scholarly journals Differential Requirements for the RAD51 Paralogs in Genome Repair and Maintenance in Human Cells

2019 ◽  
Author(s):  
Edwige B. Garcin ◽  
Stéphanie Gon ◽  
Rohit Prakash ◽  
Meghan R. Sullivan ◽  
Gregory J. Brunette ◽  
...  
Keyword(s):  
Mammary Epithelial Cells ◽  
Mutant Cell ◽  
Cell Types ◽  
Mammary Epithelial ◽  
Hek293 Cells ◽  
Growth Defects ◽  
Rad51 Paralogs ◽  
Mcf10a Cells ◽  
Different Cell Types ◽  
Sister Chromatid Recombination

ABSTRACTDeficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hypersensitivity to mitomycin C and olaparib. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers.

EGFP expression in goat mammary epithelial cells

2006 ◽  
Vol 3 (1) ◽  
pp. 71-74 ◽  
Author(s):  
Zheng Yue-Mao ◽  
An Zhi-Xing ◽  
Peng Xin-Rong ◽  
Shi Yu-Qiang ◽  
Zhang Yong
Keyword(s):  
Epithelial Cells ◽  
Fluorescent Protein ◽  
Mammary Epithelial Cells ◽  
Cell Types ◽  
Mammary Epithelial ◽  
Egfp Expression ◽  
Green Fluorescent ◽  
Mammary Gland Cells ◽  
Different Cell Types ◽  
Goat Mammary Epithelial Cells

AbstractPrimary culture of goat mammary gland cells was achieved by outgrowth of migrating cells from fragments of tissue. The fibroblast and epithelial cells were purified according to their different sensitivity to trypsin and the characteristics of goat mammary epithelial cells were observed under a light microscope. The results showed that the purified goat mammary epithelial cells retained normal characteristics until the 15th passage. The purified mammary epithelial cells propagated and formed a dome-like structure that resembled a nipple and was called a ‘milk orb’. The mammary epithelial cells could produce and secrete milk. There were different cell types: the majority were short shuttle-like or polygons, resembling a beehive; while some were large, flat and round; and others were elongated. The enhanced green fluorescent protein (EGFP) gene was transferred successfully into the goat mammary epithelial cells using electrotransfection, and its expression was observed under a fluoroscope.

scholarly journals Integrated computational and experimental identification of p53, KRAS and VHL mutant selection associated with CRISPR-Cas9 editing

2018 ◽  
Author(s):  
Sanju Sinha ◽  
Karina Barbosa Guerra ◽  
Kuoyuan Cheng ◽  
Mark DM Leiserson ◽  
David M Wilson ◽  
...  
Keyword(s):  
Gene Editing ◽  
Mutant Cell ◽  
Cell Types ◽  
Driver Mutations ◽  
Mutant Selection ◽  
Cancer Driver ◽  
Genome Wide ◽  
A Genome ◽  
Induced Changes ◽  
Different Cell Types

AbstractRecent studies have reported that CRISPR-Cas9 gene editing induces a p53-dependent DNA damage response in primary cells, which may select for cells with oncogenic p53 mutations11,12. It is unclear whether these CRISPR-induced changes are applicable to different cell types, and whether CRISPR gene editing may select for other oncogenic mutations. Addressing these questions, we analyzed genome-wide CRISPR and RNAi screens to systematically chart the mutation selection potential of CRISPR knockouts across the whole exome. Our analysis suggests that CRISPR gene editing can select for mutants of KRAS and VHL, at a level comparable to that reported for p53. These predictions were further validated in a genome-wide manner by analyzing independent CRISPR screens and patients’ tumor data. Finally, we performed a new set of pooled and arrayed CRISPR screens to evaluate the competition between CRISPR-edited isogenic p53 WT and mutant cell lines, which further validated our predictions. In summary, our study systematically charts and points to the potential selection of specific cancer driver mutations during CRISPR-Cas9 gene editing.

scholarly journals Epigenetic silencing of lncRNA MORT in 16 TCGA cancer types

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 211 ◽  
Author(s):  
Lukas Vrba ◽  
Bernard W. Futscher
Keyword(s):  
Dna Methylation ◽  
Mammary Epithelial Cells ◽  
Human Cancer ◽  
Large Fraction ◽  
Cell Types ◽  
Mammary Epithelial ◽  
The Cancer Genome Atlas ◽  
Human Mammary Epithelial ◽  
Cancer Types

We have previously described a hominid-specific long non-coding RNA, MORT (also known as ZNF667-AS1, Gene ID: 100128252), which is expressed in all normal cell types, but epigenetically silenced during cancer-associated immortalization of human mammary epithelial cells.  Initial analysis of The Cancer Genome Atlas (TCGA) showed that 15 of 17 cancer types, which represent the 10 most common cancers in women and men, display DNA methylation associated MORT silencing in a large fraction of their tumors.  In this study we analyzed MORT expression and DNA methylation state in the remaining 16 TCGA cancer types not previously reported.  Seven of the 16 cancer types showed DNA methylation linked MORT silencing in a large fraction of their tumors.  These are carcinomas (cervical cancer, and cancers of esophagus, stomach, and bile duct), and the non-epithelial tumors mesothelioma, sarcoma, and uterine carcinosarcoma.  Together with the findings from our previous report, MORT expression is silenced by aberrant DNA methylation in 22 of 33 of TCGA cancer types.  These 22 cancers include most carcinoma types, blood derived cancers and sarcomas.  In conclusion, results suggest that the MORT gene is one of the most common epigenetic aberrations seen in human cancer.  Coupled with the timing of MORT gene silencing during in vitro epithelial cell immortalization and its occurrence early in the temporal arc of human carcinogenesis, this provides strong circumstantial evidence for a tumor suppressor role for MORT.

The role of native bovine α-lactalbumin in bovine mammary epithelial cell apoptosis and casein expression

2008 ◽  
Vol 75 (3) ◽  
pp. 319-325 ◽  
Author(s):  
Lisa G Riley ◽  
Peter C Wynn ◽  
Peter Williamson ◽  
Paul A Sheehy
Keyword(s):  
Epithelial Cells ◽  
Mrna Expression ◽  
Mammary Epithelial Cells ◽  
Cell Types ◽  
Mammary Epithelial ◽  
Major Influence ◽  
Expression Levels ◽  
Bovine Mammary Epithelial Cells ◽  
Epithelial Cell Apoptosis

Folding variants of α-lactalbumin (α-la) are known to induce cell death in a number of cell types, including mammary epithelial cells (MEC). The native conformation of α-la however has not been observed to exhibit this biological activity. Here we report that native bovine α-la reduced the viability of primary bovine mammary epithelial cells (BMEC) and induced caspase activity in mammospheres, which are alveolar-like structures formed by culturing primary BMEC on extracellular matrix in the presence of lactogenic hormones. These observations suggest a possible role for bovine α-la in involution and/or maintaining the luminal space in mammary alveoli during lactation. In addition, co-incubation of bovine α-la in an in-vitro mammosphere model resulted in decreased β-casein mRNA expression and increased αs1- and κ-casein mRNA expression. This differential effect on casein expression levels is unusual and raises the possibility of manipulating expression levels of individual caseins to alter dairy processing properties. Manipulation of α-la levels could be further investigated for its potential to enhance milk protein expression and/or improve lactational persistency by influencing the balance between proliferation and apoptosis of BMEC, which has a major influence on the milk-producing capacity of the mammary gland.

Clonal analysis of morphological phenotype in cultured mammary epithelial cells from human milk

1982 ◽  
Vol 215 (1199) ◽  
pp. 231-240 ◽  
Keyword(s):  
Human Milk ◽  
Mammary Cancer ◽  
Mammary Epithelial Cells ◽  
Single Cells ◽  
Cell Types ◽  
Clonal Analysis ◽  
Mammary Epithelial ◽  
Cell Type ◽  
Open Cell ◽  
Morphological Phenotype

Three main types of colony forming epithelial cell, termed elongated, cuboidal and open, are found in cultures of human milk. Subculture of identified colonies, and cloning from single cells shows that each cell type can maintain its morphological phenotype, but in addition the cuboidal and open cell types can give rise to the elongated type. The results, which suggest a differentiation pathway starting with open cell types, are discussed in relation to differentiation studies on mammary cancer cells.

scholarly journals Breast-Specific Epigenetic Regulation of DeltaNp73 and Its Role in DNA-Damage-Response of BRCA1-Mutated Human Mammary Epithelial Cells

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2367
Author(s):  
Ayelet Avraham ◽  
Susanna Feldman ◽  
Sean Soonweng Cho ◽  
Ayala Kol ◽  
Lior Heler ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
High Risk ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Brca1 Mutation ◽  
Cell Types ◽  
Mammary Epithelial ◽  
Human Mammary Epithelial

The function of BRCA1/2 proteins is essential for maintaining genomic integrity in all cell types. However, why women who carry deleterious germline mutations in BRCA face an extremely high risk of developing breast and ovarian cancers specifically has remained an enigma. We propose that breast-specific epigenetic modifications, which regulate tissue differentiation, could team up with BRCA deficiency and affect tissue susceptibility to cancer. In earlier work, we compared genome-wide methylation profiles of various normal epithelial tissues and identified breast-specific methylated gene promoter regions. Here, we focused on deltaNp73, the truncated isoform of p73, which possesses antiapoptotic and pro-oncogenic functions. We showed that the promoter of deltaNp73 is unmethylated in normal human breast epithelium and methylated in various other normal epithelial tissues and cell types. Accordingly, deltaNp73 was markedly induced by DNA damage in human mammary epithelial cells (HMECs) but not in other epithelial cell types. Moreover, the induction of deltaNp73 protected HMECs from DNA damage-induced cell death, and this effect was more substantial in HMECs from BRCA1 mutation carriers. Notably, when BRCA1 was knocked down in MCF10A, a non-malignant breast epithelial cell line, both deltaNp73 induction and its protective effect from cell death were augmented upon DNA damage. Interestingly, deltaNp73 induction also resulted in inhibition of BRCA1 and BRCA2 expression following DNA damage. In conclusion, breast-specific induction of deltaNp73 promotes survival of BRCA1-deficient mammary epithelial cells upon DNA damage. This might result in the accumulation of genomic alterations and allow the outgrowth of breast cancers. These findings indicate deltaNp73 as a potential modifier of breast cancer susceptibility in BRCA1 mutation carriers and may stimulate novel strategies of prevention and treatment for these high-risk women.

scholarly journals A computer-guided design tool to increase the efficiency of cellular conversions

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sascha Jung ◽  
Evan Appleton ◽  
Muhammad Ali ◽  
George M. Church ◽  
Antonio del Sol
Keyword(s):  
High Efficiency ◽  
Mammary Epithelial Cells ◽  
Cell Types ◽  
Design Tool ◽  
Mammary Epithelial ◽  
Great Promise ◽  
Target Cells ◽  
Great Effort ◽  
Cell Conversion

AbstractHuman cell conversion technology has become an important tool for devising new cell transplantation therapies, generating disease models and testing gene therapies. However, while transcription factor over-expression-based methods have shown great promise in generating cell types in vitro, they often endure low conversion efficiency. In this context, great effort has been devoted to increasing the efficiency of current protocols and the development of computational approaches can be of great help in this endeavor. Here we introduce a computer-guided design tool that combines a computational framework for prioritizing more efficient combinations of instructive factors (IFs) of cellular conversions, called IRENE, with a transposon-based genomic integration system for efficient delivery. Particularly, IRENE relies on a stochastic gene regulatory network model that systematically prioritizes more efficient IFs by maximizing the agreement of the transcriptional and epigenetic landscapes between the converted and target cells. Our predictions substantially increased the efficiency of two established iPSC-differentiation protocols (natural killer cells and melanocytes) and established the first protocol for iPSC-derived mammary epithelial cells with high efficiency.

scholarly journals Induction of Endothelial PAS Domain Protein-1 by Hypoxia: Characterization and Comparison With Hypoxia-Inducible Factor-1α

Blood ◽  
1998 ◽  
Vol 92 (7) ◽  
pp. 2260-2268 ◽  
Author(s):  
M.S. Wiesener ◽  
H. Turley ◽  
W.E. Allen ◽  
C. Willam ◽  
K.-U. Eckardt ◽  
...  
Keyword(s):  
Mutant Cell ◽  
Hypoxia Inducible Factor ◽  
Cell Types ◽  
Pas Domain ◽  
Functional Studies ◽  
Hypoxia Inducible Factor 1 ◽  
Helix Loop Helix ◽  
Aryl Hydrocarbon ◽  
Domain Protein ◽  
Different Cell Types

Abstract Hypoxia results in adaptive changes in the transcription of a range of genes including erythropoietin. An important mediator is hypoxia-inducible factor-1 (HIF-1), a DNA binding complex shown to contain at least two basic helix-loop-helix PAS-domain (bHLH-PAS) proteins, HIF-1α and aryl hydrocarbon nuclear receptor translocator (ARNT). In response to hypoxia, HIF-1α is activated and accumulates rapidly in the cell. Endothelial PAS domain protein 1 (EPAS-1) is a recently identified bHLH-PAS protein with 48% identity to HIF-1α, raising the question of its role in responses to hypoxia. We developed specific antibodies and studied expression and regulation of EPAS-1 mRNA and protein across a range of human cell lines. EPAS-1 was widely expressed, and strongly induced by hypoxia at the level of protein but not mRNA. Comparison of the effect of a range of activating and inhibitory stimuli showed striking similarities in the EPAS-1 and HIF-1α responses. Although major differences were observed in the abundance of EPAS-1 and HIF-1α in different cell types, differences in the inducible response were subtle with EPAS-1 protein being slightly more evident in normoxic and mildly hypoxic cells. Functional studies in a mutant cell line (Ka13) expressing neither HIF-1α nor EPAS-1 confirmed that both proteins interact with hypoxically responsive targets, but suggest target specificity with greater EPAS-1 transactivation (relative to HIF-1α transactivation) of the VEGF promoter than the LDH-A promoter.

Jaagsiekte sheep retrovirus found in milk macrophages but not in milk lymphocytes or mammary gland epithelia of naturally infected sheep

2021 ◽  
pp. 104063872110391
Author(s):  
Marta Borobia ◽  
Marcelo De las Heras ◽  
Javier Godino ◽  
Luis M. Ferrer ◽  
Delia Lacasta ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Mammary Epithelial Cells ◽  
Surface Protein ◽  
Cell Types ◽  
Mammary Epithelial ◽  
Adherent Cells ◽  
Jaagsiekte Sheep Retrovirus ◽  
Viral Particles ◽  
Positive Results

Jaagsiekte sheep retrovirus (JSRV) causes ovine pulmonary adenocarcinoma. JSRV can be transmitted via infected colostrum or milk, which contain somatic cells (SCs) harboring JSRV provirus. Nevertheless, the cell types involved in this form of transmission and the involvement of the mammary gland remain unknown. We separated adherent cells (macrophages and monocytes) by plastic adherence, and lymphocytes (CD4+ and CD8+ T cells, and B cells) by flow cytometry, from SCs in milk samples from 12 naturally infected, PCR blood test JSRV–positive, subclinical ewes. These cell populations were tested by PCR to detect JSRV provirus. The ewes were euthanized, and mammary gland samples were analyzed immunohistochemically to detect JSRV surface protein. We did not detect JSRV provirus in any milk lymphocyte population, but milk adherent cells were positive in 3 of 12 sheep, suggesting a potential major role of this population in the lactogenic transmission of JSRV. Immunohistochemistry did not reveal positive results in mammary epithelial cells, pointing to a lack of participation of the mammary gland in the biological cycle of JSRV and reducing the probability of excretion of free viral particles in colostrum or milk.

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