Cellular arrays for large‐scale analysis of transcription factor activity

Bellis, Abigail D.; Peňalver-Bernabé, Beatriz; Weiss, Michael; Yarrington, Michael E; Barbolina, Maria V.; Pannier, Angela K.; Jeruss, Jacqueline S.; Broadbelt, Linda J.; Shea, Lonnie D.

doi:10.1002/bit.22916

Cited by 20 publications

(27 citation statements)

References 37 publications

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“…To further examine the impact of CDKi on EMT related events we used a novel transcription factor (TF) reporter cell array 21,22,31 and found that CDK2i treatment of TNBC cells led to an early increase in activity of b-catenin, Smad3, Snail, Sp1, Twist, YY1, and Zeb1 (Fig. 4).…”

Section: Discussionmentioning

confidence: 99%

“…21,22 After treating MDA-MD-231 cells with control or CDK2i, activities of 11 TFs associated with oncogenesis were measured at several time points. Factors selected for study were based on association with proliferation, apoptosis, and metastasis ( Table 2).…”

Section: Characterization Of Signaling Changes In Mda-mb-231 Cells Afmentioning

confidence: 99%

“…To form an array, MDA-MB-231 cells were transduced with lentivirus encoding TA-FLuc or one of the TF reporter genes, as previously described, 21,22,31 (25,000 PP/cell), by centrifugation, resuspension in Matrigel (BD Biosciences), and seeding into wells of a black 384-well plate (Greiner BioSciences, Monroe, NC). Cells were treated with 0.1% DMSO control or 240 nM CDK2i at indicated time points.…”

Section: Formation Of Transduced Cell Arraysmentioning

confidence: 99%

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Inhibition of CDK-mediated phosphorylation of Smad3 results in decreased oncogenesis in triple negative breast cancer cells

Tarasewicz¹,

Rivas²,

Hamdan³

et al. 2014

Cell Cycle

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Keywords: CDK, cyclin, paclitaxel, Smad3, triple negative breast cancer Abbreviations: BCSC, breast cancer stem cells; CDK, cyclin dependent kinase; CDKi, cyclin dependent kinase inhibitor; CK, cytokeratin; EGFR, epidermal growth factor receptor; EMT, epithelial-mesenchymal transition; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; Pin1, peptidyL-prolyl cis-trans isomerase NIMA-interacting 1; PR, progesterone receptor; TNBC, triple negative breast cancerBreast cancer onset and disease progression have been linked to members of the TGFb superfamily and their downstream signaling components, the Smads. Alterations in Smad3 signaling are associated with the dichotomous role of TGFb in malignancy, mediating both tumor suppressant and pro-metastatic behaviors. Overexpression of cell cycle regulators, cyclins D and E, renders cyclin-dependent kinases (CDKs) 4/2 hyperactive. Noncanonical phosphorylation of Smad3 by CDK4/2 inhibits tumor suppressant actions of Smad3. We hypothesized that CDK inhibition (CDKi) would restore Smad3 action and help promote cancer cell regression. Treatment of triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, MDA-MB-436, Hs578T) with CDK2i or CDK4i resulted in increased Smad3 activity and decreased cell migration. Transfection with a 5M Smad3 construct containing inhibitory mutations in 5 CDK phosphorylation sites also resulted in decreased TNBC cell migration and invasion. MDA-MB-231 cells treated with CDK2i or CDK4i resulted in decreased Smad3 protein phosphorylation at the CDK phosphorylation T179 site, decreased MMP2 and c-myc expression, and increased p15 and p21 expression. Using a novel transfected cell array, we found that CDK2i treatment decreased activity of the epithelial-to-mesenchymal transition related transcription factors Snail and Twist. In vivo studies in an MDA-MB-231 tumor model showed that individual and combination treatment with paclitaxel and CDK2i resulted in decreased tumor volume and Ki67 staining. Collectively, these data support further investigation of targeted CDK inhibitors as a promising therapeutic strategy for TNBC, a breast cancer subtype with limited treatment options.

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Section: Discussionmentioning

confidence: 99%

Section: Characterization Of Signaling Changes In Mda-mb-231 Cells Afmentioning

confidence: 99%

Section: Formation Of Transduced Cell Arraysmentioning

confidence: 99%

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Inhibition of CDK-mediated phosphorylation of Smad3 results in decreased oncogenesis in triple negative breast cancer cells

Tarasewicz¹,

Rivas²,

Hamdan³

et al. 2014

Cell Cycle

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“…Recently, we developed and validated a living cell array for the large-scale quantification of dynamic TF activities. This assay directly quantifies the activity of TFs, rather than abundance of mRNA or protein, and can be applied repeatedly to quantify TF activity through lineage commitment and differentiation (Bellis et al, 2011; Weiss et al, 2010). …”

Section: Introductionmentioning

confidence: 99%

Dynamic transcription factor activity profiles reveal key regulatory interactions during megakaryocytic and erythroid differentiation

Duncan

Shin²,

Wu³

et al. 2014

Biotech & Bioengineering

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The directed differentiation toward erythroid (E) or megakaryocytic (MK) lineages by the MK-E progenitor (MEP) could enhance the ex vivo generation of red blood cells and platelets for therapeutic transfusions. The lineage choice at the MEP bifurcation is controlled in large part by activity within the intracellular signal transduction network, the output of which determines the activity of transcription factors (TFs) and ultimately gene expression. Although many TFs have been implicated, E or MK differentiation is a complex process requiring multiple days, and the dynamics of TF activities during commitment and terminal maturation are relatively unexplored. Herein, we applied a living cell array for the large-scale, dynamic quantification of TF activities during MEP bifurcation. A panel of hematopoietic TFs (GATA-1, GATA-2, SCL/TAL1, FLI-1, NF-E2, PU.1, c-Myb) was characterized during E and MK differentiation of bipotent K562 cells. Dynamic TF activity profiles associated with differentiation towards each lineage were identified, and validated with previous reports. From these activity profiles, we show that GATA-1 is an important hub during early hemin- and PMA-induced differentiation, and reveal several characteristic TF interactions for E and MK differentiation that confirm regulatory mechanisms documented in the literature. Additionally, we highlight several novel TF interactions at various stages of E and MK differentiation. Furthermore, we investigated the mechanism by which nicotinamide (NIC) promoted terminal MK maturation using an MK-committed cell line, CHRF-288-11 (CHRF). Concomitant with its enhancement of ploidy, NIC strongly enhanced the activity of three TFs with known involvement in terminal MK maturation: FLI-1, NF-E2, and p53. Dynamic profiling of TF activity represents a novel tool to complement traditional assays focused on mRNA and protein expression levels to understand progenitor cell differentiation.

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“…[1][2][3][4][5][6] However, due to low transfection efficiencies, acquiring stably transfected cells requires long-term drug selection limiting the application of this approach to cell lines as opposed to primary cells. Also, the transient nature of transfection makes it difficult to follow cells for a long time (days to weeks) as required for stem cell differentiation.…”

Section: Introductionmentioning

confidence: 99%

Lentiviral Arrays for Live-cell Dynamic Monitoring of Gene and Pathway Activity During Stem Cell Differentiation

et al. 2014

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Uncovering the complexity of mesenchymal stem cell (MSC) differentiation requires novel methods to capture the dynamics of the process in a quantitative and high-throughput manner. To this end, we developed a lentiviral array (LVA) of reporters to capture the dynamics of gene and pathway activity during MSC differentiation into adipogenic, chondrogenic, and osteogenic lineages. Our results identified signature promoters and pathways with unique activation profile for each MSC lineage. In combination with chemical inhibitors, lineage-specific reporters predicted the effects of signaling pathway perturbations on MSC differentiation. Interestingly, some pathways were critical for differentiation into all lineages, while others had differential effects on each lineage. Our study suggests that when combined with large chemical or siRNA libraries, the reporter LVA can be used to uncover novel genes and signaling pathways affecting complex biological processes such as stem cell differentiation or reprogramming.

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Cellular arrays for large‐scale analysis of transcription factor activity

Cited by 20 publications

References 37 publications

Inhibition of CDK-mediated phosphorylation of Smad3 results in decreased oncogenesis in triple negative breast cancer cells

Inhibition of CDK-mediated phosphorylation of Smad3 results in decreased oncogenesis in triple negative breast cancer cells

Dynamic transcription factor activity profiles reveal key regulatory interactions during megakaryocytic and erythroid differentiation

Lentiviral Arrays for Live-cell Dynamic Monitoring of Gene and Pathway Activity During Stem Cell Differentiation

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