171 research outputs found
-fl-D-Arabinofuranosylcytosine Enhancement of Resistance to Several Antineoplastic Drugs in Mammalian Tissue Culture Cells
Simulating Properties of In Vitro Epithelial Cell Morphogenesis
How do individual epithelial cells (ECs) organize into multicellular structures? ECs are studied in vitro to help answer that question. Characteristic growth features include stable cyst formation in embedded culture, inverted cyst formation in suspension culture, and lumen formation in overlay culture. Formation of these characteristic structures is believed to be a consequence of an intrinsic program of differentiation and de-differentiation. To help discover how such a program may function, we developed an in silico analogue in which space, events, and time are discretized. Software agents and objects represent cells and components of the environment. āCellsā act independently. The āprogramā governing their behavior is embedded within each in the form of axioms and an inflexible decisional process. Relationships between the axioms and recognized cell functions are specified. Interactions between ācellsā and environment components during simulation give rise to a complex in silico phenotype characterized by context-dependent structures that mimic counterparts observed in four different in vitro culture conditions: a targeted set of in vitro phenotypic attributes was matched by in silico attributes. However, for a particular growth condition, the analogue failed to exhibit behaviors characteristic of functionally polarized ECs. We solved this problem by following an iterative refinement method that improved the first analogue and led to a second: it exhibited characteristic differentiation and growth properties in all simulated growth conditions. It is the first model to simultaneously provide a representation of nonpolarized and structurally polarized cell types, and a mechanism for their interconversion. The second analogue also uses an inflexible axiomatic program. When specific axioms are relaxed, growths strikingly characteristic of cancerous and precancerous lesions are observed. In one case, the simulated cause is aberrant matrix production. Analogue design facilitates gaining deeper insight into such phenomena by making it easy to replace low-resolution components with increasingly detailed and realistic components
Differential disruption of genomic integrity and cell cycle regulation in normal human fibroblasts by the HPV oncoproteins.
Genomic integrity is maintained by a network of cellular activities that assess the status of the genome at a given point in time, provide signals to proceed with or halt cell cycle progression, and provide for repair of damaged DNA. Mutations in any part of these pathways can have the ultimate effect of disturbing chromosomal integrity. Recent work suggests that p53 performs this integrator function in mammalian cells. Our present study demonstrates that in mortal cells, the expression of E6 and E7 viral oncoproteins of type 16 human papillomavirus each disrupts the integration of these signals by diverged pathways. Cells expressing E6 protein, which binds and degrades the p53 protein, exhibited alterations in cell cycle control when placed in drug and displayed the ability to amplify the CAD gene. The expression of E7, which binds different cellular proteins important for transformation, including Rb, led to a p53-independent alteration in cell cycle control, a widespread cytocidal response, and polyploidy as a mechanism of drug resistance. These results demonstrate that diverse perturbations of molecular pathways can have different effects on chromosomal integrity
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Changes in epithelial proportions and transcriptional state underlie major premenopausal breast cancer risks
The human breast undergoes lifelong remodeling in response to estrogen and progesterone, but hormone exposure also increases breast cancer risk. Here, we use single-cell analysis to identify distinct mechanisms through which breast composition and cell state affect hormone signaling. We show that prior pregnancy reduces the transcriptional response of hormone-responsive (HR+) epithelial cells, whereas high body mass index (BMI) reduces overall HR+ cell proportions. These distinct changes both impact neighboring cells by effectively reducing the magnitude of paracrine signals originating from HR+ cells. Because pregnancy and high BMI are known to protect against hormone-dependent breast cancer in premenopausal women, our findings directly link breast cancer risk with person-to-person heterogeneity in hormone responsiveness. More broadly, our findings illustrate how cell proportions and cell state can collectively impact cell communities through the action of cell-to-cell signaling networks
Abrogated Response to Cellular Stress Identifies DCIS Associated with Subsequent Tumor Events and Defines Basal-like Breast Tumors
SummaryApproximately 15%ā30% of women diagnosed with ductal carcinoma in situ (DCIS) develop a subsequent tumor event within 10 years after surgical lumpectomy. To date, little is known about the molecular pathways that confer this differential risk for developing subsequent disease. In this study, we demonstrate that expression of biomarkers indicative of an abrogated response to cellular stress predicts DCIS with worse outcome and is a defining characteristic of basal-like invasive tumors. Mechanistic studies identify the Rb pathway as a key regulator of this response. Conversely, biomarkers indicative of an intact response to cellular stress are strongly associated with a disease-free prognosis. Assessment of these biomarkers in DCIS begins to allow prediction of tumor formation years before it actually occurs
Regulatory network decoded from epigenomes of surface ectoderm-derived cell types
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-associated histone modifications in SE-derived cells, and for binding motifs of transcription factors important in keratinocyte and mammary gland biology. Thus, epigenomic analysis of cell types with common developmental origin reveals an epigenetic signature that underlies a shared gene regulatory network
Changes in epithelial proportions and transcriptional state underlie major premenopausal breast cancer risks
The human breast undergoes lifelong remodeling in response to estrogen and progesterone, but hormone exposure also increases breast cancer risk. Here, we use single-cell analysis to identify distinct mechanisms through which breast composition and cell state affect hormone signaling. We show that prior pregnancy reduces the transcriptional response of hormone-responsive (HR+) epithelial cells, whereas high body mass index (BMI) reduces overall HR+ cell proportions. These distinct changes both impact neighboring cells by effectively reducing the magnitude of paracrine signals originating from HR+ cells. Because pregnancy and high BMI are known to protect against hormone-dependent breast cancer in premenopausal women, our findings directly link breast cancer risk with person-to-person heterogeneity in hormone responsiveness. More broadly, our findings illustrate how cell proportions and cell state can collectively impact cell communities through the action of cell-to-cell signaling networks
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DNA hypomethylation within specific transposable element families associates with tissue-specific enhancer landscape
Introduction: Transposable element (TE) derived sequences comprise half of our genome and DNA methylome, and are presumed densely methylated and inactive. Examination of the genome-wide DNA methylation status within 928 TE subfamilies in human embryonic and adult tissues revealed unexpected tissue-specific and subfamily-specific hypomethylation signatures. Genes proximal to tissue-specific hypomethylated TE sequences were enriched for functions important for the tissue type and their expression correlated strongly with hypomethylation of the TEs. When hypomethylated, these TE sequences gained tissue-specific enhancer marks including H3K4me1 and occupancy by p300, and a majority exhibited enhancer activity in reporter gene assays. Many such TEs also harbored binding sites for transcription factors that are important for tissue-specific functions and exhibited evidence for evolutionary selection. These data suggest that sequences derived from TEs may be responsible for wiring tissue type-specific regulatory networks, and have acquired tissue-specific epigenetic regulation
p16(INK4a) Prevents Centrosome Dysfunction and Genomic Instability in Primary Cells
Aneuploidy, frequently observed in premalignant lesions, disrupts gene dosage and contributes to neoplastic progression. Theodor Boveri hypothesized nearly 100 years ago that aneuploidy was due to an increase in centrosome number (multipolar mitoses) and the resultant abnormal segregation of chromosomes. We performed immunocytochemistry, quantitative immunofluorescence, karyotypic analysis, and time-lapse microscopy on primary human diploid epithelial cells and fibroblasts to better understand the mechanism involved in the production of supernumerary centrosomes (more than two microtubule nucleating bodies) to directly demonstrate that the presence of supernumerary centrosomes in genomically intact cells generates aneuploid daughter cells. We show that loss of p16(INK4a) generates supernumerary centrosomes through centriole pair splitting. Generation of supernumerary centrosomes in human diploid epithelial cells was shown to nucleate multipolar spindles and directly drive production of aneuploid daughter cells as a result of unequal segregation of the genomic material during mitosis. Finally, we demonstrate that p16(INK4a) cooperates with p21 through regulation of cyclin-dependent kinase activity to prevent centriole pair splitting. Cells with loss of p16(INK4a) activity have been found in vivo in histologically normal mammary tissue from a substantial fraction of healthy, disease-free women. Demonstration of centrosome dysfunction in cells due to loss of p16(INK4a) suggests that, under the appropriate conditions, these cells can become aneuploid. Gain or loss of genomic material (aneuploidy) may provide the necessary proproliferation and antiapoptotic mechanisms needed for the earliest stages of tumorigenesis
Morphologically Normal-Appearing Mammary Epithelial Cells Obtained from High-Risk Women Exhibit Methylation Silencing of INK4a/ARF
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