Ectopic expression of KRASG12D and p53R167H in porcine mammary epithelial cells results in transformation and tumorigenesis

Ectopic Expression ◽

Mammary Epithelial ◽

Population Doubling ◽

Population Doubling Time ◽

Large Animal ◽

Transformed Cell ◽

We describe our initial studies in the development of an orthotopic, genetically-defined, large animal model of breast cancer, using immunocompetent pigs. Primary mammary epithelial cells were isolated from the porcine gland. Primary mammary cells were immortalized with hTERT, and then transformed cell lines were generated from these immortalized cells with oncogenic KRAS and dominant negative p53. The transformed cell lines outperformed the primary cells in terms proliferation, population doubling time, soft agar growth, 2D migration, and Matrigel invasion. Three transformed cell lines were selected based on in vitro performance, and were able to grow tumors when injected subcutaneously in nude mice, with undifferentiated morphology. Tumorigenic porcine mammary cell lines were generated in this report.

Porcine pancreatic ductal epithelial cells transformed with KRASG12D and SV40T are tumorigenic

Scientific Reports ◽

10.1038/s41598-021-92852-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Katie L. Bailey ◽

Sara B. Cartwright ◽

Neesha S. Patel ◽

Neeley Remmers ◽

Audrey J. Lazenby ◽

...

Keyword(s):

Pancreatic Cancer ◽

Animal Model ◽

Epithelial Cells ◽

Cell Line ◽

Cell Lines ◽

Large Animal Model ◽

Large Animal ◽

Transformed Cell ◽

Transformed Cell Line ◽

AbstractWe describe our initial studies in the development of an orthotopic, genetically defined, large animal model of pancreatic cancer. Primary pancreatic epithelial cells were isolated from pancreatic duct of domestic pigs. A transformed cell line was generated from these primary cells with oncogenic KRAS and SV40T. The transformed cell lines outperformed the primary and SV40T immortalized cells in terms of proliferation, population doubling time, soft agar growth, transwell migration and invasion. The transformed cell line grew tumors when injected subcutaneously in nude mice, forming glandular structures and staining for epithelial markers. Future work will include implantation studies of these tumorigenic porcine pancreatic cell lines into the pancreas of allogeneic and autologous pigs. The resultant large animal model of pancreatic cancer could be utilized for preclinical research on diagnostic, interventional, and therapeutic technologies.

Porcine pancreatic ductal epithelial cells transformed with KRAS G12D and SV40T are tumorigenic

10.21203/rs.3.rs-268379/v1 ◽

2021 ◽

Author(s):

Katie L. Bailey ◽

Sara B. Cartwright ◽

Neesha S. Patel ◽

Neeley Remmers ◽

Audrey J. Lazenby ◽

...

Keyword(s):

Pancreatic Cancer ◽

Animal Model ◽

Epithelial Cells ◽

Cell Line ◽

Cell Lines ◽

Large Animal Model ◽

Large Animal ◽

Transformed Cell ◽

Transformed Cell Line ◽

Abstract We describe our initial studies in the development of an orthotopic, genetically-defined, large animal model of pancreatic cancer. Primary pancreatic epithelial cells were isolated from pancreatic duct of domestic pigs. A transformed cell line was generated from these primary cells with oncogenic KRAS and SV40T. The transformed cell lines outperformed the primary and SV40T immortalized cells in terms of proliferation, population doubling time, soft agar growth, transwell migration and invasion. The transformed cell line grew tumors when injected subcutaneously in nude mice, forming glandular structures and staining for epithelial markers. Future work will include implantation studies of these tumorigenic porcine pancreatic cell lines into the pancreas of allogeneic and autologous pigs. The resultant large animal model of pancreatic cancer could be utilized for preclinical research on diagnostic, interventional, and therapeutic technologies.

The Effect of Bovine Colostrums IGF-I on Cultured Human Mammary Epithelial Cells Line HBL-100

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.343-344.1064 ◽

2011 ◽

Vol 343-344 ◽

pp. 1064-1069

Author(s):

Ji Xian Mo ◽

Bo Hui Zhang ◽

Xue Jun Gao

Keyword(s):

Cell Growth ◽

Epithelial Cells ◽

Doubling Time ◽

Mammary Epithelial ◽

Population Doubling ◽

Population Doubling Time ◽

Human Mammary Epithelial Cells ◽

Igf I ◽

Human Mammary Epithelial

This experimental studied the effect of the bovine colostrums IGF-I on the cultured human mammary epithelial cells line HBL-100. We selected the best optimal concentration of the bovine colostrums IGF-I on the HBL-100 and researched the cell growth curve, cell population doubling time and cell growth saturation density. Using HPLC to determine lactose content in order to reflect bovine colostrums IGF-I which would influence on the ability of lactation of cultured human mammary epithelial cells line HBL-100. The optimal IGF- I concentration was 200ng/ml, the minimum time of cell population doubling time was 13.8h, cell growth saturation density reached 8.602×105/ml; High concentrations of IGF-Ⅰhad inhibit trend to the HBL-100 cells; bovine colostrums IGF-I could promote the synthesis of the lactose of the HBL-100 significantly.

Porcine pancreatic ductal epithelial cells transformed with KRASG12D and SV40T are tumorigenic

10.1101/2021.01.15.425792 ◽

2021 ◽

Author(s):

Katie Bailey ◽

Sara B. Cartwright ◽

Neesha S. Patel ◽

Neeley Remmers ◽

Audrey J. Lazenby ◽

...

Keyword(s):

Pancreatic Cancer ◽

Animal Model ◽

Epithelial Cells ◽

Cell Line ◽

Cell Lines ◽

Large Animal Model ◽

Large Animal ◽

Transformed Cell ◽

Transformed Cell Line ◽

AbstractWe describe our initial studies in the development of an orthotopic, genetically-defined, large animal model of pancreatic cancer. Primary pancreatic epithelial cells were isolated from pancreatic duct of domestic pigs. A transformed cell line was generated from these primary cells with oncogenic KRAS and SV40T. The transformed cell lines outperformed the primary and SV40T immortalized cells in terms of proliferation, population doubling time, soft agar growth, transwell migration and invasion. The transformed cell line grew tumors when injected subcutaneously in nude mice, forming glandular structures and staining for epithelial markers. Future work will include implantation studies of these tumorigenic porcine pancreatic cell lines into the pancreas of allogeneic and autologous pigs. The resultant large animal model of pancreatic cancer could be utilized for preclinical research on diagnostic, interventional, and therapeutic technologies.

Inhibition of SIRT1 Catalytic Activity Increases p53 Acetylation but Does Not Alter Cell Survival following DNA Damage

Molecular and Cellular Biology ◽

10.1128/mcb.26.1.28-38.2006 ◽

2006 ◽

Vol 26 (1) ◽

pp. 28-38 ◽

Cited By ~ 294

Author(s):

Jonathan M. Solomon ◽

Rao Pasupuleti ◽

Lei Xu ◽

Thomas McDonagh ◽

Rory Curtis ◽

...

Keyword(s):

Dna Damage ◽

Catalytic Activity ◽

Epithelial Cells ◽

Cell Survival ◽

Cell Lines ◽

Trichostatin A ◽

Mammary Epithelial ◽

Human Mammary Epithelial Cells ◽

Human Mammary Epithelial

ABSTRACT Human SIRT1 is an enzyme that deacetylates the p53 tumor suppressor protein and has been suggested to modulate p53-dependent functions including DNA damage-induced cell death. In this report, we used EX-527, a novel, potent, and specific small-molecule inhibitor of SIRT1 catalytic activity to examine the role of SIRT1 in p53 acetylation and cell survival after DNA damage. Treatment with EX-527 dramatically increased acetylation at lysine 382 of p53 after different types of DNA damage in primary human mammary epithelial cells and several cell lines. Significantly, inhibition of SIRT1 catalytic activity by EX-527 had no effect on cell growth, viability, or p53-controlled gene expression in cells treated with etoposide. Acetyl-p53 was also increased by the histone deacetylase (HDAC) class I/II inhibitor trichostatin A (TSA). EX-527 and TSA acted synergistically to increase acetyl-p53 levels, confirming that p53 acetylation is regulated by both SIRT1 and HDACs. While TSA alone reduced cell survival after DNA damage, the combination of EX-527 and TSA had no further effect on cell viability and growth. These results show that, although SIRT1 deacetylates p53, this does not play a role in cell survival following DNA damage in certain cell lines and primary human mammary epithelial cells.

Searching for Small Molecules as Antibacterials: Non-Cytotoxic Diarylureas Analogues of Triclocarban

Antibiotics ◽

10.3390/antibiotics10020204 ◽

2021 ◽

Vol 10 (2) ◽

pp. 204

Author(s):

Alessia Catalano ◽

Domenico Iacopetta ◽

Antonio Rosato ◽

Lara Salvagno ◽

Jessica Ceramella ◽

...

Keyword(s):

Epithelial Cells ◽

Cell Lines ◽

Food Packaging ◽

Biological Evaluation ◽

Mammary Epithelial ◽

Cytotoxic Agents ◽

Over The Counter ◽

Hek 293 ◽

Human Mammary Epithelial

Triclocarban (TCC), a broad-spectrum lipophilic antimicrobial agent, is a diarylurea derivative that has been used for more than 60 years as a major ingredient of toys, clothing, food packaging materials, food industry floors, medical supplies and especially of personal care products, such as soaps, toothpaste and shampoo. In September 2016, the U.S. FDA banned nineteen antimicrobial ingredients, including TCC, in over-the-counter consumer antiseptic wash products, due to their toxicity. Withdrawal of TCC has prompted efforts to search for new antimicrobial compounds. In this paper, we present the synthesis and biological evaluation, as antibiotic and non-cytotoxic agents, of a series of diarylureas, analogues of TCC. These compounds are characterized by an intriguingly simple chemistry and can be easily synthesized. Among the synthesized compounds, 1ab and 1bc emerge as the most interesting compounds as they show the same activity of TCC (MIC = 16 µg/mL) against Staphylococcus aureus, and a higher activity than TCC against Enterococcus faecalis (MIC = 32 µg/mL versus MIC = 64 µg/mL). Moreover, 1ab and 1bc show no cytotoxicity towards the human mammary epithelial cells MCF-10A and embryonic kidney epithelial cells Hek-293, in opposition to TCC, which exhibits a marked cytotoxicity on the same cell lines and shows a good antitumor activity on a panel of cell lines tested.

Generation of tumorigenic porcine pancreatic ductal epithelial cells: toward a large animal model of pancreatic cancer

10.1101/267112 ◽

2018 ◽

Cited By ~ 3

Author(s):

Neeley Remmers ◽

Jesse L. Cox ◽

James A. Grunkemeyer ◽

Shruthi Aravind ◽

Christopher K. Arkfeld ◽

...

Keyword(s):

Pancreatic Cancer ◽

Animal Model ◽

Cell Lines ◽

Large Animal Model ◽

Large Animal ◽

Murine Models ◽

Transformed Cell ◽

AbstractBackground. A large animal model of pancreatic cancer would permit development of diagnostic and interventional technologies not possible in murine models, and also would provide a more biologically-relevant platform for penultimate testing of novel therapies, prior to human testing. Here, we describe our initial studies in the development of an autochthonous, genetically-defined, large animal model of pancreatic cancer, using immunocompetent pigs.Methods. Primary pancreatic epithelial cells were isolated from pancreatic duct of domestic pigs; epithelial origin was confirmed with immunohistochemistry. Three transformed cell lines subsequently were generated from these primary cells using expression of oncogenic KRAS and dominant negative p53, with/without knockdown of p16 and SMAD4. We tested these cell lines using in vitro and in vivo assays of transformation and tumorigenesis.Results. The transformed cell lines outperformed the primary cells in terms proliferation, population doubling time, soft agar growth, 2D migration, and Matrigel invasion, with the greatest differences observed when all four genes (KRAS, p53, p16, and SMAD4) were targeted. All three transformed cell lines grew tumors when injected subcutaneously in nude mice, demonstrating undifferentiated morphology, mild desmoplasia, and staining for both epithelial and mesenchymal markers. Injection into the pancreas of nude mice resulted in distant metastases, particularly when all four genes were targeted.Conclusions. Tumorigenic porcine pancreatic cell lines were generated. Inclusion of four genetic “hits” (KRAS, p53, p16, and SMAD4) appeared to produce the best results in our in vitro and in vivo assays. The next step will be to perform autologous or syngeneic implantation of these cell lines into the pancreas of immunocompetent pigs. We believe that the resultant large animal model of pancreatic cancer could supplement existing murine models, thus improving preclinical research on diagnostic, interventional, and therapeutic technologies.

Signaling and apoptosis differences between severe hypoxia and desferoxamine treatment of human epithelial cells

Biochemistry and Cell Biology ◽

10.1139/o08-106 ◽

2008 ◽

Vol 86 (5) ◽

pp. 425-436 ◽

Cited By ~ 4

Author(s):

Adrian Harold Box ◽

Carol Yuen ◽

Dragana Ponjevic ◽

Gordon H. Fick ◽

Douglas James Demetrick

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Epithelial Cells ◽

Cell Cycle Arrest ◽

Cell Lines ◽

Mammary Epithelial ◽

Severe Hypoxia ◽

Cycle Arrest ◽

Induced Apoptosis

The mechanisms by which cells undergo proliferation arrest or cell death in response to hypoxia are still not completely understood. Originally, we showed that HeLa and Hep3B carcinoma cells undergo different proliferation responses in hypoxia. We now show that these 2 cell lines also have different cell death responses to severe hypoxia, with HeLa showing both cell cycle arrest and apoptosis (as early as 12 h after hypoxia treatment), and Hep3B showing resistance to both. Hypoxia-induced apoptosis in Hela was associated with decreases of both phospho-S473- and -T308-AKT and loss of AKT function, whereas Hep3B cells were resistant to hypoxia-induced apoptosis and did not lose phospho-AKT or AKT function. We then decided to test if our observations were confirmed using a hypoxia mimic, desferoxamine. Desferoxamine treatment yielded cell cycle arrest in HeLa and moderate arrest in Hep3B but, surprisingly, did not induce notable apoptosis of either cell line with up to 24 h of treatment. Hypoxia-treated normal human mammary epithelial cells also showed hypoxia-induced apoptosis. Interestingly, in these cell lines, there was a complete correlation between loss of phospho-AKT and (or) total AKT, and susceptibility to hypoxia-induced apoptosis. Our data suggests a model in which regulated loss of active AKT at a precise time point in hypoxia may be associated with apoptosis in susceptible cells.

Effect of Invasion of Borrelia burgdorferi in Normal and Neoplastic Mammary Epithelial Cells

Antibiotics ◽

10.3390/antibiotics10111295 ◽

2021 ◽

Vol 10 (11) ◽

pp. 1295

Author(s):

Gauri Gaur ◽

Janhavi Y. Sawant ◽

Ankita S. Chavan ◽

Vishwa A. Khatri ◽

Yueh-Hsin Liu ◽

...

Keyword(s):

Breast Cancer ◽

Breast Carcinoma ◽

Epithelial Cells ◽

Borrelia Burgdorferi ◽

Cell Lines ◽

Carcinoma Cell ◽

Mammary Epithelial ◽

Carcinoma Cell Lines ◽

Breast Carcinoma Cell Lines

Borrelia burgdorferi, the causative agent of Lyme Disease, is known to be able to disseminate and colonize various organs and tissues of its hosts, which is very crucial for its pathogenicity and survival. Recent studies have shown the presence of B. burgdorferi DNA in various breast cancer tissues, in some with poor prognosis, which raises the question about whether B. burgdorferi can interact with mammary epithelial cells and could have any effect on their physiology, including tumorigenic processes. As the model in this study, we have used MCF 10A normal and MDA-MB-231 tumorigenic mammary epithelial cells and infected both cell lines with B. burgdorferi. Our immunofluorescence and confocal microscopy results showed that B. burgdorferi is capable of invading normal epithelial and breast carcinoma cell lines within 24 h; however, the infection rate for the breast carcinoma cell lines was significantly higher. While the infection of epithelial cells with B. burgdorferi did not cause any changes in cell proliferation rates, it showed a significant effect on the invasion and migratory capacity of the breast cancer cells, but not on the normal epithelial cells, as determined by Matrigel invasion and wound healing assays. We have also found that the levels of expression of several epithelial–mesenchymal transition (EMT) markers (fibronectin, vimentin, and Twist1/2) changed, with a significant increase in tissue remodeling marker (MMP-9) in MDA-MB-231 cells demonstrated by quantitative Western blot analyses. This observation further confirmed that B. burgdorferi infection can affect the in vitro migratory and invasive properties of MDA-MB-231 tumorigenic mammary epithelial cells. In summary, our results suggest that B. burgdorferi can invade breast cancer tumor cells and it can increase their tumorigenic phenotype, which urges the need for further studies on whether B. burgdorferi could have any role in breast cancer development.

The expression of an Ets1 transcription factor lacking its activation domain decreases uPA proteolytic activity and cell motility, and impairs normal tubulogenesis and cancerous scattering in mammary epithelial cells

Journal of Cell Science ◽

10.1242/jcs.111.11.1521 ◽

1998 ◽

Vol 111 (11) ◽

pp. 1521-1534 ◽

Cited By ~ 3

Author(s):

A. Delannoy-Courdent ◽

V. Mattot ◽

V. Fafeur ◽

W. Fauquette ◽

I. Pollet ◽

...

Keyword(s):

Cell Migration ◽

Epithelial Cells ◽

Cell Motility ◽

Cell Lines ◽

Serine Proteinase ◽

Mammary Epithelial ◽

Migration And Invasion ◽

Cell Migration And Invasion ◽

Ets Family

Cell migration and invasion play a crucial role during normal and pathological development. The expression of several members of the Ets family of transcription factors has been shown to correlate with the occurrence of these processes. In the present study, we investigated the effect of the expression of Ets1-DB, the DNA-binding domain of c-Ets1, on the functional properties of NMuMG and MMT epithelial cell lines, from normal and cancerous mouse mammary tissues, respectively. We found that stable expression of this Ets1-DB mutant inhibited, in both cell types, the gene expression and activity of urokinase type-plasminogen activator (uPA), a potential target of c-Ets1. uPA is a key serine proteinase in the proteolytic cascade leading to the degradation of the extracellular matrix. In two-dimensional cultures, expression of the Ets1-DB mutant resulted in a decrease in cell migration and invasion in both cell lines. In three-dimensional collagen gels, NMuMG cells underwent tubular morphogenesis, while MMT cells developed as scattered structures. The Ets1-DB mutant impaired the capacity of NMuMG cells to form tubules and reduced the ability of MMT cells to invade these gels. Similar inhibition of cell migration, invasion and morphogenesis were observed in non-infected NMuMG and MMT cell lines treated with aprotinin, a serine proteinase inhibitor, suggesting that the inhibition of the plasmin cascade mediates in part the biological effects induced by the Ets1-DB mutant. These results demonstrate that Ets family members are involved in the control of uPA activity, cell motility and invasion during normal tubular morphogenesis and cancerous scattering in mammary epithelial cells.