P. Gascard - Independent Researcher (original) (raw)

Papers by P. Gascard

Epithelial-Stromal Interactions in Barrett’s Esophagus Modeled in Human Organ Chips

Gastro Hep Advances

Evidence of a protective role of the Grados channel against hemolysis in murine spherocytosis

Blood, 2005

It has been shown that mice with complete deficiency of all 4.1R protein isoforms (4.1/) exhibit ... more It has been shown that mice with complete deficiency of all 4.1R protein isoforms (4.1/) exhibit moderate hemolytic anemia, with abnormal erythrocyte morphology (spherocytosis) and decreased membrane stability. Here, we characterized the Gardos channel function in vitro and in vivo in erythrocytes of 4.1/ mice. Compared with wild-type, the Gardos channel of 4.1/ erythrocytes showed an increase in Vmax (9.75 1.06 vs 6.08 0.09 mM cell minute; P < .04) and a decrease in Km (1.01 0.06 vs 1.47 1.02 M; P < .03), indicating an increased sensitivity to activation by intracellular calcium. In vivo function of the Gardos channel was assessed by the oral administration of clotrimazole, a well-characterized Gardos channel blocker. Clotrimazole treatment resulted in worsening of anemia and hemolysis, with decreased red cell survival and increased numbers of circulating hyperchromic spherocytes and microspherocytes. Clotrimazole induced similar changes in 4.2/ and band 3/ mice, indicating that these effects of the Gardos channel are shared in different models of murine spherocytosis. Thus, potassium and water loss through the Gardos channel may play an important protective role in compensating for the reduced surface-membrane area of hereditary spherocytosis (HS) erythrocytes and reducing hemolysis in erythrocytes with cytoskeletal impairments

Blood, 1994

We investigated the role of glycophorins C and D in the association of band 4.1 with the erythroc... more We investigated the role of glycophorins C and D in the association of band 4.1 with the erythrocyte membrane by measuring the binding of band 4.1 to erythrocyte inside-out vesicles stripped of endogenous band 4.1. Vesicles were prepared from either normal erythrocytes or erythrocytes completely lacking glycophorins C and D (Leach phenotype). Band 4.1 binding to vesicles from normal erythrocytes gave rise to a nonlinear Scatchard plot, indicative of two classes of binding sites: a low-capacity, high-affinity class of sites (about 10% of the total) and a high-capacity, low-affinity class of sites. Vesicles prepared from Leach erythrocytes had a binding capacity for band 4.1 that was, on average, 32% lower than that of vesicles from normal erythrocytes. This difference was caused by the complete absence of the high-affinity binding sites as well as by a decrease in the number of low-affinity binding sites. Reduction of membrane phosphatidylinositol 4,5-biphosphate (PIP2) content by ad...

Blood, 1996

Rabbit erythrocytes of progressively increasing age were isolated using an avidin-biotin affinity... more Rabbit erythrocytes of progressively increasing age were isolated using an avidin-biotin affinity technique and the activity of protein kinases and other enzymes was analyzed in cytosols and membranes from the isolated cells. The activities of cytosolic protein kinase C (PKC), cAMP-dependent kinase (PKA), and casein kinase type I and II (CKI and II) were all found to undergo an age-dependent decrease of twofold to fourfold over the 8-week lifespan of the cells. Membrane-associated tyrosine kinase showed little or no decrease, but membrane-associated CKI showed a dramatic eightfold decrease over the 8-week period. By contrast, various cytosolic enzymes, including lactate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, and acid phosphatase, showed no change in activity over the same time period. Density-separated human erythrocytes showed qualitatively similar decreases in cytosolic protein kinase activities in the densest fractions, which contain the oldest cells. Our result...

Abstract P5-18-04: HER2 overexpression in ductal carcinoma in situ: A biomarker for risk stratification and therapeutic implication

Cancer Research, 2019

Background: After initial treatment for ductal carcinoma in situ (DCIS), a subsequent clinically ... more Background: After initial treatment for ductal carcinoma in situ (DCIS), a subsequent clinically significant event (SCSE) such as another diagnosis of DCIS, atypia or invasive breast cancer (IBC), will likely lead to further surgical/medical therapy. Pathologically, DCIS is treated on the basis of estrogen (ER) and progesterone receptor (PR) status with hormonal therapy (HT), ie, tamoxifen (TMX) or aromatase inhibitor, regardless of human epidermal growth factor receptor (HER2) overexpression. Although HER2 is a well-established prognostic marker for IBC, the role of HER2 in DCIS is less appreciated. Recent studies have documented the prognostic value of p16, COX-2 and Ki67 as prognostic biomarkers for locoregional invasive recurrences due to abrogated response to cellular stress (ARCS). Notably, a high-risk population of DCIS patients lacking ER expression but over-expressing HER2, p16 and COX-2 has been recently identified. In the present study, we compared expression levels of HE...

Trans-bilayer distribution of phosphatidylinositol 4,5-bisphosphate and its role in the changes of lipid asymmetry in the human erythrocyte membrane

Biochemical Society Transactions, 1993

Biochemical Journal, 1988

Sickle-cell-anaemia erythrocytes (SS cells) are known to have a high Ca2+ content (particularly t... more Sickle-cell-anaemia erythrocytes (SS cells) are known to have a high Ca2+ content (particularly the dense cell fraction) and to take up Ca2+ on deoxygenation. It has been reported that this high Ca2+ was responsible for the activation of the Ca2+-dependent K+ loss, and of the Ca2+-sensitive polyphosphoinositide phospholipase C (PIC) in dense SS cells. We found that, either in the total population of SS cells or in the light or dense fractions, the content of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was not changed, whereas that of phosphatidylinositol 4-phosphate was increased and that of phosphatidic acid (PtdOH) was decreased compared with normal (AA) erythrocytes. Deoxygenation-induced Ca2+ entry into SS cells did not change the concentration or, in 32P-prelabelled cells, the radioactivity of polyphosphoinositides and PtdOH. It also failed to induce the formation of inositol 1,4,5-trisphosphate, the product of PtdIns(4,5)P2 hydrolysis by PIC, which was measured by an...

Phosphatidylinositol 4,5-bfsphosphate (PIP 2 ) translocation in human erythrocyte membrane is a fast and active process

Biochemical Society Transactions, 1992

Biochemical Journal, 1989

After labelling of erythrocytes with [32P]P1 for 23 h, the specific radioactivities of the phosph... more After labelling of erythrocytes with [32P]P1 for 23 h, the specific radioactivities of the phosphomonoester groups of PtdIns4P and of PtdIns(4,5)P2 approached equilibrium values which were close to that of the gamma-phosphate of ATP (78-85%), showing that almost all of these phosphate groups were metabolically active. Phosphoinositidase C (PIC) activation, using Ca2+ and the ionophore A23187, of 32P-prelabelled erythrocytes was used to investigate a possible functional heterogeneity of the phosphoinositides. Hydrolysis of PtdIns(4,5)P2, measured from its radioactivity, decreased as function of the time of prelabelling up to a constant value equal to that measured from its content. In contrast, hydrolysis of PtdIns4P, determined both from radioactivity and from content, was always the same. These data suggest that newly labelled molecules of PtdIns(4,5)P2, initially accessible to PIC, then moved towards a PIC-resistant pool. This was further confirmed by measuring the fraction of lab...

Immunological detection of phosphatidylinositol 4,5 bisphosphate (PIP) in human red blood cells (RBC) and in rat liver

Cell Biology International Reports, 1990

Structural and functional characterization of protein 4.1R-phosphatidylserine interaction: potential role in 4.1R sorting within cells

The Journal of biological chemistry, Jan 21, 2001

Erythrocyte protein 4.1R is a multifunctional protein that binds to various membrane proteins and... more Erythrocyte protein 4.1R is a multifunctional protein that binds to various membrane proteins and to phosphatidylserine. In the present study, we report two important observations concerning 4.1R-phosphatidylserine interaction. Biochemically, a major finding of the present study is that 4.1R binding to phosphatidylserine appears to be a two-step process in which 4.1R first interacts with serine head group of phosphatidylserine through the positively charged amino acids YKRS and subsequently forms a tight hydrophobic interaction with fatty acid moieties. 4.1R failed to dissociate from phosphatidylserine liposomes under high ionic strength but could be released specifically by phospholipase A(2) but not by phospholipase C or D. Biochemical analyses showed that acyl chains were associated with 4.1R released by phospholipase A(2). Importantly, the association of acyl chains with 4.1R impaired its ability to interact with calmodulin, band 3, and glycophorin C. Removal of acyl chains rest...

Regulation and post-translational modification of erythrocyte membrane and membrane-skeletal proteins

Seminars in hematology, 1992

RED CELLS-Nucleolar localization of RPS19 protein in normal cells and mislocalization due to mutations in the nucleolar localization signals in 2 Diamond-Blackfan anemia patients: Potential

Scientific Reports, 2013

A physical sciences network characterization of non-tumorigenic and metastatic cells The Physical... more A physical sciences network characterization of non-tumorigenic and metastatic cells The Physical Sciences-Oncology Centers Network* To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences-Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells' regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis. T he conversion from a non-tumorigenic state to a metastatic one is of critical interest in cancer cell biology, as most deaths from cancer occur due to metastasis 1. Typically, we think of the activation of metastasis as one of the hallmarks of cancer 2 and as a highly regulated, multistep process defined by a loss of cell adhesion due to reduced expression of cell adhesion molecules such as E-cadherin, degradation of the extracellular matrix (ECM), conversion to a motile phenotype, vascular infiltration, exit and colonization to a new organ site (i.e., intra-and extravasation), dormancy, and re-activation. From a physical sciences perspective, metastasis can be viewed as a ''phase'' transition, albeit occuring far from thermodynamic equilibrium 3. Though this transition has been the focus of much cancer biology research, there is still an incomplete understanding of this phase change, in particular, the physical biology of the metastatic state of a cell compared to its pre-malignant state. Understanding the physical forces that metastatic cells experience and overcome in their microenvironment may improve our ability to target this key step in tumor progression. The newly formed Physical Sciences-Oncology Centers (PS-OC) Network, sponsored by and under the auspices of the Office of Physical Sciences-Oncology at the National Cancer Institute (OPSO/NCI), is a multidisciplinary network of twelve research centers across the US formed, in part, to test the fundamental hypothesis that physical processes (e.g., mechanics, dynamics) play a critical role in cancer initiation and metastasis. The PS-OC Network brings analytic techniques and perspectives from the physical sciences to the interpretation of biological data and consists of physicists, engineers, mathematicians, chemists, cancer biologists, and computational scientists. The goal of the PS-OC Network is to better understand the physical and chemical forces that shape and govern the emergence and behavior of cancer at all length scales. The study described in this manuscript focused on physical changes associated with metastasis. A controlled set of comparative studies of two cell lines that are extensively used as cell models of cancer metastasis and straddle the metastatic transition was undertaken by the PS-OC Network. The cell lines analyzed were the immortalized human breast epithelial cell line MCF-10A, representing a nontumorigenic state, and the human metastatic breast cell line MDA-MB-231, representing a malignant state. Distinguishing features of the adherent, non-transformed, MCF-10A cells are their lack of tumorigenicity in nude mice, lack of anchorage-independent growth, and dependence on growth factors 4. In contrast, MDA-MB-231 cells 5 form highly malignant, invasive tumors in vivo, are resistant to chemotherapy drugs such as paclitaxel, exhibit anchorage-independent growth, and grow independently of growth factors. Although MCF-10A cells have wild-type p53 and MDA-MB-231 cells have mutant p53, both cell lines are negative for the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) 6,7. To ensure that data generated across the multiple PS-OC laboratories could be integrated, culture guidelines, common culture reagents, and the two fully characterized, karyotyped cell lines were distributed to PS-OC laboratories. This minimized phenotypic and genotypic drift. After demonstration of growth uniformity, the

Nature communications, Jan 25, 2014

Developmental history shapes the epigenome and biological function of differentiated cells. Epige... more Developmental history shapes the epigenome and biological function of differentiated cells. Epigenomic patterns have been broadly attributed to the three embryonic germ layers. Here we investigate how developmental origin influences epigenomes. We compare key epigenomes of cell types derived from surface ectoderm (SE), including keratinocytes and breast luminal and myoepithelial cells, against neural crest-derived melanocytes and mesoderm-derived dermal fibroblasts, to identify SE differentially methylated regions (SE-DMRs). DNA methylomes of neonatal keratinocytes share many more DMRs with adult breast luminal and myoepithelial cells than with melanocytes and fibroblasts from the same neonatal skin. This suggests that SE origin contributes to DNA methylation patterning, while shared skin tissue environment has limited effect on epidermal keratinocytes. Hypomethylated SE-DMRs are in proximity to genes with SE relevant functions. They are also enriched for enhancer- and promoter-asso...

European Journal of Biochemistry, 1993

Band 4.1 is a major protein of the erythrocyte membrane skeleton. It promotes the binding of spec... more Band 4.1 is a major protein of the erythrocyte membrane skeleton. It promotes the binding of spectrin to F-actin and may anchor the skeletal network to the plasma membrane via its association with integral membrane proteins. Here, we have investigated the involvement of inositol phospholipids in the binding of band 4.1 to erythrocyte membranes using membrane vesicles stripped of all peripheral proteins at alkaline pH. Trypsinization of these vesicles allows the discrimination of two classes of band 4.1 binding sites: trypsin-sensitive sites (60-65 % of the total), largely or exclusively on band 3, and trypsin-resistant sites (35-40% of the total), composed, at least in part, of the glycophorins. ATP depletion or activation of erythrocyte phosphoinositol phospholipase C led to a reduction in membrane phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] content by 20-70% in different experiments. The resulting decrease of band 4.1 binding to vesicles by was variable, but averaged about 15-20%. The same treatments led to an average decrease in the band 4.1 binding capacity of trypsinized vesicles of 55%. Since this is equivalent to a 20% decrease in the binding capacity of non-trypsinized vesicles (consistent with the above result), it indicates that PtdIns(4,5)P2 regulates the binding of band 4.1 only to trypsin-resistant binding sites (and to only a subset of these) accounting for about 1 5 2 0 % of total band 4.1 binding sites on membranes. We found that hydrolysis of > 95% of PtdIns(4,5)P2 with exogenous phospholipase C-6 (PLCG) resulted in no further decrease in band 4.1 binding to vesicles than did hydrolysis of 65-70% of PtdIns(4,5)P2 which is accessible to erythrocyte phosphoinositol phospholipase C. This suggests that only 65-70% of total membrane PtdIns(4,5)P2 is involved in regulating band 4.1 binding. Significantly, the pool of PtdIns(4,5)P, involved is the same pool which can be hydrolysed by erythrocyte phosphoinositol phospholipase C, and which has been shown to be metabolically labile in erythrocytes. The membrane binding capacity for band 4.1 found in this study (averaging 1000 pg/mg vesicle protein) is considerably higher than that found in previous studies. The results are consistent with the existence of a binding site for band 4.1 on each copy of the major transmembrane proteins (band 3 and the glycophorins). These results provide new insights into the involvement of membrane inositol phospholipids in cytoskeletal-membrane interactions. The human erythrocyte membrane is underlaid by a network of proteins that confers on the cell its characteristic biconcave shape, remarkable deformability and mechanical strength (reviewed in [I, 21). This network is mainly composed of spectrin tetramers, short oligomers of actin, and band 4.1, which self-assemble in complexes to form the skeletal lattice structure. Other proteins such as band 4.

Proceedings of the National Academy of Sciences, 2013

We identified cell surface markers associated with repression of p16 INK4a /cyclin-dependent kina... more We identified cell surface markers associated with repression of p16 INK4a /cyclin-dependent kinase inhibitor 2A(CDKN2A), a critical determinant in the acquisition of a plastic state. These cell surface markers allowed direct isolation of rare cells from healthy human breast tissue that exhibit extensive lineage plasticity. This subpopulation is poised to transcribe plasticity markers, OCT3/4, SOX2, and NANOG, at levels similar to those measured in human embryonic stem cells and to acquire a plastic state sensitive to environmental programming. In vitro, in vivo, and teratoma assays demonstrated that either a directly sorted (uncultured) or a single-cell (clonogenic) cell population from primary tissue can differentiate into functional derivatives of each germ layer, ectodermal, endodermal, and mesodermal. In contrast to other cells that express OCT3/4, SOX2, and NANOG, these human endogenous plastic somatic cells are mortal, express low telomerase activity, expand for an extensive ...

Nature Genetics, 2013

et al. 2013. "DNA hypomethylation within specific transposable element families associates with t... more et al. 2013. "DNA hypomethylation within specific transposable element families associates with tissue-specific enhancer landscape." Nature genetics 45 (7)

Journal of Biotechnology, 2005

Functional genomic analysis is a challenging step in the so-called post-genomic field. Identifica... more Functional genomic analysis is a challenging step in the so-called post-genomic field. Identification of potential targets using large-scale gene expression analysis requires functional validation to identify those that are physiologically relevant. Genetically modified cell models are often used for this purpose allowing up-or down-expression of selected targets in a well-defined and if possible highly differentiated cell type. However, the generation of such models remains time-consuming and expensive. In order to alleviate this step, we developed a strategy aimed at the rapid and efficient generation of genetically modified cell lines with conditional, inducible expression of various target genes. Efficient knock-in of various constructs, called targeted transgenesis, in a locus selected for its permissibility to the tet inducible system, was obtained through the stimulation of sitespecific homologous recombination by the meganuclease I-SceI. Our results demonstrate that targeted transgenesis in a reference inducible locus greatly facilitated the functional analysis of the selected recombinant cells. The efficient screening strategy we have designed makes possible automation of the transfection and selection steps. Furthermore, this strategy could be applied to a variety of highly differentiated cells.