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Article ; Online: A bioluminescent and homogeneous assay for monitoring GPCR-mediated cAMP modulation and PDE activity.

Mikheil, Dareen / Larsen, Matthew A / Hsiao, Kevin / Murray, Nathan H / Ugo, Tim / Wang, Hui / Goueli, Said A

2024 Volume 14, Issue 1, Page(s) 4440

Abstract: 3',5'-Cyclic adenosine monophosphate (cAMP), the first identified second messenger, is implicated in diverse cellular processes involving cellular metabolism, cell proliferation and differentiation, apoptosis, and gene expression. cAMP is synthesized by ... ...

Abstract	3',5'-Cyclic adenosine monophosphate (cAMP), the first identified second messenger, is implicated in diverse cellular processes involving cellular metabolism, cell proliferation and differentiation, apoptosis, and gene expression. cAMP is synthesized by adenylyl cyclase (AC), which converts ATP to cAMP upon activation of G
MeSH term(s)	Cyclic AMP/metabolism ; Signal Transduction ; Adenylyl Cyclases/metabolism ; Cell Differentiation ; Drug Discovery
Chemical Substances	Cyclic AMP (E0399OZS9N) ; Adenylyl Cyclases (EC 4.6.1.1)
Language	English
Publishing date	2024-02-23
Publishing country	England
Document type	Journal Article
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-024-55038-0
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Article: Notch Signaling Suppresses Melanoma Tumor Development in BRAF/Pten Mice.

Mikheil, Dareen / Prabhakar, Kirthana / Ng, Tun Lee / Teertam, Sireesh / Longley, B Jack / Newton, Michael A / Setaluri, Vijayasaradhi

Cancers

2023 Volume 15, Issue 2

Abstract: Both oncogenic and tumor suppressor roles have been assigned to Notch signaling in melanoma. In clinical trials, Notch inhibitors proved to be ineffective for melanoma treatment. Notch signaling has also been implicated in melanoma transdifferentiation, ... ...

Abstract	Both oncogenic and tumor suppressor roles have been assigned to Notch signaling in melanoma. In clinical trials, Notch inhibitors proved to be ineffective for melanoma treatment. Notch signaling has also been implicated in melanoma transdifferentiation, a prognostic feature in primary melanoma. In this study, we investigated the role of Notch signaling in melanoma tumor development and growth using the genetic model of mouse melanoma by crossing BRAFCA/+/Pten+/+/Tyr-CreER+ (B) and BRAFCA/+/Pten-/-/Tyr-CreER + (BP) mice with Notch1 or Notch2 floxed allele mice. The topical application of tamoxifen induced tumors in BP mice but not in B mice with or without the deletion of either Notch1 or Notch2. These data show that the loss of either Notch1 nor Notch2 can substitute the tumor suppressor function of Pten in BRAFV600E-induced melanomagenesis. However, in Pten-null background, the loss of either Notch1 or Notch2 appeared to accelerate BRAFV600E-induced tumor development, suggesting a tumor suppressor role for Notch1 and Notch2 in BRAFV600E/Pten-null driven melanomagenesis. Quantitative immunochemical analysis of a human cutaneous melanoma tissue microarray that consists of >100 primary tumors with complete clinical history showed a weak to moderate correlation between NOTCH protein levels and clinical and pathological parameters. Our data show that Notch signaling is involved during melanomagenesis and suggest that the identification of genes and signaling pathways downstream of Notch could help devise strategies for melanoma prevention.
Language	English
Publishing date	2023-01-14
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2527080-1
ISSN	2072-6694
ISSN	2072-6694
DOI	10.3390/cancers15020519
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Article ; Online: A Homogeneous Bioluminescent System to Monitor Cyclic Guanosine Monophosphate.

Murray, Nathan H / Larsen, Matthew A / Hsiao, Kevin / Mikheil, Dareen / Vidugiris, Gediminas / Wang, Hui / Goueli, Said A

ACS pharmacology & translational science

2023 Volume 6, Issue 12, Page(s) 1851–1858

Abstract: Cyclic guanosine monophosphate (cGMP) is a critical second messenger involved in various physiological processes, such as vasodilation and phototransduction. Its synthesis is stimulated by nitric oxide and natriuretic hormones, while its breakdown is ... ...

Abstract	Cyclic guanosine monophosphate (cGMP) is a critical second messenger involved in various physiological processes, such as vasodilation and phototransduction. Its synthesis is stimulated by nitric oxide and natriuretic hormones, while its breakdown is mediated through highly regulated phosphodiesterase activities. cGMP metabolism has been targeted for the treatment of several diseases, including erectile dysfunction, hypertension, and heart failure. As more drugs are being sought, it will be critical to develop assays that accurately determine cGMP levels. Here, we present cGMP Lumit, a sensitive and specific bioluminescent assay to detect cGMP. We demonstrate the utility of the detection system in enzyme assays, cell-based assays, and high-throughput screening formats. It is anticipated that this assay will be of significant value to aid in further understanding the role of cGMP in physiology and support further drug discovery efforts toward the treatment of human disease.
Language	English
Publishing date	2023-11-21
Publishing country	United States
Document type	Journal Article
ISSN	2575-9108
ISSN (online)	2575-9108
DOI	10.1021/acsptsci.3c00176
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Article ; Online: Monitoring PROTAC interactions in biochemical assays using Lumit immunoassays.

Crummy, Ellen K / Caine, Elizabeth A / Mikheil, Dareen / Corona, Cesear / Riching, Kristin M / Hosfield, Chris / Urh, Marjeta

Methods in enzymology

2022 Volume 681, Page(s) 81–113

Abstract: The discovery of new PROTAC molecules is dependent on robust and high-throughput assays to measure PROTAC-protein interactions and ternary complex formation. Here we present the optimization and execution of Lumit Immunoassays to measure PROTAC binding ... ...

Abstract	The discovery of new PROTAC molecules is dependent on robust and high-throughput assays to measure PROTAC-protein interactions and ternary complex formation. Here we present the optimization and execution of Lumit Immunoassays to measure PROTAC binding and ternary complex formation in a biochemical format. We demonstrate how Lumit can be used to rank order affinities of small molecules and PROTACs to BRD4(BD1, BD2) and how to measure PROTAC-mediated ternary complex formation of BRD4(BD1, BD2) and E3 Ligase VHL. Results from both biochemical assays correlate with live and lytic cell assays, indicating that Lumit Immunoassays can be used as a high-throughput compatible screening methodology to test new small molecules.
MeSH term(s)	Nuclear Proteins/metabolism ; Transcription Factors/metabolism ; Small Molecule Libraries/chemistry ; Ubiquitin-Protein Ligases/metabolism ; Immunoassay ; Proteolysis
Chemical Substances	Nuclear Proteins ; Transcription Factors ; Small Molecule Libraries ; Ubiquitin-Protein Ligases (EC 2.3.2.27)
Language	English
Publishing date	2022-12-12
Publishing country	United States
Document type	Journal Article
ISSN	1557-7988
ISSN (online)	1557-7988
DOI	10.1016/bs.mie.2022.08.055
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Article ; Online: Notch signaling activation induces cell death in MAPKi-resistant melanoma cells.

Mikheil, Dareen M / Prabhakar, Kirthana / Arshad, Ayyan / Rodriguez, Carlos I / Newton, Michael A / Setaluri, Vijayasaradhi

Pigment cell & melanoma research

2019 Volume 32, Issue 4, Page(s) 528–539

Abstract: The role of Notch signaling in melanoma drug resistance is not well understood. In this study, we show that although NOTCH proteins are upregulated in metastatic melanoma cell lines, Notch signaling inhibition had no effect on cell survival, growth, ... ...

Abstract	The role of Notch signaling in melanoma drug resistance is not well understood. In this study, we show that although NOTCH proteins are upregulated in metastatic melanoma cell lines, Notch signaling inhibition had no effect on cell survival, growth, migration or the sensitivity of BRAFV600E-melanoma cells to MAPK inhibition (MAPKi). We found that NOTCH1 is downregulated in melanoma cell lines with intrinsic and acquired resistance to MAPKi. Forced expression of NICD1, the active form of Notch1, caused apoptosis of the NOTCH
MeSH term(s)	Apoptosis/drug effects ; Cell Death/drug effects ; Cell Line, Tumor ; Down-Regulation/drug effects ; Drug Resistance, Neoplasm/drug effects ; Endothelin-1/genetics ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Ligands ; Melanoma/enzymology ; Melanoma/pathology ; Mitogen-Activated Protein Kinases/antagonists & inhibitors ; Mitogen-Activated Protein Kinases/metabolism ; Mutation/genetics ; Protein Kinase Inhibitors/pharmacology ; Proto-Oncogene Proteins B-raf/genetics ; Proto-Oncogene Proteins c-jun/metabolism ; Receptors, Notch/metabolism ; Signal Transduction/drug effects ; Transcription Factor HES-1/metabolism ; Transcriptome/genetics ; Up-Regulation/drug effects ; Up-Regulation/genetics
Chemical Substances	Endothelin-1 ; Ligands ; Protein Kinase Inhibitors ; Proto-Oncogene Proteins c-jun ; Receptors, Notch ; Transcription Factor HES-1 ; HES1 protein, human (149348-15-2) ; BRAF protein, human (EC 2.7.11.1) ; Proto-Oncogene Proteins B-raf (EC 2.7.11.1) ; Mitogen-Activated Protein Kinases (EC 2.7.11.24)
Language	English
Publishing date	2019-02-03
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2409570-9
ISSN	1755-148X ; 1600-0749 ; 0893-5785 ; 1755-1471
ISSN (online)	1755-148X ; 1600-0749
ISSN	0893-5785 ; 1755-1471
DOI	10.1111/pcmr.12764
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Article ; Online: Melanoma Progression Inhibits Pluripotency and Differentiation of Melanoma-Derived iPSCs Produces Cells with Neural-like Mixed Dysplastic Phenotype.

Castro-Pérez, Edgardo / Rodríguez, Carlos I / Mikheil, Dareen / Siddique, Shakir / McCarthy, Alexandra / Newton, Michael A / Setaluri, Vijayasaradhi

Stem cell reports

2019 Volume 13, Issue 1, Page(s) 177–192

Abstract: Melanomas are known to exhibit phenotypic plasticity. However, the role cellular plasticity plays in melanoma tumor progression and drug resistance is not fully understood. Here, we used reprogramming of melanocytes and melanoma cells to induced ... ...

Abstract	Melanomas are known to exhibit phenotypic plasticity. However, the role cellular plasticity plays in melanoma tumor progression and drug resistance is not fully understood. Here, we used reprogramming of melanocytes and melanoma cells to induced pluripotent stem cell (iPSCs) to investigate the relationship between cellular plasticity and melanoma progression and mitogen-activated protein kinase (MAPK) inhibitor resistance. We found that melanocyte reprogramming is prevented by the expression of oncogenic BRAF, and in melanoma cells harboring oncogenic BRAF and sensitive to MAPK inhibitors, reprogramming can be restored by inhibition of the activated oncogenic pathway. Our data also suggest that melanoma tumor progression acts as a barrier to reprogramming. Under conditions that promote melanocytic differentiation of fibroblast- and melanocyte-derived iPSCs, melanoma-derived iPSCs exhibited neural cell-like dysplasia and increased MAPK inhibitor resistance. These data suggest that iPSC-like reprogramming and drug resistance of differentiated cells can serve as a model to understand melanoma cell plasticity-dependent mechanisms in recurrence of aggressive drug-resistant melanoma.
MeSH term(s)	Biomarkers, Tumor ; Cell Differentiation ; Cell Line, Tumor ; Cell Plasticity ; Cellular Reprogramming/drug effects ; Cellular Reprogramming/genetics ; Disease Progression ; Drug Resistance, Neoplasm/genetics ; Gene Expression ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Induced Pluripotent Stem Cells/pathology ; Melanocytes/metabolism ; Melanoma/genetics ; Melanoma/metabolism ; Melanoma/pathology ; Mitogen-Activated Protein Kinases/antagonists & inhibitors ; Mutation ; Neoplastic Stem Cells/metabolism ; Neoplastic Stem Cells/pathology ; Phenotype ; Protein Kinase Inhibitors/pharmacology
Chemical Substances	Biomarkers, Tumor ; Protein Kinase Inhibitors ; Mitogen-Activated Protein Kinases (EC 2.7.11.24)
Language	English
Publishing date	2019-06-20
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2720528-9
ISSN	2213-6711 ; 2213-6711
ISSN (online)	2213-6711
ISSN	2213-6711
DOI	10.1016/j.stemcr.2019.05.018
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Article ; Online: Role of miR-214 in regulation of β-catenin and the malignant phenotype of melanoma.

Prabhakar, Kirthana / Rodrίguez, Carlos I / Jayanthy, Ashika S / Mikheil, Dareen M / Bhasker, Aishwarya Iyer / Perera, Ranjan J / Setaluri, Vijayasaradhi

Molecular carcinogenesis

2019 Volume 58, Issue 11, Page(s) 1974–1984

Abstract: Wnt/β-catenin signaling plays an important role in melanocyte biology, especially in the early stages of melanocyte transformation and melanomagenesis. β-catenin, encoded by the gene CTNNB1, is an intracellular signal transducer of Wnt signaling and ... ...

Abstract	Wnt/β-catenin signaling plays an important role in melanocyte biology, especially in the early stages of melanocyte transformation and melanomagenesis. β-catenin, encoded by the gene CTNNB1, is an intracellular signal transducer of Wnt signaling and activates transcription of genes important for cell proliferation and survival. Wnt/β-catenin signaling is frequently activated in melanoma through oncogenic mutations of β-catenin and elevated β-catenin levels are positively correlated with melanoma aggressiveness. Molecular mechanisms that regulate β-catenin expression in melanoma are not fully understood. MicroRNA-214 is known to function as a tumor suppressor by targeting β-catenin in several types of cancer cells. Here, we investigated the regulation of β-catenin by miR-214 and its role in melanoma. We show that β-catenin mRNA levels are negatively correlated with miR-214 in melanoma. However, overexpression of miR-214 paradoxically increased β-catenin protein levels and promoted malignant properties of melanoma cells including resistance to mitogen-activated protein kinase inhibitors (MAPKi). RNA-seq analysis revealed that melanoma cells predominantly express a β-catenin mRNA isoform lacking miR-214 target site. Using matched miRNA and mRNA-seq and bioinformatics analysis, we identified novel miR-214 targets, ankyrin repeat domain 6 (ANKRD6) and C-terminal binding protein 1 (CTBP1), that are involved in negative regulation of Wnt signaling. Overexpression of miR-214 or knockdown of the novel miR-214 targets, ANKRD6 or CTBP1, increased melanoma cell proliferation, migration, and decreased sensitivity to MAPKi. Our data suggest that in melanoma cells β-catenin is not regulated by miR-214 and the functions of miR-214 in melanoma are mediated partly by regulating proteins involved in attenuation of Wnt/β-catenin signaling.
MeSH term(s)	Alcohol Oxidoreductases/genetics ; Cell Line, Tumor ; Cell Movement/genetics ; Cell Proliferation/genetics ; Cytoskeletal Proteins/genetics ; DNA-Binding Proteins/genetics ; Gene Expression Regulation, Neoplastic/genetics ; Humans ; Melanoma/genetics ; Melanoma/pathology ; MicroRNAs/genetics ; RNA, Messenger/genetics ; Wnt Signaling Pathway/genetics ; beta Catenin/genetics
Chemical Substances	ANKRD6 protein, human ; CTNNB1 protein, human ; Cytoskeletal Proteins ; DNA-Binding Proteins ; MIRN214 microRNA, human ; MicroRNAs ; RNA, Messenger ; beta Catenin ; Alcohol Oxidoreductases (EC 1.1.-) ; C-terminal binding protein (EC 1.1.1.-)
Language	English
Publishing date	2019-07-24
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	1004029-8
ISSN	1098-2744 ; 0899-1987
ISSN (online)	1098-2744
ISSN	0899-1987
DOI	10.1002/mc.23089
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Article ; Online: Role of the flagellar basal-body protein, FlgC, in the binding of Salmonella enterica serovar Enteritidis to host cells.

Shippy, Daniel C / Eakley, Nicholas M / Mikheil, Dareen M / Fadl, Amin A

Current microbiology

2014 Volume 68, Issue 5, Page(s) 621–628

Abstract: Salmonella enterica serovar Enteritidis (SE) infection in humans is often associated with the consumption of contaminated poultry products. Binding of the bacterium to the intestinal mucosa is a major pathogenic mechanism of Salmonella in poultry. ... ...

Abstract	Salmonella enterica serovar Enteritidis (SE) infection in humans is often associated with the consumption of contaminated poultry products. Binding of the bacterium to the intestinal mucosa is a major pathogenic mechanism of Salmonella in poultry. Transposon mutagenesis identified flgC as a potential binding mutant of SE. Therefore, we hypothesize FlgC which plays a significant role in the binding ability of SE to the intestinal mucosa of poultry. To test our hypothesis, we created a mutant of SE in which flgC was deleted. We then tested the in vitro and in vivo binding ability of ∆flgC when compared to the wild-type SE strain. Our data showed a significant decrease in the binding ability of ∆flgC to intestinal epithelial cells as well as in the small intestine and cecum of poultry. Furthermore, the decrease in binding correlated to a defect in invasion as shown by a cell culture model using intestinal epithelial cells and bacterial recovery from the livers and spleens of chickens. Overall, these studies indicate FlgC is a major factor in the binding ability of Salmonella to the intestinal mucosa of poultry.
MeSH term(s)	Animals ; Bacterial Adhesion ; Bacterial Load ; Bacterial Proteins/genetics ; Bacterial Proteins/metabolism ; Basal Bodies/physiology ; Cecum/microbiology ; Chickens ; Epithelial Cells/microbiology ; Flagella/genetics ; Flagella/physiology ; Gene Deletion ; Humans ; Intestine, Small/microbiology ; Liver/microbiology ; Salmonella enteritidis/genetics ; Salmonella enteritidis/physiology ; Spleen/microbiology ; Virulence Factors/genetics ; Virulence Factors/metabolism
Chemical Substances	Bacterial Proteins ; Virulence Factors
Language	English
Publishing date	2014-01-14
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	134238-1
ISSN	1432-0991 ; 0343-8651
ISSN (online)	1432-0991
ISSN	0343-8651
DOI	10.1007/s00284-014-0521-z
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Article: Role of StdA in adhesion of Salmonella enterica serovar Enteritidis phage type 8 to host intestinal epithelial cells.

Shippy, Daniel C / Eakley, Nicholas M / Mikheil, Dareen M / Fadl, Amin A

Gut pathogens

2013 Volume 5, Issue 1, Page(s) 43

Abstract: Background: Salmonella is often implicated in foodborne outbreaks, and is a major public health concern in the United States and throughout the world. Salmonella enterica serovar Enteritidis (SE) infection in humans is often associated with the ... ...

Abstract	Background: Salmonella is often implicated in foodborne outbreaks, and is a major public health concern in the United States and throughout the world. Salmonella enterica serovar Enteritidis (SE) infection in humans is often associated with the consumption of contaminated poultry products. Adhesion to epithelial cells in the intestinal mucosa is a major pathogenic mechanism of Salmonella in poultry. Transposon mutagenesis identified stdA as a potential adhesion mutant of SE. Therefore, we hypothesize StdA plays a significant role in adhesion of SE to the intestinal mucosa of poultry. Methods and results: To test our hypothesis, we created a mutant of SE in which stdA was deleted. Growth and motility were assayed along with the in vitro and in vivo adhesion ability of the ∆stdA when compared to the wild-type SE strain. Our data showed a significant decrease in motility in ∆stdA when compared to the wild-type and complemented strain. A decrease in adhesion to intestinal epithelial cells as well as in the small intestine and cecum of poultry was observed in ∆stdA. Furthermore, the lack of adhesion correlated to a defect in invasion as shown by a cell culture model using intestinal epithelial cells and bacterial recovery from the livers and spleens of chickens. Conclusions: These studies suggest StdA is a major contributor to the adhesion of Salmonella to the intestinal mucosa of poultry.
Language	English
Publishing date	2013-12-24
Publishing country	England
Document type	Journal Article
ZDB-ID	2478277-4
ISSN	1757-4749
ISSN	1757-4749
DOI	10.1186/1757-4749-5-43
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Article: Role of the Flagellar Basal-Body Protein, FlgC, in the Binding of Salmonella enterica Serovar Enteritidis to Host Cells

Shippy, Daniel C / Eakley, Nicholas M / Mikheil, Dareen M / Fadl, Amin A

Current microbiology. 2014 May, v. 68, no. 5

2014

Abstract	Salmonella enterica serovar Enteritidis (SE) infection in humans is often associated with the consumption of contaminated poultry products. Binding of the bacterium to the intestinal mucosa is a major pathogenic mechanism of Salmonella in poultry. Transposon mutagenesis identified flgC as a potential binding mutant of SE. Therefore, we hypothesize FlgC which plays a significant role in the binding ability of SE to the intestinal mucosa of poultry. To test our hypothesis, we created a mutant of SE in which flgC was deleted. We then tested the in vitro and in vivo binding ability of ∆flgC when compared to the wild-type SE strain. Our data showed a significant decrease in the binding ability of ∆flgC to intestinal epithelial cells as well as in the small intestine and cecum of poultry. Furthermore, the decrease in binding correlated to a defect in invasion as shown by a cell culture model using intestinal epithelial cells and bacterial recovery from the livers and spleens of chickens. Overall, these studies indicate FlgC is a major factor in the binding ability of Salmonella to the intestinal mucosa of poultry.
Keywords	Salmonella enteritidis ; bacteria ; cecum ; cell culture ; chickens ; epithelial cells ; humans ; intestinal mucosa ; models ; mutagenesis ; mutants ; poultry products ; small intestine ; transposons
Language	English
Dates of publication	2014-05
Size	p. 621-628.
Publishing place	Springer-Verlag
Document type	Article
ZDB-ID	134238-1
ISSN	1432-0991 ; 0343-8651
ISSN (online)	1432-0991
ISSN	0343-8651
DOI	10.1007/s00284-014-0521-z
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