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Viable biobanking of primary head and neck squamous cell carcinoma.
Godoy JM, Sewell A, Johnston B, Brown BT, Lu X, Sinard RJ, Rohde S, Mannion K, Netterville JL, Yarbrough WG
(2013) Laryngoscope 123: 641-5
MeSH Terms: Carcinoma, Squamous Cell, Cryopreservation, Feasibility Studies, Head and Neck Neoplasms, Humans, Immunohistochemistry, Keratins, Laryngeal Neoplasms, Mouth Neoplasms, Oropharyngeal Neoplasms, Squamous Cell Carcinoma of Head and Neck, Tissue Banks, Transplantation, Heterologous, Tumor Cells, Cultured
Show Abstract · Added March 7, 2014
OBJECTIVES/HYPOTHESIS - To determine the feasibility of viable storage of head and neck squamous cell carcinoma (HNSCC) for regrowth of cells in culture.
STUDY DESIGN - Laboratory-based translational study.
METHODS - Methods for intermediate-term frozen storage of viable HNSCC were explored using small pieces of primary tumor and dissociated HNSCC cells after short-term culture. Viable cells after freezing were confirmed by adherence to tissue culture plates, cell morphology, and increased cell or colony density. Two cultures were immunostained for cytokeratin to confirm epithelial origin of viable cultured cells after freezing.
RESULTS - Six primary HNSCCs (two oral cavity, three larynx, one oropharynx) and two HNSCCs that had been passaged through a xenograft (two oral cavity) were dissociated to single cells and grown in short-term cell culture for 0 to 12 passages. After short-term culture, cells were frozen for up to 8 months, thawed, and replated. Frozen cells derived from all tumors (six primary and two xenografts) were successfully replated with cultures lasting >7 days with seven of eight tumors presenting increased colony or cell density over 1 week of growth after freezing. In total, 15 of 15 tested samples derived from six primary and two xenografted HNSCCs were viable after freezing.
CONCLUSIONS - In the current study, we show that biopreservation of primary or xenografted HNSCC using short-term cell culture is feasible. Initial short-term cell culture was required for successful storage and viability of frozen cells. These proof-of-principle studies, if more widely implemented, could improve preclinical testing of new therapies for HNSCC.
Copyright © 2013 The American Laryngological, Rhinological, and Otological Society, Inc.
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14 MeSH Terms
Luminal cytokeratin expression profiles of breast papillomas and papillary carcinomas and the utility of a cytokeratin 5/p63/cytokeratin 8/18 antibody cocktail in their distinction.
Reisenbichler ES, Balmer NN, Adams AL, Pfeifer JD, Hameed O
(2011) Mod Pathol 24: 185-93
MeSH Terms: Adult, Biomarkers, Tumor, Breast Neoplasms, Carcinoma, Papillary, Diagnosis, Differential, Female, Humans, Immunohistochemistry, Keratins, Membrane Proteins, Papilloma, Sensitivity and Specificity
Show Abstract · Added March 5, 2014
Luminal cytokeratin (CK) expression in breast papillary lesions, and its potential diagnostic utility among other markers in distinguishing between papillomas and papillary carcinomas, has not been previously evaluated. Such expression was determined in 42 papillary lesions (18 papillary carcinomas and 24 papillomas) by immunostaining with a CK5/p63/CK8/18 antibody cocktail. The mean CK8/18 intensity score and percentage of positive cells were significantly higher in papillary carcinomas (227 and 95%, respectively, vs 86 and 42% in papillomas; both P-values <0.0001), whereas the mean CK5 intensity score and percentage of positive cells were significantly lower (7 and 5%, respectively, vs 107 and 58% in papillomas; both P-values <0.0001). Half (9/18) of the papillary carcinomas expressed p63 vs all (24/24) of the papillomas (P = 0.0001). P63 expression in papillary carcinoma was always (9/9; 100%) focal/limited in nature (expression in <10% of cells), whereas focal expression was seen in only four (17%) papillomas (P<0.0001). Both differential CK (CK8/18 and CK5) expression and p63 were equally sensitive (100%) for the diagnosis of papillary carcinoma, but differential CK expression was more specific (96 vs 83%), resulting in a greater accuracy. However, the best discriminatory power in the distinction from papilloma was achieved when all three markers were used in combination, resulting in 100% sensitivity and specificity values. It is concluded that breast papillary lesions have differential CK expression profiles that, especially in combination with p63, can be useful for their stratification, potentially also in needle biopsy material, in which more accurate and reproducible characterization is needed.
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12 MeSH Terms
DKK1 mediated inhibition of Wnt signaling in postnatal mice leads to loss of TEC progenitors and thymic degeneration.
Osada M, Jardine L, Misir R, Andl T, Millar SE, Pezzano M
(2010) PLoS One 5: e9062
MeSH Terms: Animals, Apoptosis, Cell Count, Cell Proliferation, Doxycycline, Epithelial Cells, Female, Gene Expression Regulation, In Situ Hybridization, Intercellular Signaling Peptides and Proteins, Keratins, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Microscopy, Confocal, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction, Stem Cells, Tetracycline, Thymus Gland, Trans-Activators, Wnt Proteins
Show Abstract · Added January 30, 2013
BACKGROUND - Thymic epithelial cell (TEC) microenvironments are essential for the recruitment of T cell precursors from the bone marrow, as well as the subsequent expansion and selection of thymocytes resulting in a mature self-tolerant T cell repertoire. The molecular mechanisms, which control both the initial development and subsequent maintenance of these critical microenvironments, are poorly defined. Wnt signaling has been shown to be important to the development of several epithelial tissues and organs. Regulation of Wnt signaling has also been shown to impact both early thymocyte and thymic epithelial development. However, early blocks in thymic organogenesis or death of the mice have prevented analysis of a role of canonical Wnt signaling in the maintenance of TECs in the postnatal thymus.
METHODOLOGY/PRINCIPAL FINDINGS - Here we demonstrate that tetracycline-regulated expression of the canonical Wnt inhibitor DKK1 in TECs localized in both the cortex and medulla of adult mice, results in rapid thymic degeneration characterized by a loss of DeltaNP63(+) Foxn1(+) and Aire(+) TECs, loss of K5K8DP TECs thought to represent or contain an immature TEC progenitor, decreased TEC proliferation and the development of cystic structures, similar to an aged thymus. Removal of DKK1 from DKK1-involuted mice results in full recovery, suggesting that canonical Wnt signaling is required for the differentiation or proliferation of TEC populations needed for maintenance of properly organized adult thymic epithelial microenvironments.
CONCLUSIONS/SIGNIFICANCE - Taken together, the results of this study demonstrate that canonical Wnt signaling within TECs is required for the maintenance of epithelial microenvironments in the postnatal thymus, possibly through effects on TEC progenitor/stem cell populations. Downstream targets of Wnt signaling, which are responsible for maintenance of these TEC progenitors may provide useful targets for therapies aimed at counteracting age associated thymic involution or the premature thymic degeneration associated with cancer therapy and bone marrow transplants.
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23 MeSH Terms
Novel association of APC with intermediate filaments identified using a new versatile APC antibody.
Wang Y, Azuma Y, Friedman DB, Coffey RJ, Neufeld KL
(2009) BMC Cell Biol 10: 75
MeSH Terms: Adenomatous Polyposis Coli, Antibodies, Cell Line, Chromatography, High Pressure Liquid, Colon, Epithelial Cells, Fluorescent Antibody Technique, Humans, Immunoblotting, Immunoprecipitation, Intermediate Filament Proteins, Keratins, Lamin Type B, RNA Interference, Tandem Mass Spectrometry, beta Catenin
Show Abstract · Added August 12, 2010
BACKGROUND - As a key player in suppression of colon tumorigenesis, Adenomatous Polyposis Coli (APC) has been widely studied to determine its cellular functions. However, inconsistencies of commercially available APC antibodies have limited the exploration of APC function. APC is implicated in spindle formation by direct interactions with tubulin and microtubule-binding protein EB1. APC also interacts with the actin cytoskeleton to regulate cell polarity. Until now, interaction of APC with the third cytoskeletal element, intermediate filaments, has remained unexamined.
RESULTS - We generated an APC antibody (APC-M2 pAb) raised against the 15 amino acid repeat region, and verified its reliability in applications including immunoprecipitation, immunoblotting, and immunofluorescence in cultured cells and tissue. Utilizing this APC-M2 pAb, we immunoprecipitated endogenous APC and its binding proteins from colon epithelial cells expressing wild-type APC. Using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), we identified 42 proteins in complex with APC, including beta-catenin and intermediate filament (IF) proteins lamin B1 and keratin 81. Association of lamin B1 with APC in cultured cells and human colonic tissue was verified by co-immunoprecipitation and colocalization. APC also colocalized with keratins and remained associated with IF proteins throughout a sequential extraction procedure.
CONCLUSION - We introduce a versatile APC antibody that is useful for cell/tissue immunostaining, immunoblotting and immunoprecipitation. We also present evidence for interactions between APC and IFs, independent of actin filaments and microtubules. Our results suggest that APC associates with all three major components of the cytoskeleton, thus expanding potential roles for APC in the regulation of cytoskeletal integrity.
1 Communities
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16 MeSH Terms
Markers of epithelial-mesenchymal transition and epithelial differentiation in sarcomatoid carcinoma: utility in the differential diagnosis with sarcoma.
Cates JM, Dupont WD, Barnes JW, Edmunds HS, Fasig JH, Olson SJ, Black CC
(2008) Appl Immunohistochem Mol Morphol 16: 251-62
MeSH Terms: Biomarkers, Tumor, Carcinoma, Transitional Cell, Cell Differentiation, Diagnosis, Differential, Epithelial Cells, Gene Expression, Humans, Immunohistochemistry, Keratins, Mesenchymal Stem Cells, Sarcoma, Snail Family Transcription Factors, Transcription Factors, Twist-Related Protein 1
Show Abstract · Added March 15, 2013
The distinction between sarcomatoid carcinoma (SC) and bona fide sarcoma can be difficult using conventional immunohistochemical markers. Epithelial-mesenchymal transition (EMT) has been proposed as a histogenetic mechanism for the development of SC. Expression of selected markers of EMT (Twist and Slug) was compared with other markers of epithelial differentiation in SC and spindle cell sarcoma to determine the utility of these antigens in this differential diagnosis. Twenty-seven cases of SC (excluding those of gynecologic origin) were stained by immunohistochemistry for cytokeratins (AE1/AE3, 5D3, CK5/6, and 34betaE12), p63, claudin-1, claudin-7, epithelial cadherin, placental cadherin, epithelial cell adhesion molecule/epithelial-specific antigen, 14-3-3sigma, Twist, and Slug. A comparison group of 21 spindle or pleomorphic spindle cell sarcomas was also studied. Immunohistochemical stains were scored in a semiquantitative manner and subsequent exploratory analyses were performed using logistic regression and chi2 tests. Only cytokeratin AE1/AE3 specifically labeled SC in a statistically significant manner. Other epithelial-specific markers tested did not distinguish SC from sarcoma primarily owing to low sensitivity. However, when positive, immunostains such as CK5/6, membranous epithelial cadherin, and nuclear p63 may aid in the distinction of SC from sarcoma. EMT markers were expressed in most cases of both SC and sarcoma, and were not useful in making a differential diagnosis between these neoplasms.
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14 MeSH Terms
The miRNA-processing enzyme dicer is essential for the morphogenesis and maintenance of hair follicles.
Andl T, Murchison EP, Liu F, Zhang Y, Yunta-Gonzalez M, Tobias JW, Andl CD, Seykora JT, Hannon GJ, Millar SE
(2006) Curr Biol 16: 1041-9
MeSH Terms: Animals, Hair, Hair Follicle, Hedgehog Proteins, Keratin-15, Keratins, Mice, MicroRNAs, Morphogenesis, Oncogene Proteins, Receptor, Notch1, Ribonuclease III, Sequence Deletion, Skin, Skin Abnormalities, Stem Cells, Trans-Activators, Zinc Finger Protein GLI1
Show Abstract · Added January 30, 2013
The discovery that microRNAs (miRNAs) play important roles in regulating gene expression via posttranscriptional repression has revealed a previously unsuspected mechanism controlling development and progenitor-cell function (reviewed in ); however, little is known of miRNA functions in mammalian organogenesis. Processing of miRNAs and their assembly into the RNA-induced silencing (RISC) complex requires the essential multifunctional enzyme Dicer . We found that Dicer mRNA and multiple miRNAs are expressed in mouse skin, suggesting roles in skin- and hair-follicle biology. In newborn mice carrying an epidermal-specific Dicer deletion, hair follicles were stunted and hypoproliferative. Hair-shaft and inner-root-sheath differentiation was initiated, but the mutant hair follicles were misoriented and expression of the key signaling molecules Shh and Notch1 was lost by postnatal day 7. At this stage, hair-follicle dermal papillae were observed to evaginate, forming highly unusual structures within the basal epidermis. Normal hair shafts were not produced in the Dicer mutant, and the follicles lacked stem cell markers and degenerated. In contrast to decreased follicular proliferation, the epidermis became hyperproliferative. These results reveal critical roles for Dicer in the skin and implicate miRNAs in key aspects of epidermal and hair-follicle development and function.
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18 MeSH Terms
Stratified epithelium in prostatic adenocarcinoma: a mimic of high-grade prostatic intraepithelial neoplasia.
Hameed O, Humphrey PA
(2006) Mod Pathol 19: 899-906
MeSH Terms: Adenocarcinoma, Aged, Aged, 80 and over, Biopsy, Needle, Diagnosis, Differential, Epithelium, Humans, Immunohistochemistry, Keratins, Male, Membrane Proteins, Middle Aged, Neoplasm Invasiveness, Prostate-Specific Antigen, Prostatic Intraepithelial Neoplasia, Prostatic Neoplasms, Racemases and Epimerases, Retrospective Studies
Show Abstract · Added March 21, 2014
Typically glands of prostatic adenocarcinoma have a single cell lining, although stratification can be seen in invasive carcinomas with a cribriform architecture, including ductal carcinoma. The presence and diagnostic significance of stratified cells within non-cribriform carcinomatous prostatic glands has not been well addressed. The histomorphological features and immunohistochemical profile of cases of non-cribriform prostatic adenocarcinoma with stratified malignant glandular epithelium were analyzed. These cases were identified from needle biopsy cases from the consultation files of one of the authors and from a review of 150 consecutive in-house needle biopsy cases of prostatic adenocarcinoma. Immunohistochemistry was performed utilizing antibodies reactive against high molecular weight cytokeratin (34betaE12), p63 and alpha-methylacyl-coenzyme-A racemase (AMACR). A total of 8 cases were identified, including 2 from the 150 consecutive in-house cases (1.3%). In 4 cases, the focus with glands having stratified epithelium was the sole carcinomatous component in the biopsy, while such a component represented 5-30% of the invasive carcinoma seen elsewhere in the remaining cases. The main attribute in all these foci was the presence of glandular profiles lined by several layers of epithelial cells with cytological and architectural features resembling flat or tufted high-grade prostatic intraepithelial neoplasia, but lacking basal cells as confirmed by negative 34betaE12 and/or p63 immunostains in all cases. The AMACR staining profile of the stratified foci was variable, with 4 foci showing positivity, and 3 foci being negative, including two cases that displayed AMACR positivity in adjacent non-stratified prostatic adenocarcinoma. Prostatic adenocarcinoma with stratified malignant glandular epithelium can be identified in prostate needle biopsy samples harboring non-cribriform prostatic adenocarcinoma and resembles glands with high-grade prostatic intraepithelial neoplasia. These 'PIN-like' carcinomas can present in pure form. Recognition of this pattern of prostatic adenocarcinoma is necessary to correctly diagnose such cases as invasive carcinoma.
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18 MeSH Terms
The requirement for perp in postnatal viability and epithelial integrity reflects an intrinsic role in stratified epithelia.
Marques MR, Ihrie RA, Horner JS, Attardi LD
(2006) J Invest Dermatol 126: 69-73
MeSH Terms: Animals, Cell Adhesion, Cell Proliferation, Epidermal Cells, Epidermis, Genes, Lethal, Heart, Keratin-15, Keratin-5, Keratins, Membrane Proteins, Mice, Mice, Knockout, Myocardium
Show Abstract · Added August 21, 2012
Mice lacking the desmosome protein Perp exhibit blistering in their stratified epithelia and display postnatal lethality. However, it is unclear if these phenotypes are strictly related to Perp function in stratified epithelia, as Perp expression is not restricted to these tissues during embryogenesis, and certain desmosomal blistering diseases such as pemphigus vulgaris and pemphigus foliaceus have non-cell-intrinsic bases. Furthermore, we show here that Perp is expressed in the heart, raising the possibility that defects in heart function could account for lethality in the Perp-deficient mice. To determine conclusively if Perp function in stratified epithelia is crucial for postnatal survival and epithelial adhesion, we specifically ablated Perp in stratified epithelia by breeding conditional Perp knockout mice to keratin 5 (K5)-Cre transgenic mice. We found that the majority of mice lacking Perp in stratified epithelia die within 10 days after birth, accompanied by blistering and hyperproliferation in the epithelia, similar to the constitutive Perp null mice. Together, these findings indicate that Perp's requirement for both viability and epithelial integrity reflects a role in the stratified epithelial compartment.
1 Communities
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14 MeSH Terms
Keratin 19 gene drives Cre recombinase expression throughout the early postimplantation mouse embryo.
Means AL, Chytil A, Moses HL, Coffey RJ, Wright CV, Taketo MM, Grady WM
(2005) Genesis 42: 23-7
MeSH Terms: Animals, Animals, Genetically Modified, Embryonic Development, Female, Gene Expression Regulation, Developmental, Integrases, Keratins, Male, Mice, Recombination, Genetic, beta-Galactosidase
Show Abstract · Added February 17, 2014
The development of Cre-lox technology has created new opportunities for studying the tissue-specific functions of genes in vivo during development and disease. We analyzed the spatial and temporal activity of Cre recombinase whose coding sequence was inserted into the endogenous locus for keratin 19. Rather than providing epithelial-specific recombination during organogenesis, this K19cre allele allows unexpected recombination in early embryonic development, resulting in recombination of a loxP-flanked allele throughout all tissues of the mouse, but with sparing of the extraembryonic endoderm, including the anterior visceral endoderm.
2005 Wiley-Liss, Inc.
2 Communities
4 Members
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11 MeSH Terms
Negative regulation of the expressions of cytokeratins 8 and 19 by SLUG repressor protein in human breast cells.
Tripathi MK, Misra S, Chaudhuri G
(2005) Biochem Biophys Res Commun 329: 508-15
MeSH Terms: Breast Neoplasms, Cell Line, Tumor, Gene Expression Regulation, Neoplastic, Humans, Keratins, Repressor Proteins, Snail Family Transcription Factors, Transcription Factors
Show Abstract · Added June 14, 2013
Invasiveness of tumor cells is often determined by the profile of their expressed genes. To determine the gene expression differences between an invasive and a non-invasive human breast tumor cells, we selected BT-549 (invasive) and MDA-MB-468 (non-invasive) cells, and compared their transcriptomes by cDNA microarray analysis. Among the significant differences in gene expressions, notable are the up-regulation of cytokeratins 8 and 19, and down-regulation of metallothioneins 1G and IL in MDA-MB-468 cells. Since MDA-MB-468 cells do not express SLUG, a member of a small family of E2-box-binding zinc finger silencer proteins, we studied whether the cytokeratin gene overexpressions in these cells are due to the absence of SLUG. Inducible expression of SLUG in MDA-MB-468 cells inhibited the expressions of the cytokeratin 8 and 19 but not others as was revealed by microarray analysis. Similarly, siRNA knock down of SLUG in BT-549 cells increased the expressions of those cytokeratin mRNAs. SLUG levels in the cell regulated the function of cytokeratins 8 and 19 gene promoters. We conclude that the expressions of cytokeratins and metallothioneins may be associated with the differential invasive behaviors of these breast tumor cells and SLUG may have regulatory roles in this process.
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8 MeSH Terms