Cell lines with extended in vitro growth potential from human renal proximal tubule: Characterization, response to inducers, and comparison with established cell lines

Racusen, Lorraine C.; Monteil, Christelle; Sgrignoli, Anita R.; Lucskay, Margit; Marouillat, Sylviane; Rhim, John G.S.; Morin, Jean Paul

doi:10.1016/s0022-2143(97)90180-3

Cited by 221 publications

(165 citation statements)

References 20 publications

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“…Cell Culture-The renal tubular epithelial cell line HKC, obtained from Dr. L. Racusen (22), and HK-2 obtained from the ATCC (Manassas, VA) were cultured in Dulbecco's modified Eagle's medium/F-12 supplemented with 10% fetal bovine serum, penicillin/streptomycin, amphotericin B, HEPES buffer, and glutamine. Human primary cultures of neonatal foreskin fibroblasts were a generous gift from Dr. J. Varga, established as previously described (23).…”

Section: Methodsmentioning

confidence: 99%

Role of SARA (SMAD Anchor for Receptor Activation) in Maintenance of Epithelial Cell Phenotype

Runyan

Hayashida

Hubchak

et al. 2009

Journal of Biological Chemistry

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By inducing epithelial-to-mesenchymal transition (EMT), transforming growth factor-␤ (TGF-␤) promotes cancer progression and fibrosis. Here we show that expression of the TGF-␤ receptor-associated protein, SARA (Smad anchor for receptor activation), decreases within 72 h of exposure to TGF-␤ and that this decline is both required and sufficient for the induction of several markers of EMT. It has been suggested recently that expression of the TGF-␤ signaling mediators, Smad2 and Smad3, may have different functional effects, with Smad2 loss being more permissive for EMT progression. We find that the loss of SARA expression leads to a concomitant decrease in Smad2 expression and a disruption of Smad2-specific transcriptional activity, with no effect on Smad3 signaling or expression. Further, the effects of inducing the loss of Smad2 mimic those of the loss of SARA, enhancing expression of the EMT marker, smooth muscle ␣-actin. Smad2 mRNA levels are not affected by the loss of SARA. However, the ubiquitination of Smad2 is increased in SARA-deficient cells. We therefore examined the E3 ubiquitin ligase Smurf2 and found that although Smurf2 expression was unaltered in SARA-deficient cells, the interaction of Smad2 and Smurf2 was enhanced. These results describe a significant role for SARA in regulating cell phenotype and suggest that its effects are mediated through modification of the balance between Smad2 and Smad3 signaling. In part, this is achieved by enhancing the association of Smad2 with Smurf2, leading to Smad2 degradation.

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Section: Methodsmentioning

confidence: 99%

Role of SARA (SMAD Anchor for Receptor Activation) in Maintenance of Epithelial Cell Phenotype

Runyan

Hayashida

Hubchak

et al. 2009

Journal of Biological Chemistry

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“…Human renal proximal tubular epithelial cells (RPTEC) were characterized by and obtained from Dr. Lorraine Racusen (Johns Hopkins University School of Medicine, Baltimore, MD) (14). Cells were grown in Renal Epithelial Cell Basal Medium (REBM, Clonetics, San Diego, CA) supplemented with REGM complex (0.5 l/ml hydrocortisone, 10 pg/ml hEGF, 0.5 g/ml epinephrine, 6.5 pg/ml triiodothyronine, 10 g/ml transferrin, 5 g/ml insulin, 1 g/ml gentamicin sulfate, and 2% FBS).…”

Section: Cell Culturesmentioning

confidence: 99%

VEGF Expression in Hypoxia and Hyperglycemia

Kim

Chen

Weinstein

et al. 2002

Journal of the American Society of Nephrology

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ABSTRACT. Vascular endothelial growth factor (VEGF), an angiogenic factor for endothelial cells, is produced by glomerular and tubular epithelia. Using immunoelectron microscopy, VEGF expression by podocytes (GEC) and the proximal tubular epithelium of rat kidney was confirmed. To elucidate the mechanisms of VEGF production and its physiologic consequences, studies were performed in cultured GEC and proximal tubular epithelial cells (RPTEC). Both GEC and RPTEC expressed VEGF-120 and 164 mRNA, as detected by quantitative RT-PCR. Hypoxia resulted in an increase in mRNA abundance, more robust in RPTEC than in GEC, and an increase in VEGF expression by 1.9- and 1.6-fold, respectively. 30 mM d-glucose, but not 30 mM l-glucose, resulted in the elevation of VEGF mRNA in RPTEC, but not in GEC, although both cell types showed a comparable modest increase in VEGF expression. Combined treatment (hypoxia and 30 mM d-glucose) resulted in an increase of VEGF mRNA only in RPTEC; however, an enhanced protein expression was detectable in both cell types. To investigate the role of VEGF in branching angiogenesis, “sandwich” co-cultures were applied with endothelial cells and capillary tube formation was compared under the above conditions. Both RPTEC and GEC induced VEGF-dependent capillary tube formation by co-cultured endothelial cells and in both cell types hypoxia further augmented angiogenesis. In contrast, 30 mM d-glucose suppressed angiogenesis in co-cultures with both cell types despite increased mRNA for VEGF receptors 1 and 2. This study shows (1) that VEGF produced by GEC and RPTEC is necessary for branching angiogenesis and (2) that hypoxic environment stimulates VEGF production by both epithelial cell types and augments branching angiogenesis, whereas (3) hyperglycemic microenvironment, although also stimulatory for VEGF production, fails to augment angiogenesis.

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“…These enzymes are markers for proximal tubule differentiation. Their activities were affected most severely in human RPTEC clones established previously by the expression of SV40 Tag alone (Racusen et al, 1997). Assays for these enzyme activities at several points in time (passage 5, 18, 37 and 54) showed comparable results.…”

Section: Biochemical Characterization Of the Immortalized Rptecsmentioning

confidence: 66%

“…Continuous proliferation of the immortalized cells in culture frequently downregulates the expression of tissuespecific genes when compared with their primary cell counterparts (Racusen et al, 1997;Kim et al, 2000). The presence of genes for cell immortalization also precludes the application of these cells in cell-based therapies due to safety concerns.…”

Section: Introductionmentioning

confidence: 99%

Cre-mediated reversible immortalization of human renal proximal tubular epithelial cells

Kowolik

Liang

et al. 2004

Oncogene

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Primary human renal proximal tubule epithelial cells (RPTECs) are of limited use for basic research and for clinical applications due to their limited lifespan in culture. Here we used two lentivirus vectors carrying the human telomerase (hTERT) and the SV40T antigen (Tag) flanked by loxP sites to reversibly immortalize RPTECs. Transduced RPTEC clones continued to proliferate while retaining biochemical and functional characteristics of primary cells. The clones exhibited contact-inhibited, anchorage-and growth factor-dependent growth and did not form tumors in nude mice, suggesting that the cells were not transformed. Transient Cre expression in these cells led to efficient proviral deletion, upregulation of some renal specific activities, and decreased growth rates. Ultimately, the cells underwent replicative senescence, indicating intact cell cycle control. Thus, reversible immortalization allows the expansion of human RPTECs, leading to large production of RPTECs that retain most tissue-specific properties.

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Cell lines with extended in vitro growth potential from human renal proximal tubule: Characterization, response to inducers, and comparison with established cell lines

Cited by 221 publications

References 20 publications

Role of SARA (SMAD Anchor for Receptor Activation) in Maintenance of Epithelial Cell Phenotype

Role of SARA (SMAD Anchor for Receptor Activation) in Maintenance of Epithelial Cell Phenotype

VEGF Expression in Hypoxia and Hyperglycemia

Cre-mediated reversible immortalization of human renal proximal tubular epithelial cells

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