ABSTRACT
Glucocorticoids (GCs), including dexamethasone (dex), are a central component of combination chemotherapy for childhood B-cell precursor acute lymphoblastic leukemia (B-ALL). GCs work by activating the GC receptor (GR), a ligand-induced transcription factor, which in turn regulates genes that induce leukemic cell death. Which GR-regulated genes are required for GC cytotoxicity, which pathways affect their regulation, and how resistance arises are not well understood. Here, we systematically integrate the transcriptional response of B-ALL to GCs with a next-generation short hairpin RNA screen to identify GC-regulated "effector" genes that contribute to cell death, as well as genes that affect the sensitivity of B-ALL cells to dex. This analysis reveals a pervasive role for GCs in suppression of B-cell development genes that is linked to therapeutic response. Inhibition of phosphatidylinositol 3-kinase δ (PI3Kδ), a linchpin in the pre-B-cell receptor and interleukin 7 receptor signaling pathways critical to B-cell development (with CAL-101 [idelalisib]), interrupts a double-negative feedback loop, enhancing GC-regulated transcription to synergistically kill even highly resistant B-ALL with diverse genetic backgrounds. This work not only identifies numerous opportunities for enhanced lymphoid-specific combination chemotherapies that have the potential to overcome treatment resistance, but is also a valuable resource for understanding GC biology and the mechanistic details of GR-regulated transcription.
Subject(s)
Glucocorticoids/therapeutic use , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cells, B-Lymphoid/drug effects , Precursor Cells, B-Lymphoid/pathology , Cell Death/drug effects , Cell Death/genetics , Cell Line, Tumor , Class I Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Class I Phosphatidylinositol 3-Kinases/genetics , Class I Phosphatidylinositol 3-Kinases/metabolism , Dexamethasone/pharmacology , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation/drug effects , Humans , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor Cells, B-Lymphoid/metabolism , Proto-Oncogene Proteins c-bcr/genetics , Proto-Oncogene Proteins c-bcr/metabolism , RNA, Small Interfering/genetics , Receptors, Glucocorticoid/drug effects , Signal TransductionABSTRACT
The telomere repeat amplification protocol (TRAP) for the human reverse transcriptase, telomerase, is a PCR-based assay developed two decades ago and is still used for routine determination of telomerase activity. The TRAP assay can only reproducibly detect â¼ 2-fold differences and is only quantitative when compared to internal standards and reference cell lines. The method generally involves laborious radioactive gel electrophoresis and is not conducive to high-throughput analyzes. Recently droplet digital PCR (ddPCR) technologies have become available that allow for absolute quantification of input deoxyribonucleic acid molecules following PCR. We describe the reproducibility and provide several examples of a droplet digital TRAP (ddTRAP) assay for telomerase activity, including quantitation of telomerase activity in single cells, telomerase activity across several common telomerase positive cancer cells lines and in human primary peripheral blood mononuclear cells following mitogen stimulation. Adaptation of the TRAP assay to digital format allows accurate and reproducible quantification of the number of telomerase-extended products (i.e. telomerase activity; 57.8 ± 7.5) in a single HeLa cell. The tools developed in this study allow changes in telomerase enzyme activity to be monitored on a single cell basis and may have utility in designing novel therapeutic approaches that target telomerase.
Subject(s)
Enzyme Assays/methods , Polymerase Chain Reaction/methods , Telomerase/analysis , Animals , Cell Line , Cell Line, Tumor , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , HeLa Cells , Humans , Lymphocytes/enzymology , MiceABSTRACT
During the process of branching morphogenesis, the mammary gland undergoes distinct phases of remodeling to form an elaborate ductal network that ultimately produces and delivers milk to newborn animals. These developmental events rely on tight regulation of critical cellular pathways, many of which are probably disrupted during initiation and progression of breast cancer. Transgenic mouse and in vitro organoid models previously identified growth factor signaling as a key regulator of mammary branching, but the functional downstream targets of these pathways remain unclear. Here, we used purified primary mammary epithelial cells stimulated with fibroblast growth factor-2 (FGF2) to model mammary branching morphogenesis in vitro. We employed a forward chemical genetic approach to identify modulators of this process and describe a potent compound, 1023, that blocks FGF2-induced branching. In primary mammary epithelial cells, we used lentivirus-mediated knockdown of the aryl hydrocarbon receptor (AHR) to demonstrate that 1023 acts through AHR to block branching. Using 1023 as a tool, we identified desmosomal adhesion as a novel target of AHR signaling and show that desmosomes are critical for AHR agonists to block branching. Our findings support a functional role for desmosomes during mammary morphogenesis and also in blocking FGF-induced invasion.
Subject(s)
Desmosomes/metabolism , Gene Expression Regulation, Neoplastic , Mammary Glands, Animal/metabolism , Mammary Neoplasms, Animal/genetics , Mammary Neoplasms, Animal/metabolism , Animals , Cell Adhesion , Cells, Cultured , Collagen/chemistry , Down-Regulation , Drug Combinations , Epithelial Cells/cytology , Female , Fibroblast Growth Factor 2/metabolism , Fibroblast Growth Factors/metabolism , Genetic Techniques , Laminin/chemistry , Mammary Glands, Animal/physiology , Mice , Morphogenesis , Proteoglycans/chemistry , RNA, Small Interfering/metabolism , Receptors, Aryl Hydrocarbon/metabolism , Signal TransductionABSTRACT
INTRODUCTION: High failure rates of new investigational drugs have impaired the development of breast cancer therapies. One challenge is that excellent activity in preclinical models, such as established cancer cell lines, does not always translate into improved clinical outcomes for patients. New preclinical models, which better replicate clinically-relevant attributes of cancer, such as chemoresistance, metastasis and cellular heterogeneity, may identify novel anti-cancer mechanisms and increase the success of drug development. METHODS: Metastatic breast cancer cells were obtained from pleural effusions of consented patients whose disease had progressed. Normal primary human breast cells were collected from a reduction mammoplasty and immortalized with human telomerase. The patient-derived cells were characterized to determine their cellular heterogeneity and proliferation rate by flow cytometry, while dose response curves were performed for chemotherapies to assess resistance. A screen was developed to measure the differential activity of small molecules on the growth and survival of patient-derived normal breast and metastatic, chemoresistant tumor cells to identify selective anti-cancer compounds. Several hits were identified and validated in dose response assays. One compound, C-6, was further characterized for its effect on cell cycle and cell death in cancer cells. RESULTS: Patient-derived cells were found to be more heterogeneous, with reduced proliferation rates and enhanced resistance to chemotherapy compared to established cell lines. A screen was subsequently developed that utilized both tumor and normal patient-derived cells. Several compounds were identified, which selectively targeted tumor cells, but not normal cells. Compound C-6 was found to inhibit proliferation and induce cell death in tumor cells via a caspase-independent mechanism. CONCLUSIONS: Short-term culture of patient-derived cells retained more clinically relevant features of breast cancer compared to established cell lines. The low proliferation rate and chemoresistance make patient-derived cells an excellent tool in preclinical drug development.
Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Small Molecule Libraries , Animals , Breast Neoplasms/drug therapy , Caspases/metabolism , Cell Death , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Drug Evaluation, Preclinical , Female , Humans , Immunophenotyping , Neoplasm Metastasis , Phenotype , Poly(ADP-ribose) Polymerases/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Chronic myelogenous leukemia (CML) is a hematopoietic stem cell malignancy that accounts for 15-20% of all cases of leukemia. CML is caused by a translocation between chromosomes 9 and 22 which creates an abnormal fusion gene, BCR::ABL1. The amount of BCR::ABL1 transcript RNA is a marker of disease progression and the effectiveness of tyrosine kinase inhibitor (TKI) treatment. This study determined the analytical and clinical performance of a droplet digital PCR based assay (QXDx BCR-ABL %IS Kit; Bio-Rad) for BCR::ABL1 quantification. The test has a limit of detection of MR4.7 (0.002%) and a linear range of MR0.3-4.7 (50-0.002%IS). Reproducibility of results across multiple sites, days, instruments, and users was evaluated using panels made from BCR::ABL1 positive patient samples. Clinical performance of the assay was evaluated on patient samples and compared to an existing FDA-cleared test. The reproducibility study noted negligible contributions to variance from site, instrument, day, and user for samples spanning from MR 0.7-4.2. The assay demonstrated excellent clinical correlation with the comparator test using a Deming regression with a Pearson R of 0.99, slope of 1.037 and intercept of 0.1084. This data establishes that the QXDx™ BCR-ABL %IS Kit is an accurate, precise, and sensitive system for the diagnosis and monitoring of CML.
Subject(s)
Fusion Proteins, bcr-abl , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Fusion Proteins, bcr-abl/genetics , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Polymerase Chain Reaction/methods , Protein Kinase Inhibitors/therapeutic use , Reproducibility of Results , United States , United States Food and Drug AdministrationABSTRACT
Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene seem to underlie the initiation of many colorectal carcinomas. Loss of APC function results in accumulation of beta-catenin and activation of beta-catenin/TCF-dependent transcription. Recent studies have implicated APC in controlling retinoic acid biosynthesis during normal intestinal development through a WNT-independent mechanism. Paradoxically, however, previous studies found that dietary supplementation of Apc(MIN) mice with retinoic acid failed to abrogate adenoma formation. While investigating the above finding, we found that expression of CYP26A1, a major retinoic acid catabolic enzyme, was up-regulated in Apc(MIN) mouse adenomas, human FAP adenomas, human sporadic colon carcinomas, and in the intestine of apc(mcr) mutant zebrafish embryos. Mechanistically, cyp26a1 induction following apc mutation is dependent on WNT signaling as antisense morpholino knockdown of tcf4 or injection of a dnLEF construct into apc(mcr) mutant zebrafish suppressed expression of cyp26a1 along with known WNT target genes. In addition, injection of stabilized beta-catenin or dnGSK3beta into wild-type embryos induced cyp26a1 expression. Genetic knockdown or pharmacologic inhibition of cyp26a1 in apc(mcr) mutant zebrafish embryos rescued gut differentiation defects such as expression of intestinal fatty acid-binding protein and pancreatic trypsin. These findings support a novel role for APC in balancing retinoic acid biosynthesis and catabolism through WNT-independent and WNT-dependent mechanisms.
Subject(s)
Adenomatous Polyposis Coli Protein/deficiency , Colonic Neoplasms/metabolism , Cytochrome P-450 Enzyme System/biosynthesis , Intestines/pathology , Wnt Proteins/metabolism , Adenomatous Polyposis Coli Protein/genetics , Animals , Cell Differentiation/drug effects , Cell Differentiation/physiology , Colonic Neoplasms/enzymology , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/genetics , Gene Expression Regulation, Enzymologic , Humans , Intestines/drug effects , Intestines/enzymology , Mice , Morpholines/pharmacology , Oligonucleotides/genetics , Oligonucleotides/pharmacology , Retinoic Acid 4-Hydroxylase , Signal Transduction , Tretinoin/metabolism , Tretinoin/pharmacology , Up-Regulation , Zebrafish , Zebrafish ProteinsABSTRACT
Melanoma lacks a clinically useful blood-based biomarker of disease activity to help guide patient management. To determine whether measurements of circulating, cell-free, tumor-associated BRAF(mutant) and NRAS(mutant) DNA (ctDNA) have a higher sensitivity than LDH to detect metastatic disease prior to treatment initiation and upon disease progression we studied patients with unresectable stage IIIC/IV metastatic melanoma receiving treatment with BRAF inhibitor therapy or immune checkpoint blockade and at least 3 plasma samples obtained during their treatment course. Levels of BRAF(mutant) and NRAS(mutant) ctDNA were determined using droplet digital PCR (ddPCR) assays. Among patients with samples available prior to treatment initiation ctDNA and LDH levels were elevated in 12/15 (80%) and 6/20 (30%) (p = 0.006) patients respectively. In patients with RECIST scores <5 cm prior to treatment initiation, ctDNA levels were elevated in 5/7 (71%) patients compared to LDH which was elevated in 1/13 (8%) patients (p = 0.007). Among all disease progression events the modified bootstrapped sensitivities for ctDNA and LDH were 82% and 40% respectively, with a median difference in sensitivity of 42% (95% confidence interval, 27%-58%; P < 0.001). In addition, ctDNA levels were elevated in 13/16 (81%) instances of non-RECIST disease progression, including 10/12 (83%) instances of new brain metastases. In comparison LDH was elevated 8/16 (50%) instances of non-RECIST disease progression, including 6/12 (50%) instances of new brain metastases. Overall, ctDNA had a higher sensitivity than LDH to detect disease progression, including non-RECIST progression events. ctDNA has the potential to be a useful biomarker for monitoring melanoma disease activity.
Subject(s)
Biomarkers, Tumor/blood , DNA, Neoplasm/blood , GTP Phosphohydrolases/genetics , Melanoma/blood , Membrane Proteins/genetics , Mutation , Proto-Oncogene Proteins B-raf/genetics , Cell-Free System , Disease Progression , Humans , L-Lactate Dehydrogenase/blood , Melanoma/pathology , Neoplasm Metastasis , Polymorphism, Single NucleotideABSTRACT
The recent introduction of Droplet Digital PCR (ddPCR) has provided researchers with a tool that permits direct quantification of nucleic acids from a wide range of samples with increased precision and sensitivity versus RT-qPCR. The sample interdependence of RT-qPCR stemming from the measurement of Cq and ΔCq values is eliminated with ddPCR which provides an independent measure of the absolute nucleic acid concentration for each sample without standard curves thereby reducing inter-well and inter-plate variability. Well-characterized RNA purified from H275-wild type (WT) and H275Y-point mutated (MUT) neuraminidase of influenza A (H1N1) pandemic 2009 virus was used to demonstrate a ddPCR optimization workflow to assure robust data for downstream analysis. The ddPCR reaction mix was also tested with RT-qPCR and gave excellent reaction efficiency (between 90% and 100%) with the optimized MUT/WT duplexed assay thus enabling the direct comparison of the two platforms from the same reaction mix and thermal cycling protocol. ddPCR gave a marked improvement in sensitivity (>30-fold) for mutation abundance using a mixture of purified MUT and WT RNA and increased precision (>10 fold, p<0.05 for both inter- and intra-assay variability) versus RT-qPCR from patient samples to accurately identify residual mutant viral population during recovery.
Subject(s)
Antiviral Agents/pharmacology , Drug Resistance, Viral , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H1N1 Subtype/genetics , Oseltamivir/pharmacology , Polymerase Chain Reaction/methods , Viral Load/methods , Child, Preschool , Humans , Influenza A Virus, H1N1 Subtype/classification , Influenza A Virus, H1N1 Subtype/isolation & purification , Influenza, Human/virology , Male , Neuraminidase/genetics , Reproducibility of Results , Sensitivity and Specificity , Viral Proteins/geneticsABSTRACT
In mammals, lactation is a rich source of nutrients and antibodies for newborn animals. However, millions of mothers each year experience an inability to breastfeed. Exposure to several environmental toxicants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has been strongly implicated in impaired mammary differentiation and lactation. TCDD and related polyhalogenated aromatic hydrocarbons are widespread industrial pollutants that activate the aryl hydrocarbon receptor (AHR). Despite many epidemiological and animal studies, the molecular mechanism through which AHR signaling blocks lactation remains unclear. We employed in vitro models of mammary differentiation to recapitulate lactogenesis in the presence of toxicants. We demonstrate AHR agonists directly block milk production in isolated mammary epithelial cells. Moreover, we define a novel role for the aryl hydrocarbon receptor repressor (AHRR) in mediating this response. Our mechanistic studies suggest AHRR is sufficient to block transcription of the milk gene ß-casein. As TCDD is a prevalent environmental pollutant that affects women worldwide, our results have important public health implications for newborn nutrition.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/agonists , Basic Helix-Loop-Helix Transcription Factors/drug effects , Environmental Pollutants/toxicity , Epithelial Cells/drug effects , Lactation/drug effects , Mammary Glands, Animal/drug effects , Polychlorinated Dibenzodioxins/toxicity , Receptors, Aryl Hydrocarbon/agonists , Repressor Proteins/drug effects , Animals , Aryl Hydrocarbon Receptor Nuclear Translocator/drug effects , Aryl Hydrocarbon Receptor Nuclear Translocator/genetics , Aryl Hydrocarbon Receptor Nuclear Translocator/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Caseins/genetics , Caseins/metabolism , Cells, Cultured , Down-Regulation , Epithelial Cells/metabolism , Epithelial Cells/pathology , Female , Mammary Glands, Animal/metabolism , Mammary Glands, Animal/pathology , Mammary Glands, Animal/physiopathology , Mice , RNA Interference , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism , Repressor Proteins/metabolism , Signal Transduction/drug effects , Transcription, Genetic/drug effects , TransfectionABSTRACT
OBJECTIVE: To determine whether human blastocysts secrete microRNA (miRNAs) into culture media and whether these reflect embryonic ploidy status and can predict in vitro fertilization (IVF) outcomes. DESIGN: Experimental study of human embryos and IVF culture media. SETTING: Academic IVF program. PATIENT(S): 91 donated, cryopreserved embryos that developed into 28 tested blastocysts, from 13 couples who had previously completed IVF cycles. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Relative miRNA expression in IVF culture media. RESULT(S): Blastocysts were assessed by chromosomal comparative genomic hybridization analysis, and the culture media from 55 single-embryo transfer cycles was tested for miRNA expression using an array-based quantitative real-time polymerase chain reaction analysis. The expression of the identified miRNA was correlated with pregnancy outcomes. Ten miRNA were identified in the culture media; two were specific to spent media (miR-191 and miR-372), and one was only present in media before the embryos had been cultured (miR-645). MicroRNA-191 was more highly concentrated in media from aneuploid embryos, and miR-191, miR-372, and miR-645 were more highly concentrated in media from failed IVF/non-intracytoplasmic sperm injection cycles. Additionally, miRNA were found to be more highly concentrated in ICSI and day-5 media samples when compared with regularly inseminated and day-4 samples, respectively. CONCLUSION(S): MicroRNA can be detected in IVF culture media. Some of these miRNA are differentially expressed according to the fertilization method, chromosomal status, and pregnancy outcome, which makes them potential biomarkers for predicting IVF success.
Subject(s)
Embryo Culture Techniques , Embryo Implantation/genetics , MicroRNAs/metabolism , Biomarkers/metabolism , Cohort Studies , Cryopreservation/methods , Embryo Culture Techniques/methods , Embryo Transfer/methods , Female , Humans , Male , MicroRNAs/biosynthesis , Pregnancy , Pregnancy Rate/trendsABSTRACT
OBJECTIVE: To determine the most highly expressed microRNAs (miRNAs) in human blastocysts and to compare miRNAs in euploid versus aneuploid embryos and in male versus female embryos. DESIGN: Experimental study of human embryos: 14 blastocysts (four male, five female, and five aneuploid) were evaluated for miRNA expression with the use of an array-based quantitative real-time polymerase chain reaction (qPCR). Highly expressed and differentially expressed miRNAs were confirmed with the use of qPCR in an expanded set of 27 blastocysts (seven male, eleven female, and nine aneuploid). SETTING: Academic IVF program. PATIENT(S): Thirteen couples donated 91 cryopreserved embryos for this study. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Relative miRNA expression in individual blastocysts. RESULT(S): The most highly expressed miRNA in euploid embryos was miR-372. Many of the highly expressed miRNAs have been shown to be critical to mammalian embryo development and to maintenance of stem cell pluripotency. Several differentially expressed miRNAs were discovered based on chromosomal makeup, including sex of the embryo. CONCLUSION(S): Human blastocysts express miRNAs, which may be important to their survival. Differential miRNA expression based on sex implies some degree of differentiation at the blastocyst stage of development. Differential miRNA expression between euploid and aneuploid embryos may be an early indicator of their prognosis or a mechanism behind their eventual fate.
Subject(s)
Blastocyst/physiology , Embryonic Development/genetics , Gene Expression Regulation, Developmental/genetics , MicroRNAs/genetics , Transcriptome/genetics , Aneuploidy , Cryopreservation , Embryo Culture Techniques , Female , Fertilization in Vitro , Humans , Male , Real-Time Polymerase Chain Reaction , Sex CharacteristicsABSTRACT
Human breast cancer is a heterogeneous disease composed of different histologies and molecular subtypes, many of which are not replicated in animal models. Here, we report a mouse model of breast cancer that generates unique tumor histologies including tubular, adenosquamous, and lipid-rich carcinomas. Utilizing a nononcogenic variant of polyoma middle T oncogene (PyMT) that requires a spontaneous base-pair deletion to transform cells, in conjunction with lentiviral transduction and orthotopic transplantation of primary mammary epithelial cells, this model sporadically induces oncogene expression in both the luminal and myoepithelial cell lineages of the normal mouse mammary epithelium. Microarray and hierarchical analyses using an intrinsic subtype gene set revealed that lentiviral PyMT generates both luminal and basal-like tumors. Cumulatively, these results show that low-level expression of PyMT in a broad range of cell types significantly increases tumor heterogeneity and establishes a mouse model of several rare human breast cancer subtypes.
ABSTRACT
Endometrial carcinoma is the most common gynecologic cancer, yet the mechanisms underlying this disease process are poorly understood. We hypothesized that Lef1 is required for endometrial gland formation within the uterus and is overexpressed in endometrial cancer. Using Lef1 knockout (KO) mice, we compared uterine gland development to wild-type (WT) controls, with respect to both morphology and expression of the Lef1 targets, cyclin D1 and MMP7. We characterized the dynamics of Lef1 protein expression during gland development and the mouse estrus cycle, by immunostaining and Western blot. Finally, we investigated the roles of cyclin D1 and MMP7 in gland and cancer formation in the mouse, and assessed the relevance of Lef1 to human cancer by comparing expression levels in cancerous and normal endometrial tissues. Lef1 upregulation in mouse endometrium correlates with the proliferative stages of the estrus cycle and gland development during the neonatal period. WT mice endometrial glands began to develop by day 5 and were easily identified by day 9, whereas Lef1 KO mice endometrial glands had not developed by day 9 although the endometrial lining was intact. We found that during gland development cyclin D1 is elevated and localized to the gland buds, and that this requires the presence of Lef1. We also noted that Lef1 protein was expressed at higher levels in endometrial cancers within mice and humans when compared to normal endometrium. Our loss-of-function data indicate that Lef1 is required for the formation of endometrial glands in the mouse uterus. Lef1 protein elevation corresponds to gland formation during development, and varies cyclically with the mouse estrus cycle, in parallel with gland regeneration. Finally, Lef1 is overexpressed in human and mouse endometrial tumors, consistent with it playing a role in gland proliferation.
Subject(s)
Adenocarcinoma/metabolism , Endometrial Neoplasms/metabolism , Endometrium/growth & development , Lymphoid Enhancer-Binding Factor 1/metabolism , Lymphoid Enhancer-Binding Factor 1/physiology , Adenocarcinoma/chemically induced , Adult , Aged , Aged, 80 and over , Animals , Cell Proliferation , Cell Transformation, Neoplastic , Cyclin D1/genetics , Cyclin D1/metabolism , Endometrial Neoplasms/chemically induced , Endometrium/metabolism , Endometrium/pathology , Estrous Cycle/metabolism , Female , Gene Expression Regulation, Developmental , Humans , Lymphoid Enhancer-Binding Factor 1/genetics , Male , Methylnitrosourea , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Knockout , Middle AgedABSTRACT
The mammary gland consists of an epithelial ductal tree embedded in a fat pad. Adult mammary epithelium has been demonstrated to have outstanding regenerative potential, consistent with the presence of resident, adult stem cells. However, there are currently no bona fide markers to identify these cells within their tissue context. Here, we introduce long-term label retention as a method to investigate the location of quiescent cells (a property attributed to adult stem cells) in situ. Long-term label retaining cells divide actively during tissue development and remain quiescent at homeostasis. These two properties have been attributed to adult stem cells. Therefore, label-retaining cells can be used to identify populations that contain stem cells. We describe the materials and methods necessary to identify and image mammary label-retaining cells, to carry out morphometric analysis on these cells and to map their distribution of the mammary epithelium. The morphometric and spatial analyses described here are generally applicable to any mammary cell populations, and will therefore be useful to characterize mammary stem cells once bona fide mammary stem cell markers become available.
Subject(s)
Mammary Glands, Animal/cytology , Staining and Labeling , Animals , Biomarkers/metabolism , Bromodeoxyuridine/metabolism , Female , Formaldehyde/metabolism , Mammary Glands, Animal/metabolism , Mice , Microscopy, Fluorescence , Molecular Imaging , Time FactorsABSTRACT
The regenerative potential of mammary epithelium facilitates assessment of the "stemness" of any epithelial subpopulation in transplantation assays. Thus, mammary tissue can be dissociated into single cells, stained for cell surface markers of interest and classified using fluorescence-activated cell sorting. The selected cells can then be transplanted into epithelium-devoided fat pads from recipient hosts. Recent publications have described markers that enrich for mammary repopulating potential. Here, we describe the materials and methods necessary to sort cells according to these markers. This approach can be used interchangeably with other cell surface markers with slight variation to the protocol.
Subject(s)
Flow Cytometry/methods , Mammary Glands, Animal/cytology , Stem Cells/metabolism , Animals , Biomarkers/metabolism , Mice , Staining and LabelingABSTRACT
Mammary reconstitution assays can be used to measure the stem cell frequency within an epithelial population by transplanting increasingly diluted single-cell preparations of the population of interest. There are fundamental steps in the single-cell isolation protocol which are directly related to the number of single epithelial cells obtained. Once single-cell suspensions have been obtained, serial dilutions are prepared and transplanted into the cleared fat pads of the host mice. After 8-10 weeks, the transplanted fat pads are reevaluated for the presence of epithelial outgrowths. Based on the frequency of no outgrowth for each one of the transplanted dilutions, it is possible to estimate the frequency of mammary repopulating cells present in a given cell population. Here, we give details on how to carry out all these steps.
Subject(s)
Cell Transplantation/methods , Mammary Glands, Animal/cytology , Stem Cells/cytology , Adipose Tissue/cytology , Adipose Tissue/transplantation , Animals , Cell Proliferation , Cell Separation , Centrifugation , Epithelial Cells/transplantation , Mice , Staining and LabelingABSTRACT
Mutations in adenomatous polyposis coli (APC) underlie the earliest stages of colorectal carcinogenesis. Consequences of APC mutation include stabilization of beta-catenin, dysregulation of cyclooxygenase-2 (COX-2) expression, and loss of retinoic acid production, events with poorly defined interactions. Here we showed that treatment of zebrafish expressing a truncated form of Apc with either retinoic acid or a selective COX-2 inhibitor decreased beta-catenin protein levels and downstream signaling events. Interestingly, the destruction of beta-catenin in apc mutant embryos following Cox-2 inhibition required the presence of truncated Apc. These findings support roles for retinoic acid and Cox-2 in regulating the stability of beta-catenin following Apc loss. Furthermore, truncated Apc appears to retain the ability to target beta-catenin for destruction, but only in the absence of Cox-2 activity. This novel function of truncated Apc may provide a molecular basis for the efficacy of COX-2 inhibitors in the treatment of colon cancer.
Subject(s)
Adenomatous Polyposis Coli/metabolism , Cyclooxygenase 2/metabolism , Gene Expression Regulation , Mutation , Tretinoin/pharmacology , beta Catenin/metabolism , Animals , Dinoprostone/metabolism , Down-Regulation , Immunoblotting , In Situ Hybridization , RNA/metabolism , Signal Transduction , Zebrafish , beta Catenin/antagonists & inhibitorsABSTRACT
Mutations in the adenomatous polyposis coli (APC) gene result in uncontrolled proliferation of intestinal epithelial cells and are associated with the earliest stages of colorectal carcinogenesis. Cyclooxygenase-2 (COX-2) is elevated in human colorectal cancers and plays an important role in colorectal tumorigenesis; however, the mechanisms by which APC mutations result in increased COX-2 expression remain unclear. We utilized APC mutant zebrafish and human cancer cells to investigate how APC modulates COX-2 expression. We report that COX-2 is up-regulated in APC mutant zebrafish because of a deficiency in retinoic acid biosynthesis. Treatment of both APC mutant zebrafish and human carcinoma cell lines with retinoic acid significantly reduces COX-2 expression. Retinoic acid regulates COX-2 levels by a mechanism that involves participation of the transcription factor C/EBP-beta. APC mutant zebrafish express higher levels of C/EBP-beta than wild-type animals, and retinoic acid supplementation reduces C/EBP-beta expression to basal levels. Both morpholino knockdown of C/EBP-beta in APC mutant zebrafish and silencing of C/EBP-beta using small interfering RNA in HT29 colon cancer cells robustly decrease COX-2 expression. Our findings support a sequence of events in which mutations in APC result in impaired retinoic acid biosynthesis, elevated levels of C/EBP-beta, up-regulation of COX-2, increased prostaglandin E(2) accumulation, and activation of Wnt target genes.
Subject(s)
Cyclooxygenase 2/genetics , Tretinoin/physiology , Alternative Splicing , Animals , Base Sequence , Cell Line , Cell Line, Tumor , DNA Primers , Dinoprostone/metabolism , Gene Expression Regulation, Enzymologic , Genes, APC , Humans , Morpholines , RNA, Messenger/genetics , Wnt Proteins/genetics , Zebrafish , beta Catenin/physiologyABSTRACT
Congenital hypertrophy/hyperplasia of the retinal pigmented epithelium is an ocular lesion found in patients harboring mutations in the adenomatous polyposis coli (APC) tumor suppressor gene. We report that Apc-deficient zebrafish display developmental abnormalities of both the lens and retina. Injection of dominant-negative Lef reduced Wnt signaling in the lens but did not rescue retinal differentiation defects. In contrast, treatment of apc mutants with all-trans retinoic acid rescued retinal differentiation defects but had no apparent effect on the lens. We identified Rdh5 as a retina-specific retinol dehydrogenase controlled by APC. Morpholino knockdown of Rdh5 phenocopied the apc mutant retinal differentiation defects and was rescued by treatment with exogenous all-trans retinoic acid. Microarray analyses of apc mutants and Rdh5 morphants revealed a profound overlap in the transcriptional profile of these embryos. These findings support a model wherein Apc serves a dual role in regulating Wnt and retinoic acid signaling within the eye and suggest retinoic acid deficiency as an explanation for APC mutation-associated retinal defects such as congenital hypertrophy/hyperplasia of the retinal pigmented epithelium.
Subject(s)
Adenomatous Polyposis Coli Protein/metabolism , Eye/embryology , Tretinoin/metabolism , Wnt Proteins/metabolism , Zebrafish/embryology , Adenomatous Polyposis Coli Protein/deficiency , Adenomatous Polyposis Coli Protein/genetics , Animals , Eye/drug effects , Gene Expression Regulation, Developmental , Homozygote , Microinjections , Molecular Sequence Data , Mutation , Oligonucleotides, Antisense/pharmacology , Tretinoin/pharmacology , Zebrafish/geneticsABSTRACT
Retinoic acid (RA) is a potent signaling molecule that plays important roles in multiple and diverse developmental processes. The contribution of retinoic acid to promoting the development and differentiation of the vertebrate intestine and the factors that regulate RA production in the gut remain poorly defined. Herein, we report that the novel retinol dehydrogenase, rdh1l, is required for proper gut development and differentiation. rdh1l is expressed ubiquitously during early development but becomes restricted to the gut by 3 days postfertilization. Knockdown of rdh1l results in a robust RA-deficient phenotype including lack of intestinal differentiation, which can be rescued by the addition of exogenous retinoic acid. We report that adenomatous polyposis coli (APC) mutant zebrafish harbor an RA-deficient phenotype including aberrant intestinal differentiation and that these mutants can be rescued by treatment with retinoic acid or injection of rdh1l mRNA. Further, we have found that although APC mutants are deficient in rdh1l expression, they harbor increased expression of raldh2 suggesting the control of RA production by APC is via retinol dehydrogenase activity. These results provide genetic evidence that retinoic acid is required for vertebrate gut development and that the tumor suppressor APC controls the production of RA in the gut by regulating the expression of the retinol dehydrogenase, rdh1l.