Estrogen and Vitamin D Control of Transcription in MCF-7 Cells

Abstract The Extracellular Signal-Regulated Kinase (ERK) is part of a key, signaling pathway that regulates both transcription and translation in many cell types. Increases in intracellular calcium levels results in the CaM Kinase-dependent activation of ERK and cell growth in MCF-7 breast cancer cells. ERK has also been shown to play a role in the regulation of MCF-7 cell proliferation through control of downstream transcription factors including Elk-1. The hormone, Vitamin D has been suggested to play an inhibitory role on cancer cells by blocking ERK activation. Our goal was to evaluate the ability of E2 to activate Elk-1, through a CaM Kinase/ERK dependent pathway, in MCF-7 cells. We also examined Vitamin D’s inhibitory regulation of ERK and Elk-1 activation. Interestingly, E2 stimulation of MCF-7 cells triggered Elk-1 phosphorylation an effect that was blocked by inhibiting either CaM KK or ERK. Similarly, E2 treatment of MCF-7 cells also triggered a significant increase in Elk-1-dependent luciferase activity. siRNA inhibition of CaM KK or ERK blocked E2-stimulated Elk-1 luciferase activity. Additionally, E2 triggered a sustained increase in ERK and Elk-1 phosphorylation, both of which were blocked by Vitamin D treatment. Vitamin D treatment of cells also inhibited Elk-1 luciferase activity downstream of E2 stimulation. In summary, our data suggests that E2 utilizes both CaM KK and ERK to activate Elk-1 transcriptional activity an effect that is blocked by the hormone Vitamin D

TRP Channel Regulation of Estrogen Signaling

Abstract Calcium regulates numerous cell functions including growth and development. Calcium can enter cells through transient receptor potential channels (TRPCs). Previous studies in MCF-7 cells have suggested that the expression of one particular TRPC, TRPC6, correlates with cell transformation and disease progression. Calcium has several cellular targets including the Calcium/Calmodulin-dependent protein kinases (CaM Ks) and ERK. Previous work has shown that estrogen (E2) may utilize CaM Ks and ERK to promote breast cancer cell proliferation, however the possible involvement of TRPCs in this pathway is currently unknown. Our objective was to understand which E2 receptor is used in our system and if TRPCs participate in the control of CaM Kinase activation of ERK in MCF-7 cells. Specifically, we wanted to explore if E2 may utilize TRPCs particularly, TRPC6, upstream of the ERK pathway in MCF-7 cells. E2 stimulation of MCF-7 cells and the estrogen receptor alpha (α) inhibitor, MPP, completely blocked ERK activity. In contrast, MPP did not block EGF stimulation of ERK. MCF-7 cells express endogenous TRPC6 protein and TRPC inhibitors, APB and SK&F, both blocked ERK activation downstream of E2. In addition, neither APB or SK&F inhibited EGF activation of ERK. Results from these studies suggest that E2 is capable of activating ERK through the specifically through the alpha form of the estrogen receptor and TRPCs

Transcription Factor Regulation of ERK and Estrogen in MCF-7 Cells

Abstract ERK is activated by increased intracellular calcium downstream of the hormone estrogen (E2). E2 activates ERK via the CaM Kinases, specifically CaM KK and CaM KI in MCF-7 cells. ERK may control cell growth and proliferation through Elk-1, Rsk, SRF, CREB, and numerous other molecules and nuclear targets. Vitamin D, a hormone, has proven to be an effective antagonist of ERK and MCF-7 breast cancer cell growth. Our goal was to evaluate if the E2 pathway working through CaM KK and ERK regulated the transcription factors Elk-1, CREB, and SRF. We also examined the ability of vitamin D to antagonize ERK activation of its downstream targets. Interestingly, E2 stimulation of MCF-7 cells activated both ERK and Elk-1 an effect that was blocked by inhibiting both CaM KK and ERK. E2 treatment of MCF-7 cells also triggered a significant increase in SRF and CREB phosphorlation in a CaM KK- and ERK-dependent manner. Dimerization of transcription factors may enhance DNA binding and gene expression. E2 stimulation of MCF-7 cells promoted the formation of a molecular complex between endogenous Elk-1 and SRF. Finally, E2 triggered a prolonged increased in ERK and Elk-1 phosphorylation, both of which were blocked by vitamin D treatment. Taken together our data demonstrates several transcriptional targets for E2 working through CaM KK and their inhibition by vitamin D signaling

Estrogen Regulation of Jun and Fos in MCF-7 Cells

Abstract C-Fos and c-Jun are transcription factors that form the dimer Activator Protein 1 (AP-1) and bind DNA to initiate transcription. C-Fos, c-Jun are targets of the Extracellular Signal-Regulated Kinase (ERK) in multiple cell types, including MCF-7 breast cancer cells. The hormone estrogen (E2) can increase intracellular calcium levels which activates calcium/calmodulin-dependent kinase (CaM Kinase) proteins, which control ERK and gene transcription. Our goal was to evaluate the ability of E2 to activate c-Fos and c-Jun and induce their dimerization, via CaM KK and ERK, in MCF-7 cells. Interestingly, E2 stimulation of MCF-7 cells triggered phosphorylation of c-Jun and c-Fos an effect that was blocked with STO-609 and U0126, which target CaM KK and ERK, respectively. siRNA inhibition of CaM KK and ERK blocked E2-stimulated c-Jun and c-Fos phosphorylation. Additionally, E2 triggered AP-1 directed luciferase activity in MCF-7 cells that was blocked by inhibiting either CaM KK or ERK with siRNA. In summary, our data suggests that E2 utilizes both CaM KK and ERK to phosphorylate c-Jun and c-Fos and regulate their transcriptional activity in breast cancer cells

Experimental comparison of coherent polarization-switched QPSK to polarization-multiplexed QPSK for 10 x 100 km WDM transmission

Polarization-switched quadrature phase-shift keying has been demonstrated experimentally at 40.5Gb/s with a coherent receiver and digital signal processing. Compared to polarization-multiplexed QPSK at the same bit rate, its back-to-back sensitivity at 10-3 bit-error-ratio shows 0.9dB improvement, and it tolerates about 1.6dB higher launch power for 10 × 100km, 50GHz-spaced WDM transmission allowing 1dB penalty in required optical-signal-to-noise ratio relative to back-to-back

Beyond the Invisible Barriers of the Classroom: iEngage and Civic Praxis

Research literature suggests students need to engage in actual civic experiences; however, in most cases, teachers feel unwilling or unable to facilitate experiences beyond the formal classroom setting. In this project, we sought to understand the relationship between social studies teachers’ civic ideology, pedagogical approaches, and instructional decision-making through their engagement in an action civics camp. The project is part of a more significant effort to help critically minded teachers engage in more activist praxis by moving past the often-limiting ideological barriers of the classroom. By activist praxis, we refer to the ways a teacher’s ideology informs pedagogy related to the ways they are able and willing to extend civic engagement into the material and social world. Activist praxis is part of a teacher’s continual engagement in efforts to create the conditions for a more just and equitable public sphere

Using Artificial Intelligence to Identify Perpetrators of Technology Facilitated Coercive Control.

This study investigated the feasibility of using Artificial Intelligence to identify perpetrators of coercive control through digital data held on mobile phones. The research also sought the views of the police and victim/survivors of domestic abuse to using technology in this way

Change Talk During Brief Motivational Intervention With Young Adult Males: Strength Matters.

Client change talk (CT) during motivational interviewing (MI) has been described as a predictor of change in alcohol use. We examined the predictive validity of different strength levels of CT within a brief MI session for 174 young men from the general population screened as hazardous drinkers. CT was measured using the MI Skill Code (MISC 2.1) and categorized with positive (toward change) and negative (against change) valence and 3 strength levels (1=low, 2=medium, 3=high). Analyses included linear regression models predicting drinking at 3-month follow-up, while controlling for baseline drinking. Frequency of overall negative CT (i.e., sum of -1, -2, -3) significantly predicted poorer drinking outcomes. In a multivariate model entering frequency of CT utterances at each level of strength (i.e. +1, +2, +3, -1, -2, -3), the directionality of negative strength ratings was consistently in the expected direction, but only CT-2 was statistically significant. In contrast, overall CT positive (i.e., sum of +1, +2, +3) was not a significant predictor of less alcohol use, but the multivariate model showed that the presence of CT+3 significantly predicted less drinking at 3-month follow-up. Averaged strength summary score (i.e. on the scale from -3 to +3) was a significant predictor of better outcome, while percent positive CT was not. Moderation analyses showed that young men with lower baseline readiness to change or lower alcohol problem severity had higher follow-up drinking when they expressed more CT+1 or CT+2, while the opposite pattern was observed with those reporting higher baseline readiness to change or higher alcohol problem severity. Mixed findings for varying levels of positive CT strength might explain previous studies showing poor predictive validity of positive client language in MI. Together with other studies in similar settings, these findings suggest the importance of advanced MI techniques to shape client language to soften negative change talk (also known as sustain talk) and elicit positive CT verbalized with high intensity

Predicate Abstraction for Linked Data Structures

We present Alias Refinement Types (ART), a new approach to the verification of correctness properties of linked data structures. While there are many techniques for checking that a heap-manipulating program adheres to its specification, they often require that the programmer annotate the behavior of each procedure, for example, in the form of loop invariants and pre- and post-conditions. Predicate abstraction would be an attractive abstract domain for performing invariant inference, existing techniques are not able to reason about the heap with enough precision to verify functional properties of data structure manipulating programs. In this paper, we propose a technique that lifts predicate abstraction to the heap by factoring the analysis of data structures into two orthogonal components: (1) Alias Types, which reason about the physical shape of heap structures, and (2) Refinement Types, which use simple predicates from an SMT decidable theory to capture the logical or semantic properties of the structures. We prove ART sound by translating types into separation logic assertions, thus translating typing derivations in ART into separation logic proofs. We evaluate ART by implementing a tool that performs type inference for an imperative language, and empirically show, using a suite of data-structure benchmarks, that ART requires only 21% of the annotations needed by other state-of-the-art verification techniques