cells, but in the absence of VHL, loss signaling integration involves three parallel routes: 1) hypoxia-induced reactive oxygen species production and decreased DUSP2 expression, leading to enhanced mitogen-activated protein kinase (MAPK) cascade activation; 2) reactive oxygen species-induced diacylglycerol production by phospholipase Cγ, leading to protein kinase C activation and increased protein phosphatase- 2A activity, thereby suppressing HGF-induced Akt activation; and 3) a profound shift from HGF-enhanced, proliferation- oriented metabolism to autophagy-dependent invasion and suppression of proliferation. This tripartite signaling integration was not unique to RCC or HGF; in RCC cells, invasive synergy induced by the combination of hypoxia and epidermal growth factor occurred through the same mechanism, and in estrogen receptor-positive breast cancer cells, this mechanism was suppressed in the absence of estrogen. These results define the molecular basis of growth factor and hypoxia invasive synergy in VHL-competent papillary RCC cells, illustrate the plasticity of invasive and proliferative tumor cell states, and provide signaling profiles by which they may be predicted.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::b76b02fc5a26a57c1a0fabb53b4793a8Test
https://pubmed.ncbi.nlm.nih.gov/24914205Test

المؤلفون: Bethanie L. Morrison, Joe J. Mitala, Joseph M. Reilly, Kathryn M. Headley, Kevin A. Murray, Federico Bernal, Amanda L. Whiting

المصدر: Cancer Research. 74:3234-3234

مصطلحات موضوعية: chemistry.chemical_classification, Cancer Research, Peptide, Plasma protein binding, Amino acid, chemistry.chemical_compound, Residue (chemistry), Oncology, chemistry, Biochemistry, Covalent bond, Protein purification, Azide, DNA

الوصف: Mutations in the DNA binding region of tumor suppressor p53 render it transcriptionally inactive (dominant negative) but can also manifest in transcriptionally-independent gain-of-function (GOF) effects, possibly due to mutp53 interacting with other, as yet, unknown targets. Identification of these targets would help to elucidate the pathways and vulnerabilities involved in mutp53 GOF. Covalently linking a known handle to these unknown proteins is one method to aid in their isolation and identification. A hydrocarbon-stapled alpha helical peptide of the p53 transactivation domain (residues 14-29), SAH-p53-8, has been shown to strongly interact with known p53 targets that utilize this domain for binding (HDM2, Kd = 55 nM, HDMX, Kd = 2.3 nM). Substitution of a key interacting residue for the unnatural amino acid benzoylphenylalanine (Bpa) results in a stapled peptide that retains the biochemical properties of SAH-p53-8 (affinities of 38.1 nM and 58.3 nM, respectively) and can covalently capture known targets via photochemical reaction. The introduction of an azido group - a versatile, biochemically-inert capping group - to the N-terminus of this photo-reactive peptide allows for the attachment of various tags to the covalently bound protein(s) via a copper-catazlyzed azide alkyne cycloaddition. Tags include both fluorophores for visualization and affinity tags for protein isolation. This poster will cover the biochemical proof-of-principle of this protein isolation method and exploration into more complex cell lysate environments. Citation Format: Amanda L. Whiting, Joe J. Mitala, Kathryn M. Headley, Joe Reilly, Bethanie L. Morrison, Kevin A. Murray, Federico Bernal. Covalent capture of protein binding partners using an azide-tagged, photo-reactive stapled alpha helical p53 peptide. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3234. doi:10.1158/1538-7445.AM2014-3234

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::86d3cde1af4eb34117427b20cbf88587Test
https://doi.org/10.1158/1538-7445.am2014-3234Test

المؤلفون: Amanda L. Whiting, Bethanie L. Morrison, Federico Bernal

المصدر: Cancer Research. 74:4071-4071

مصطلحات موضوعية: Cancer Research, Transactivation, Stress fiber, Oncology, biology, Endosome, Cancer cell, Mutant, biology.protein, Focal adhesion assembly, Cell migration, Platelet-derived growth factor receptor, Cell biology

الوصف: The mutation of p53 is critical for the increased metastatic potential of cancer cells. As such, it is essential not only to understand the mechanisms underlying the functional implications of p53 mutation, but also to identify ways to regulate their impact on cancer cell physiology. I have found that a hydrocarbon-stapled p53 transactivation domain peptide (SAH-p53) designed to disrupt the interaction of p53 with its negative regulators HDM2 and HDMX retains its function in cells harboring a mutant TP53. A common gain-of-function activity of the mutant p53 protein (mtp53) is the enhancement of cell migration and invasion; however, I found that SAH-p53 has the ability to inhibit the migration and invasion of HS578T and MDA-MB-231 breast cancer cells (mtp53), while exhibiting no change in proliferative ability at the same dose. This effect was accompanied by sustained alterations in actin stress fiber dynamics and inhibition of focal adhesion assembly and signaling. Furthermore, treatment of MDA-MB-231 cells with various growth factors (EGF, TGF-β, PDGF, HGF) was unable to rescue the migratory ability of cells in the presence of SAH-p53. The combination of SAH-p53 and TGF-β resulted in a decrease in the total expression levels of both EGFR and FAK. Moreover, it was shown that the combined treatment of SAH-p53 and TGF-β enhanced the localization of EGFR to early endosomes and eventually to lysosomes, prompting the use of fluorophore-capped SAH-p53 to track the localization of the peptide in relation to EGFR. It was determined that over time SAH-53 localizes to vesicles containing EGFR, but not other RTKs (ie. PDGFRβ). SAH-p53 was able to retain its ability to inhibit migration in the absence of functional HDMX/HDM2 or mutant p53, suggesting the possibility of an alternative target. We are currently using a photoactivatable and capped SAH-p53 that can cross-link to a target protein in the presence of UV in order to determine the identity of the target of SAH-p53 responsible for the anti-migratory effects seen in mutant p53- or p53-null cells. These data show that the use of a stapled peptide provides a novel avenue to study the molecular interactions that drive metastatic behavior in cancer cells. Citation Format: Bethanie L. Morrison, Amanda L. Whiting, Federico Bernal. SAH-p53-mediated inhibition of cell migration via alteration of actin dynamics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4071. doi:10.1158/1538-7445.AM2014-4071

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::99c5f4f9ac6456eac7c54489d0798a50Test
https://doi.org/10.1158/1538-7445.am2014-4071Test

المؤلفون: Brian J. Smith, Bethanie L. Morrison, Trevor Glaros, Luke H. Stockwin, Dianne L. Newton, Michael E. Mullendore

المصدر: Cancer Chemotherapy and Pharmacology. 70:763-764

مصطلحات موضوعية: Pharmacology, Cancer Research, Oncology, business.industry, Pharmacology toxicology, Survivin, Cancer research, Medicine, Pharmacology (medical), Damage response, Toxicology, business

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::cfdf62b97e6640602c177287c7486570Test
https://doi.org/10.1007/s00280-012-1971-2Test

المؤلفون: Federico Bernal, Bethanie L. Morrison, Elisabeth A. Russell, Joseph J. Mitala

المصدر: Cancer Research. 73:839-839

مصطلحات موضوعية: Cancer Research, Programmed cell death, Autophagy, Cell, Biology, Cell biology, Cell membrane, medicine.anatomical_structure, Oncology, Apoptosis, Cytoplasm, Immunology, Cancer cell, medicine, Intracellular

الوصف: Resistance to chemotherapeutic agents is often a rate-limiting event in the treatment of metastatic cancers. Mutations of TP53 or dysfunctions in the p53 pathway are known to play primary roles in the resistance to apoptosis-inducing agents. A stapled alpha-helical p53 peptide (SAH-p53) capable of disrupting the p53-HDM2 and p53-HDMX complexes and restoring p53 function has been found to induce a death response in breast, melanoma and choriocarcinoma cancer cells independent of p53 mutational status. We have recently discovered that the mechanism of death induced by SAH-p53 does not exhibit all of the characteristics of classical apoptosis, necrosis or autophagy independently. The binding of SAH-p53 to HDM2, HDMX, or a yet unidentified target results in a mechanism that leads to the intracellular aggregation of proteins and lipids that are extruded from the cell, resulting in catastrophic damage to the integrity of the cell membrane and culminating in cell death. This type of cell death involves the loss of cytoplasm, generation of phase-clear vacuole-like structures, apparent degradation of cellular membrane components, and the significant release of uniform non-vesicular cellular components. The cellular debris, while yet undefined, displays orange staining with acridine orange as well as accumulation of MitoTracker, suggesting that the debris contains hydrophobic regions. Thus far it has been established that there is a lack of any membrane blebbing, no chromatin condensation, no apoptosis signaling events or typical necrotic bubble formation after treatment with SAH-p53 in cells harboring wild-type p53, mutant p53, or null p53, suggesting an alternative mechanism of cell death. Data suggest the manifestation of early autophagy within 6 hours of SAH-p53 treatment as evidenced by LC3 accumulation, yet this phenotype is lost within 24 hours post-treatment. In addition, the pretreatment of the cells with chloroquine does not inhibit the release of debris and changes in morphology characteristic of SAH-p53 treatment, suggesting that the mechanism of death is independent of classical autophagy. We have begun to uncover the precise mechanism of cell death through the identification and validation of the transcriptional program regulated by SAH-p53 in both wild-type and mutant p53-containing cells via microarray analysis. The complete understanding and characterization of this novel mechanism may be exploited to overcome resistance to apoptosis and thus lead to a more effective way of treating chemotherapy-resistant metastatic cancers. Citation Format: Bethanie L. Morrison, Elisabeth Russell, Joseph Mitala, Federico Bernal. An uncharacterized mechanism of cell death independent of p53 mutation status. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 839. doi:10.1158/1538-7445.AM2013-839

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::36e2967ca2fd403cd7b8c764c207549fTest
https://doi.org/10.1158/1538-7445.am2013-839Test

المؤلفون: Bethanie L. Morrison, Federico Bernal

المصدر: Cancer Research. 72:4329-4329

مصطلحات موضوعية: Oncology, Cancer Research, medicine.medical_specialty, biology, medicine.medical_treatment, CD44, Cancer, medicine.disease, Stem cell marker, Targeted therapy, Breast cancer, Internal medicine, Cancer cell, biology.protein, medicine, Cancer research, Cell adhesion, Triple-negative breast cancer

الوصف: The mutation of p53 generally occurs during the later stages of cancer and leads to the increased metastatic potential of triple negative (ER_/PR_/Her2_) breast cancer. The low survival rates of advanced metastatic cancer make it critical not only to understand the mechanisms underlying the functional implications of p53 mutation, but also to identify ways to regulate its impact in cancer cell physiology. We have previously described the design, synthesis, and evaluation of a stabilized α-helix of p53 (SAH-p53) capable of inhibiting the p53-HDM2 and p53-HDMX interactions in order to restore the functionality of p53. Despite having low expression of both HDM2 and HDMX and mutations in p53, SAH-p53 treatment of the triple negative breast cancer cell lines MDA-MB-231 and HS578T results in a modest decrease in proliferative activity. While mutant p53 is known to promote metastatic behavior of breast cancer cells, the propensity of triple negative breast cancer cells to migrate and invade through Matrigel in the presence of EGF was decreased by treatment with SAH-p53. The anti-proliferative and anti-migratory effects of SAH-p53 treatment are independent of each other, yet both are accompanied by an alteration in cell morphology that includes the loss of cell membrane integrity and actin cytoskeletal organization. The surface level of β1 integrin, which is typically kept constant by mutant p53, is decreased in the presence of SAH-p53, pointing to possible alterations in integrin recycling. SAH-p53 also causes a change in the localization of the adhesion protein and stem cell marker CD44. Moreover, it has been suggested that mutant p53 enhances invasive properties by interacting with and inhibiting isoforms of p63, which have been implicated in the regulation of cell adhesion. Treatment of the triple negative breast cancer cells with a stabilized α-helix of p63 showed a significant decrease in cell viability as well as an enhanced inhibition of migration when treated simultaneously with SAH-p53. Targeted therapy for triple negative breast cancer is currently very limited and patients must rely on standard chemotherapeutic regimens. These data show that the use of SAH-p53 and SAH-p63 peptides provides a novel avenue to study the molecular interactions that drive metastatic behavior originated by p53 gain-of-function mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4329. doi:1538-7445.AM2012-4329

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::c480202afa9dccf1ffe265b8bd40ad29Test
https://doi.org/10.1158/1538-7445.am2012-4329Test

المؤلفون: Suzanne Borgel, Bingnan Han, Michael E. Mullendore, Melinda G. Hollingshead, Bingliang Fang, Luke H. Stockwin, Bethanie L. Morrison, Howard Stotler, Dianne L. Newton

المصدر: Cancer Research. 72:2810-2810

مصطلحات موضوعية: Cancer Research, biology, Microarray analysis techniques, p38 mitogen-activated protein kinases, Oncology, Cell culture, Apoptosis, Tyrosine kinase 2, Immunology, biology.protein, Cancer research, Phosphorylation, STAT3, GADD45A

الوصف: Several indole-3-carbinol analogs are under preclinical evaluation at the National Cancer Institute. One analog, NSC743380 (1-[(3-chlorophenyl)-methyl]-1H-indole-3-carbinol), is selectively toxic to a subset of NCI 60 cell lines in vitro and has undergone extensive in vivo testing. In vivo, NSC743380 produced complete regressions in A498 renal xenograft models at doses as low as 45 mg/kg when administered intraperitoneally. Additional studies demonstrated that NSC743380 is orally bioavailable. To extend knowledge regarding the mechanism of action, two pairs of resistant and sensitive cell lines [A498/ACHN (renal) and NCI-H226/A549 (NSCLC), sensitive/resistant, respectively] were used. Results showed that 5-10 min following NSC743380 treatment, phosphorylation of p38 and JNK were enhanced in sensitive but not resistant lines. Two hrs of treatment resulted in an inhibition of transcription and translation (45% and 75% inhibition, respectively) and by 4 hrs NSC743380 induced caspase-dependent apoptosis with loss of c-FLIP. Pathway-specific inhibitors were then used to gain mechanistic insight. Diverse antioxidants and NSAIDs, inhibitors of JNK, RAS/RAF, PPAR, lipid 2nd messenger signaling, transcription and actin polymerization were shown to completely inhibit NSC743380 activity. Microarray analysis of three sensitive cell lines using Affymetrix U133 Plus 2 chipset identified several trends in the transcriptome notably the upregulation of diverse immediate-early genes (IEGs) including; transcriptional regulators [EGR1, FOS/FOSB, HES1, MAFF and SOX9], secreted factors [HBEGF, IL-8 and GDF-15] and several general IEGs [ARC, ERRFI1, GADD45A/B, GEM and IER2]. A second noteworthy trend involved increased expression of the IL-6 family members, IL-6, IL-11 and LIF. These data led to an exploration as to whether enhanced JAK/STAT signaling was responsible for the above effects. Short time course lysates from 3 sensitive (A498, CAKI-1 and NCI-H226) cell lines and 1 resistant (A549) cell line showed that in sensitive lines only, NSC743380 rapidly (5 minutes) enhanced phosphorylation of the JAK family member Tyk2 at Y1054/55 and STAT3 at Y705. Additionally several phosphostate changes were observed in a subset of sensitive cell lines such as JAK1 phosphorylation in A498 cells and transient increase in pSTAT1 in A498 and CAKI-1 cell lines. Furthermore, two inhibitors of JAK/STAT signaling, AG490 and resveratrol, completely inhibited NSC743380 activity and p38/JNK phosphorylation. Subsequent siRNA knockdown experiments showed that Tyk2 siRNA inhibited NSC743380 activity whereas STAT3 and JAK1 siRNA along with scrambled siRNA had no effect. These data suggest that signaling through the JAK family member Tyk2 contributes towards NSC743380 activity and the data are being evaluated to determine how best to move forward. Funded by NCI Contract No. HHSN261200800001E. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2810. doi:1538-7445.AM2012-2810

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::03f6e14bed6e9b72f8a68075d974e523Test
https://doi.org/10.1158/1538-7445.am2012-2810Test