Publications by authors named "Samuel C Wolff"

CDK4/6 inhibitors such as palbociclib block cell cycle progression and improve outcomes for many ER+/HER2- breast cancer patients. Unfortunately, many patients are initially resistant to the drug or develop resistance over time in part due to heterogeneity among individual tumor cells. To better understand these mechanisms of resistance, we used multiplex, single-cell imaging to profile cell cycle proteins in ER+ breast tumor cells under increasing palbociclib concentrations.

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Article Synopsis
  • Regulated cell cycle progression is crucial for maintaining cellular balance and preventing cancer, with the E3 ubiquitin ligase APC/C playing a key role in preventing premature entry into the S phase for proliferating cells.
  • Research shows that APC/C activity is necessary for maintaining cell cycle arrest when CDK4/6 is inhibited, indicating that protein degradation is vital for effective cell cycle regulation.
  • The study suggests that cancers with high levels of EMI1 may evade CDK4/6 inhibition, leading to unregulated S phase entry and increased genome instability due to improper licensing of DNA replication.
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Single-cell technologies can measure the expression of thousands of molecular features in individual cells undergoing dynamic biological processes. While examining cells along a computationally-ordered pseudotime trajectory can reveal how changes in gene or protein expression impact cell fate, identifying such dynamic features is challenging due to the inherent noise in single-cell data. Here, we present DELVE, an unsupervised feature selection method for identifying a representative subset of molecular features which robustly recapitulate cellular trajectories.

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The CDK4/6 inhibitor palbociclib blocks cell cycle progression in Estrogen receptor-positive, human epidermal growth factor 2 receptor-negative (ER+/HER2-) breast tumor cells. Despite the drug's success in improving patient outcomes, a small percentage of tumor cells continues to divide in the presence of palbociclib-a phenomenon we refer to as fractional resistance. It is critical to understand the cellular mechanisms underlying fractional resistance because the precise percentage of resistant cells in patient tissue is a strong predictor of clinical outcomes.

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It is well known that clonal cells can make different fate decisions, but it is unclear whether these decisions are determined during, or before, a cell's own lifetime. Here, we engineered an endogenous fluorescent reporter for the pluripotency factor OCT4 to study the timing of differentiation decisions in human embryonic stem cells. By tracking single-cell OCT4 levels over multiple cell cycle generations, we found that the decision to differentiate is largely determined before the differentiation stimulus is presented and can be predicted by a cell's preexisting OCT4 signaling patterns.

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Article Synopsis
  • The Cell Cycle Browser (CCB) is an interactive web tool that allows users to visualize and manipulate data related to the cell cycle in human cells, based on real-time reporting.
  • Users can simulate different cell cycle scenarios by adjusting molecular activity levels and relationships, predicting impacts on cell behavior.
  • The CCB enhances our understanding of cell cycle dynamics and supports hypothesis generation through virtual experiments and familiar outputs like growth curves.
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The P2Y(4) receptor is selectively targeted to the apical membrane in polarized epithelial cell lines and has been shown to play a key role in intestinal chloride secretion. In this study, we delimit a 23 amino acid sequence within the P2Y(4) receptor C-tail that directs its apical targeting. Using a mutagenesis approach, we found that four hydrophobic residues near the COOH-terminal end of the signal are necessary for apical sorting, whereas two basic residues near the NH(2)-terminal end of the signal are involved to a lesser extent.

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The P2Y(1) receptor is localized to the basolateral membrane of polarized Madin-Darby canine kidney (MDCK) cells. In the present study, we identified a 25-residue region within the C-terminal tail (C-tail) of the P2Y(1) receptor that directs basolateral sorting. Deletion of this sorting signal caused redirection of the receptor to the apical membrane, indicating that the region from the N-terminus to transmembrane domain 7 (TM7) contains an apical-sorting signal that is overridden by a dominant basolateral signal in the C-tail.

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The P2Y(2) receptor, which is activated by UTP, ATP, and dinucleotides, was studied as a prototypical nucleotide-activated GPCR. A combination of receptor mutagenesis, determination of its effects on potency and efficacy of agonists and antagonists, homology modeling, and chemical experiments was applied. R272 (extracellular loop EL3) was found to play a gatekeeper role, presumably responsible for recognition and orientation of the nucleotides.

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Asymmetries in muscarinic receptor binding were investigated in the hippocampus of female rats by in vitro autoradiography. Coronal sections from 18 brains were incubated with the muscarinic receptor antagonist [3H]quinuclidinyl benzilate, the muscarinic M1 receptor antagonist [3H]pirenzepine, or the muscarinic M2 receptor antagonist [3H]AF-DX 384. Binding of these radioligands was higher on the right than the left side of CA1, CA3, and dentate gyrus in almost every brain confirming hemispheric asymmetry at the neurochemical level.

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P2Y2 and P2Y4 receptors, which have 52% sequence identity, are both expressed at the apical membrane of Madin-Darby canine kidney cells, but the locations of their apical targeting signals are distinctly different. The targeting signal of the P2Y2 receptor is located between the N terminus and 7TM, whereas that of the P2Y4 receptor is present in its C-terminal tail. To identify the apical targeting signal in the P2Y2 receptor, regions of the P2Y2 receptor were progressively substituted with the corresponding regions of the P2Y4 receptor lacking its targeting signal.

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Eight human G protein-coupled P2Y receptors (P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(11), P2Y(12), P2Y(13), and P2Y(14)) that respond to extracellular nucleotides have been molecularly identified and characterized. P2Y receptors are widely expressed in epithelial cells and play an important role in regulating epithelial cell function. Functional studies assessing the capacity of various nucleotides to promote increases in short-circuit current (I(sc)) or Ca(2+) mobilization have suggested that some subtypes of P2Y receptors are polarized with respect to their functional activity, although these results often have been contradictory.

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Nucleotide stimulation of G(q)-coupled P2Y receptors expressed in Xenopus laevis oocytes produces the activation of an endogenous voltage-gated ion channel, previously identified as the transient inward (T(in)) channel. Expression of human P2Y(1), human P2Y(2), rat P2Y(6), human P2Y(11), or skate P2Y receptors in oocytes resulted in modulation of the voltage dependence and inactivation gating of the channel. Expression of the human P2Y(4) receptor, rat M(1)-muscarinic receptor, and human B(1)-bradykinin receptor did not alter the properties of the T(in) channel.

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