Neurogenic and pericytic plasticity of conditionally immortalized cells derived from renal erythropoietin‐producing cells

The growth factor/receptor combination of hepatocyte growth factor (HGF)/c-met has been postulated to be critical for mesenchymal-to-epithelial conversion and tubule formation in the developing kidney. We therefore isolated and immortalized cells from embryonic kidneys of met -/- transgenic mice to determine whether these cells were epithelial and able to chemotax and form tubules in vitro. The cells were immortalized with retrovirus expressing human papillomavirus 16 (HPV 16) E6/E7 genes. Two rapidly dividing clones were isolated and found to express the epithelial cell markers cytokeratin, zonula occludens-1, and E-cadherin but not to express the fibroblast marker vimentin. The met -/- cells were able to chemotax in response to epidermal growth factor and transforming growth factor-alpha (TGF-alpha) and form tubules in vitro in response to TGF-alpha but not HGF. These experiments suggest that the HGF/c-met axis is not essential for epithelial cell development in the embryonic kidney and demonstrate that other growth factors are capable of supporting early tubulogenesis.

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Neuroprotective Potential of a Small Molecule RET Agonist in Cultured Dopamine Neurons and Hemiparkinsonian Rats

Journal of Parkinson s Disease ◽

10.3233/jpd-202400 ◽

2021 ◽

pp. 1-24

Author(s):

Juho-Matti Renko ◽

Arun Kumar Mahato ◽

Tanel Visnapuu ◽

Konsta Valkonen ◽

Mati Karelson ◽

...

Keyword(s):

Mapk Signaling ◽

Dopamine Neurons ◽

Dopamine Depletion ◽

Wild Type ◽

Disease Modifying Therapy ◽

Immortalized Cells ◽

Midbrain Dopamine ◽

6 Hydroxydopamine

Background: Parkinson’s disease (PD) is a progressive neurological disorder where loss of dopamine neurons in the substantia nigra and dopamine depletion in the striatum cause characteristic motor symptoms. Currently, no treatment is able to halt the progression of PD. Glial cell line-derived neurotrophic factor (GDNF) rescues degenerating dopamine neurons both in vitro and in animal models of PD. When tested in PD patients, however, the outcomes from intracranial GDNF infusion paradigms have been inconclusive, mainly due to poor pharmacokinetic properties. Objective: We have developed drug-like small molecules, named BT compounds that activate signaling through GDNF’s receptor, the transmembrane receptor tyrosine kinase RET, both in vitro and in vivo and are able to penetrate through the blood-brain barrier. Here we evaluated the properties of BT44, a second generation RET agonist, in immortalized cells, dopamine neurons and rat 6-hydroxydopamine model of PD. Methods: We used biochemical, immunohistochemical and behavioral methods to evaluate the effects of BT44 on dopamine system in vitro and in vivo. Results: BT44 selectively activated RET and intracellular pro-survival AKT and MAPK signaling pathways in immortalized cells. In primary midbrain dopamine neurons cultured in serum-deprived conditions, BT44 promoted the survival of the neurons derived from wild-type, but not from RET knockout mice. BT44 also protected cultured wild-type dopamine neurons from MPP +-induced toxicity. In a rat 6-hydroxydopamine model of PD, BT44 reduced motor imbalance and could have protected dopaminergic fibers in the striatum. Conclusion: BT44 holds potential for further development into a novel, possibly disease-modifying therapy for PD.

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Highly efficient establishment of immortalized cells from adult human liver

Methods in Cell Science ◽

10.1007/bf00986655 ◽

1995 ◽

Vol 17 (2) ◽

pp. 83-89 ◽

Cited By ~ 13

Author(s):

A. M. A. Pfeifer ◽

K. Mace ◽

Y. Tromvoukis ◽

M. M. Lipsky

Keyword(s):

Human Liver ◽

Adult Human ◽

Highly Efficient ◽

Immortalized Cells

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Myc and Human Papillomavirus Type 16 E7 Genes Cooperate To Immortalize Human Keratinocytes

Journal of Virology ◽

10.1128/jvi.00669-07 ◽

2007 ◽

Vol 81 (22) ◽

pp. 12689-12695 ◽

Cited By ~ 27

Author(s):

Xuefeng Liu ◽

Gary L. Disbrow ◽

Hang Yuan ◽

Vjekoslav Tomaić ◽

Richard Schlegel

Keyword(s):

Transcriptional Activation ◽

High Efficiency ◽

P53 Protein ◽

Significant Proportion ◽

Telomerase Activity ◽

Human Papillomaviruses ◽

Mrna Levels ◽

Crisis Period ◽

Cell Immortalization ◽

Immortalized Cells

ABSTRACT The E6 protein of the oncogenic human papillomaviruses (HPVs), in combination with the E7 protein, is essential for the efficient immortalization of human foreskin keratinocytes (HFKs). Since we recently demonstrated that E6 activates the human telomerase reverse transcriptase (hTERT) promoter via a Myc-dependent mechanism, we speculated that overexpressed Myc might be able to substitute for E6 in cell immortalization. Myc (similar to E6) was unable to immortalize HFKs when transduced alone, despite inducing high levels of telomerase activity. However, when transduced with E7, Myc immortalized HFKs following a brief but detectable crisis period. In contrast to E6 + E7-immortalized cells, the Myc + E7-immortalized cells expressed high levels of p53 protein as well as two p53-regulated proteins, p21 and hdm-2. The increase in p21 and hdm-2 proteins correlated directly with their mRNA levels, suggesting transcriptional activation of the respective genes by the overexpressed p53 protein. Interestingly, a significant proportion of the p53 protein in the Myc + E7-immortalized cells was localized to the cytoplasm, potentially due to interactions with the overexpressed hdm-2 protein. Regardless, cell immortalization by the Myc + E7 genes occurred independently of p53 degradation. Since we have already observed high-efficiency cell immortalization with the hTERT + E7 or E6 mutant (p53 degradation-defective) + E7 genes (i.e., no crisis period) that proceeds in the presence of high levels of p53, we hypothesize that the crisis period in the Myc + E7 cells is due not to the levels of the p53 protein but rather to unique properties of the Myc protein. The common factor in cell immortalization by the three gene sets (E6 + E7, Myc + E7, and hTERT + E7 genes) is the induction of telomerase activity.

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Viral Entry Properties Required for Fitness in Humans Are Lost through Rapid Genomic Change during Viral Isolation

mBio ◽

10.1128/mbio.00898-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 9

Author(s):

Sho Iketani ◽

Ryan C. Shean ◽

Marion Ferren ◽

Negar Makhsous ◽

Dolly B. Aquino ◽

...

Keyword(s):

Primary Culture ◽

Parainfluenza Virus ◽

Clinical Samples ◽

Genomic Change ◽

Viral Isolation ◽

Dimer Interface ◽

Genome Wide ◽

A Genome ◽

Immortalized Cells ◽

Human Parainfluenza Virus

ABSTRACTHuman parainfluenza viruses cause a large burden of human respiratory illness. While much research relies upon viruses grown in cultured immortalized cells, human parainfluenza virus 3 (HPIV-3) evolves in culture. Cultured viruses differ in their properties compared to clinical strains. We present a genome-wide survey of HPIV-3 adaptations to culture using metagenomic next-generation sequencing of matched pairs of clinical samples and primary culture isolates (zero passage virus). Nonsynonymous changes arose during primary viral isolation, almost entirely in the genes encoding the two surface glycoproteins—the receptor binding protein hemagglutinin-neuraminidase (HN) or the fusion protein (F). We recovered genomes from 95 HPIV-3 primary culture isolates and 23 HPIV-3 strains directly from clinical samples. HN mutations arising during primary viral isolation resulted in substitutions at HN’s dimerization/F-interaction site, a site critical for activation of viral fusion. Alterations in HN dimer interface residues known to favor infection in culture occurred within 4 days (H552 and N556). A novel cluster of residues at a different face of the HN dimer interface emerged (P241 and R242) and imply a role in HPIV-3-mediated fusion. Functional characterization of these culture-associated HN mutations in a clinical isolate background revealed acquisition of the fusogenic phenotype associated with cultured HPIV-3; the HN-F complex showed enhanced fusion and decreased receptor-cleaving activity. These results utilize a method for identifying genome-wide changes associated with brief adaptation to culture to highlight the notion that even brief exposure to immortalized cells may affect key viral properties and underscore the balance of features of the HN-F complex required for fitness by circulating viruses.IMPORTANCEHuman parainfluenza virus 3 is an important cause of morbidity and mortality among infants, the immunocompromised, and the elderly. Using deep genomic sequencing of HPIV-3-positive clinical material and its subsequent viral isolate, we discover a number of known and novel coding mutations in the main HPIV-3 attachment protein HN during brief exposure to immortalized cells. These mutations significantly alter function of the fusion complex, increasing fusion promotion by HN as well as generally decreasing neuraminidase activity and increasing HN-receptor engagement. These results show that viruses may evolve rapidly in culture even during primary isolation of the virus and before the first passage and reveal features of fitness for humans that are obscured by rapid adaptation to laboratory conditions.

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Safety evaluation of conditionally immortalized cells for renal replacement therapy

Oncotarget ◽

10.18632/oncotarget.27152 ◽

2019 ◽

Vol 10 (51) ◽

pp. 5332-5348 ◽

Cited By ~ 1

Author(s):

Milos Mihajlovic ◽

Sam Hariri ◽

Koen C.G. Westphal ◽

Manoe J. Janssen ◽

Miriam J. Oost ◽

...

Keyword(s):

Renal Replacement Therapy ◽

Replacement Therapy ◽

Safety Evaluation ◽

Immortalized Cells

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c-Myc Overexpression Uncouples DNA Replication from Mitosis

Molecular and Cellular Biology ◽

10.1128/mcb.19.8.5339 ◽

1999 ◽

Vol 19 (8) ◽

pp. 5339-5351 ◽

Cited By ~ 78

Author(s):

Qing Li ◽

Chi V. Dang

Keyword(s):

Mitotic Spindle ◽

Cyclin Dependent Kinase ◽

Wild Type ◽

Apoptotic Pathway ◽

Chromosomal Segregation ◽

Cdk2 Activity ◽

Murine Fibroblasts ◽

Immortalized Cells ◽

Primary Fibroblasts ◽

Massive Apoptosis

ABSTRACT c-myc has been shown to regulate G1/S transition, but a role for c-myc in other phases of the cell cycle has not been identified. Exposure of cells to colcemid activates the mitotic spindle checkpoint and arrests cells transiently in metaphase. After prolonged colcemid exposure, the cells withdraw from mitosis and enter a G1-like state. In contrast to cells in G1, colcemid-arrested cells have decreased G1 cyclin-dependent kinase activity and show hypophosphorylation of the retinoblastoma protein. We have found that overexpression of c-myc causes colcemid-treated human and rodent cells to become either apoptotic or polyploid by replicating DNA without chromosomal segregation. Although c-myc-induced polyploidy is not inhibited by wild-type p53 in immortalized murine fibroblasts, overexpression of c-myc in primary fibroblasts resulted in massive apoptosis of colcemid-treated cells. We surmise that additional genes are altered in immortalized cells to suppress the apoptotic pathway and allow c-myc-overexpressing cells to progress forward in the presence of colcemid. Our results also suggest that c-myc induces DNA rereplication in this G1-like state by activating CDK2 activity. These observations indicate that activation of c-myc may contribute to the genomic instability commonly found in human cancers.

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Establishment and characterization of conditionally immortalized cells from the mouse urogenital ridge

Journal of Cell Science ◽

10.1242/jcs.109.5.899 ◽

1996 ◽

Vol 109 (5) ◽

pp. 899-909 ◽

Cited By ~ 2

Author(s):

B. Capel ◽

J.R. Hawkins ◽

E. Hirst ◽

D. Kioussis ◽

R. Lovell-Badge

Keyword(s):

Primordial Germ Cells ◽

Cell Types ◽

T Antigen ◽

Temperature Sensitive ◽

Sv40 Large T Antigen ◽

In Vitro Morphogenesis ◽

Immortalized Cells ◽

Clonal Lines

Cell cultures from the urogenital ridge have been established to facilitate the study of the regulation and downstream interactions of Sry in mammalian sex determination. Cells have been explanted from transgenic mice carrying a temperature sensitive SV40 large T-antigen, and established in ongoing cultures. Analysis of the cells in these cultures at the electron microscope level reveals multiple cell types that compare to the cell types found in vivo during this period of development. Primordial germ cells, that are simultaneously explanted in the course of these experiments, also survive in culture. The explants undergo a morphogenetic organization into branching cord-like structures when cells are trypsinized and plated in extracellular matrix (Matrigel). We analyzed the expression of a number of molecular markers of the fetal gonad during monolayer culture, during in vitro morphogenesis in Matrigel, and in clonal lines derived from the complex explants. This analysis included Sry which is found to be expressed in some cultures from XY urogenital ridges that have been maintained for as long as 8 months.

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